In Vitro Assessment of Macleaya Cordata Crude Extract Bioactivity and Anticancer
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Experimental and Toxicologic Pathology 65 (2013) 775–787 Contents lists available at SciVerse ScienceDirect Experimental and Toxicologic Pathology jo urnal homepage: www.elsevier.de/etp In vitro assessment of Macleaya cordata crude extract bioactivity and anticancer properties in normal and cancerous human lung cells a,b,∗ b a a,c a Min Liu , Yu-ling Lin , Xuan-Ren Chen , Chi-Cheng Liao , Wak-Kim Poo a Department of Life Science, Chinese Culture University, Taipei, Taiwan, ROC b Graduate Institute of Biotechnology, Chinese Culture University, Taipei, Taiwan, ROC c Graduate Institute of Earth Science, Chinese Culture University, Taipei, Taiwan, ROC a r t i c l e i n f o a b s t r a c t Article history: The purpose of this study is to assess the bioactivity and anticancer properties of Macleaya cordata crude Received 20 August 2012 extract in vitro using normal fetal lung fibroblast MRC5 and adenocarcinomic epithelial cell A549 as Accepted 8 November 2012 model systems,. Treatment of extract induced cell detachment, rounding, and irregularity in shape, in both normal and adenocarcinomic human lung cells, in accompanied of significant reduction in cell pro- Keywords: liferation. The data indicated that necrosis appeared to be involved in compromising cell growth in both Macleaya cordata types of lung cells since membrane permeability and cell granularity were elevated. Although apoptosis Human lung cancers was evident, the responses were differential in normal and diseased lung cells. Viability of treated MRC5 A549 MRC5 cells was reduced in a dose-dependent manner, demonstrating that the normal lung cells are sensitive Cytotoxicity to the extract. Surprisingly, A549 viability was slightly elevated in response to extract exposure at low Anticancer concentration, implying that cells survived were metabolically active; the viability was reduced accord- ingly to treatment at higher concentrations. The present findings demonstrate that the crude extract of M. cordata contains agents affecting the functioning of normal and diseased lung cells in vitro. The observed cytotoxic effects against adenocarcinomic lung cells validate the potential of using M. cordata as herbal intervention in combined with conventional chemotherapy for lung cancer treatment. © 2012 Elsevier GmbH. All rights reserved. 1. Introduction thought to be highly poisonous. Historically, the plant was used as a common medication to temporarily relieve muscle pain and Macleaya cordata (Willd.) R. Br. is a deciduous perennial plant to treat inflamed wounds, as well as bee sting. In the modern in the family Papaveraceae, which flowers from July to August. The days, M. cordata is extensively used in traditional Chinese medicine plant is found almost anywhere in temperate North America east for the treatment of wound, arthritis, rheumatism arthralgia, and of the Mississippi River at elevations below 1000 m sea level and trichomonas vaginalis (Jiangsu, 1986). M. cordata is also a tradi- is native to temperate eastern Asia (e.g. China and Japan). M. cor- tional medicinal plant in North America, Europe to manage insect data contains quaternary benzo[c]phenanthridine alkaloids (QBA) bites (Grieve, 1984) and ringworm infection (Duke and Ayenus, and protopine type alkaloids, which are important classes of alka- 1984). As a folk medicine, the roots of M. cordata are given to can- loids in the isoquinoline alkaloids family. Literature reports that cer patients as an alternative therapy in China (Xu et al., 1997). the fraction of QBA and protopine type alkaloids found in M. cordata Experimental studies show that this plant has a wide spectrum of are mainly composed of sanguinarine (SG), chelerythrine (CH), pro- biological activities such as anti-microbial (Walterova et al., 1995), topine (PR), and allocryptopine (AL) (Kosina et al., 2010). In China, anti-fungal (Newman et al., 1999), pesticidal (Yang, 2003), anti- M. cordata is a wild plant and commonly distributed in Shanxi, inflammatory, as well as anti-tumor properties (Pang et al., 2005). Guizhou, Yunnan provinces. This plant was first described as a M. cordata is on the European Food Safety Authority (EFSA) list of medicinal plant in Ben Cao Shi Yi (“A Supplement to Materia Med- plants exploited as a component in feed additives in animal pro- ica”), which is one of the most important Chinese medical material duction (Franz et al., 2005), and the powered mixture of leaves, books written by Chen Can-Qi. M. cordata was first recorded in capsules, and seeds is the main ingredient of the feed additive this book and prescribed for external use only since the plant was Sangrovit for rearing pigs, broilers and dairy cattle (Psotova et al., 2006; Stiborova et al., 2008; Vieira et al., 2008). This commercially available product has been reported to increase intestinal secre- ∗ tion of enzymes in livestock and possess bacteriostatic, mucolytic Corresponding author at: Department of Life Science, Chinese Culture Univer- and anti-inflammatory effects (Lindermayer, 2005). As reported in sity, Taipei, Taiwan, ROC. Tel.: +886 228610511; fax: +886 228623724. E-mail address: [email protected] (M. Liu). safety assessment studies, administration of M. cordata extract to 0940-2993/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.etp.2012.11.004 776 M. Liu et al. / Experimental and Toxicologic Pathology 65 (2013) 775–787 experimental animals did not affect animal’s body weight, organ recognition among scientific field and the acceptance of herbal weight, organ morphological structure, hematological parameters, medicine as an therapeutic option by the West is now emerging. as well as oxidative stress parameters (Zdarilova et al., 2008). Historically, medicine derived from several herbal plants such as The extract was also demonstrated to be non-genotoxic in model Platycodon grandiflorum (Campanulaceae), Morus alba (Moraceae), animals (Psotova et al., 2006; Stiborova et al., 2008). Moreover, Prunusarmenica (Rosaceae) and Rhus verniciflua (Anacardiaceae), rats with acute hepatic injuries caused by tetrachloromethane or Perilla frutescens (Labiatae), Stemona japonica (Stemonaceae), Tussi- galactosamine not only showed improved liver and lymphocyte lago farfara (Compositae) and Draba nemorosa (Brassicaceae) have functions but also reduced mortality following the treatment of M. been frequently used for lung diseases, including cancer, as folk cordata extract (Yang et al., 1999), in support of the use of Sangrovit remedies and medicines (Jeong et al., 2011). Generally, anti-cancer in live stock. However, cytotoxicity to human and porcine hepato- phytochemicals exhibited their pharmacological effects by induc- cytes was reported when cells were exposed to the extract in vitro ing apoptosis and/or inhibiting angiogenesis and metastasis by (Ulrichova et al., 2001). Elevation in the expression of two apoptotic targeting molecules modulating these biological processes. How- markers, Bcl-2 and Bax proteins was evident in cultures of rodent ever, a proper in vitro assessment of any potential phytochemical myocardial cells (Zhang et al., 2009). In China and Russia, M. cordata agents using both normal and diseased model systems should be is widely cultivated and used as a primary source for the production done first to correctly identify any curative properties and possible of QBA, such as sanguinarine and chelerythrine. These two alkaloids side-effects of tested substances in initial attempts. Perturbation in are most well studied QBAs found in this plant, and the compo- cell proliferation and metabolic activities in both normal and malig- nents are reported to be the main active ingredients responsible nant tissues caused by the administration of tested anti-cancer the reported pharmacological effects (Franz et al., 2005). A prior agents should also be carefully examined to better predict the ther- study showed that sanguinarine and chelerythrine have inhibitory apeutic outcomes. effect on the activity of rat liver l-alanine-2-oxoglutarate amino- The development of anti-cancer agents is attractive for lung can- transferase, which is essential in regulating cellular metabolism cer therapy. Based on the preceding views of application of natural (Walterova et al., 1981). Interestingly, these two QBAs impaired products as alternative therapy, it is to our great interest to discover the functioning of aminopeptidase N and dipeptidyl peptidase IV, new plants and of which the extract with pharmacological poten- which are enzymologically important in the processes of cell activa- tial, especially in treating lung cancer. In the present study, normal tion and differentiation (Sedo et al., 2002). The observed inhibitory human fetal lung fibroblast cell line MRC5 and adenocarcinomic effects were so potent that the efficiency was comparable to that human alveolar basal epithelial cell line A549 with NSCLC origin of aminopeptidase N inhibitors Bestatin and Amastatin. were exposed to the crude extract of M. cordata and the effects on Herbal products and other supplements derived from plants are cell proliferation, viability, metabolism, as well as cell death were widely consumed in many countries. The use of herbal intervention assessed in vitro. is common in all regions of the developing world and rapidly grow- ing in developed countries (Cassileth, 1999; Cassileth and Vickers, 2. Materials and methods 2005). The use of substances with natural origin as medicines, also known as phytotherapy or complementary and alternative therapy 2.1.