This article was downloaded by: [University of Jordan] On: 08 October 2011, At: 23:50 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Natural Product Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20 Phytochemical screening and biological activities of Eminium spiculatum (Blume) Kuntze (family Araceae) Fatma U. Afifi a & Rana Abu-Dahab b a Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Jordan, Queen Rania Street, Amman 11942, Jordan b Department of Clinical Pharmacy, Faculty of Pharmacy, University of Jordan, Queen Rania Street, Amman 11942, Jordan Available online: 02 Aug 2011 To cite this article: Fatma U. Afifi & Rana Abu-Dahab (2011): Phytochemical screening and biological activities of Eminium spiculatum (Blume) Kuntze (family Araceae), Natural Product Research, DOI:10.1080/14786419.2011.565558 To link to this article: http://dx.doi.org/10.1080/14786419.2011.565558 PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: http://www.tandfonline.com/page/terms-and- conditions This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material. Natural Product Research 2011, 1–5, iFirst SHORT COMMUNICATION Phytochemical screening and biological activities of Eminium spiculatum (Blume) Kuntze (family Araceae) Fatma U. Afifia* and Rana Abu-Dahabb aDepartment of Pharmaceutical Sciences, Faculty of Pharmacy, University of Jordan, Queen Rania Street, Amman 11942, Jordan; bDepartment of Clinical Pharmacy, Faculty of Pharmacy, University of Jordan, Queen Rania Street, Amman 11942, Jordan (Received 24 June 2010; final version received 19 February 2011) Eminium spiculatum (Blume) Kuntze (Araceae) is an indigenous plant in Jordan used as an anticancer agent. The objective of this study was to determine the chemical composition of E. spiculatum and its antimicrobial, antiplatelet and antiproliferative activities. After isolation, antimicrobial activity was tested using well-in-agar method and in vitro platelet aggregation by aggrometric method. Antiproliferative activity was evaluated by Sulphorhodamine B assay. Luteolin, luteolin-7-O-glucoside, isoorientin, vitexin, chrysoeriol-7-O-glucoside and - sitosterol were isolated and their structures determined. Luteolin exhibited moderate antibacterial activity against Escherichia coli and resistant strains of Staphylococcus aureus in 1 mgmLÀ1 concentration. Luteolin, luteolin-7-O-gluco- side and vitexin inhibited ADP and collagen-induced platelet aggregation in a concentration-dependent manner. For the determination of the antiproliferative activity, MCF-7 and T47D were used. Luteolin demonstrated the highest À1 inhibitory activity with IC50 values of 14.92 and 18.49 mmol L for MCF-7 and T47D, respectively. This research provided evidence about the chemical compo- sition and biological activity of E. spiculatum. Keywords: Eminium spiculatum; flavonoids; antimicrobial; antiproliferative; antiplatelet 1. Introduction Family Araceae is represented in Jordan by three genera and five species, namely Arum palestinum Boiss., Arum dioscoridis Sm., Arum hygrophilum Boiss., Biarum angustatum (Hook. F.) N.E.Br. and Eminium spiculatum (Blume) Kuntze. In previous years, Downloaded by [University of Jordan] at 23:50 08 October 2011 A. palestinum was investigated in our laboratories and different flavone C-glycosides were isolated and their antimicrobial activities established (Afifi, Shervington, & Darwish, 1997). Recently, the antiproliferative activity of the same plant was evaluated using MCF- 7 and A549 cell lines (Abu Dahab & Afifi-Yazar, 2007). A literature survey indicated that no studies were carried out or published on E. spiculatum, the only representative of the genus Eminium grown wild in Jordan. The few published references (from 1967 to 1976) deal primarily with the amino acid and protein composition of this plant. The only report about the isolation of some flavonoids from the leaves of E. spiculatum grown in Greece dates back to 1988 (Shammas & Couladi, 1988). *Corresponding author. Email: [email protected] ISSN 1478–6419 print/ISSN 1478–6427 online ß 2011 Taylor & Francis http://dx.doi.org/10.1080/14786419.2011.565558 http://www.tandfonline.com 2 F.U. Afifi and R. Abu-Dahab In the course of our search for active compounds of plant origin, this phytochemical and biological study with E. spiculatum was initiated with two aims: (1) to identify and isolate the constituents of this species; and (2) determine the antimicrobial, antiplatelet and antiproliferative activities of E. spiculatum for the crude extract, as well as for the isolated compounds. To the best of our knowledge, this is the first report on the screening of E. spiculatum for different biological activities. 2. Results and discussion Numerous studies describe the different biological activities of the flavonoids including antimicrobial, antioxidant, antiplatelet and anticancer activities. Flavonoids have been demonstrated to affect the activity of many mammalian enzyme systems in vitro and in vivo (Middleton, Kandaswami, & Theoharides, 2000). In this study, from the flowering aerial parts of E. spiculatum five flavonoids – one flavone aglycon (luteolin), two flavone- O-glucosides (luteolin-7-O-glucoside and chrysoeriol-7-O-glucoside) and two flavone- C-glucosides (vitexin and isoorientin) and -sitosterol were isolated using conventional column chromatography. The occurrence of vitexin, isoorientin, luteolin-7-O-glucoside and chrysoeriol-7-O-glucoside has been reported in an earlier study carried out with E. spiculatum grown in Greece (Shammas & Couladi, 1988). This is the first report of isolation of luteolin and -sitosterol from this species. The structures of the isolated compounds were determined using their physical properties and the different spectroscopic data (UV, IR, 1H-NMR, 13C-NMR). The obtained results were in agreement with the values reported for luteolin, luteolin- 7-O-glucoside, vitexin, isoorientin, chrysoeriol-7-O-glucoside and -sitosterol (Aggrawal, 1989; Gupta & Singh, 1991; Karioti, Skaltsa, Heilmann, & Sticher, 2003; Rabanal, Valverde, Martin-Lomas, Rodriguez, & Chari, 1982). For all isolated compounds, melting points and mixed melting points with the reference substances were determined and confirmed. No pronounced antibacterial activity was observed with either the crude extract or with the isolated compounds. Luteolin exhibited moderate antibacterial activity against Staphylococcus aureus in concentrations of 0.625 mg mLÀ1, while negligible weak antibacterial activity (41mgmLÀ1) was observed for vitexin and isoorientin. These findings are in agreement with those of Afifi et al. (1997) and Li, Henry, and Seeram (2009) and in contrast to those of Yamomoto and Ogawa (2002) and Kuete et al. (2008), who found that luteolin was potent against oral pathogenic bacteria and against Downloaded by [University of Jordan] at 23:50 08 October 2011 different pathogenic microorganisms. Luteolin-7-O-glucoside and chrysoeriol-7-O-gluco- side were inactive against all tested microorganism strains. The weak antibacterial activity observed can be explained by the absence of OH at C-3 of these flavonoids since it is well accepted that one of the main requirements of the antibacterial activity for the flavonoids is the presence of a free OH at C-3 (Mori, Nishino, Enoki, & Tawata, 1987). Table 1 shows the results of the antiplatelet activity of the ethanol extract and of the three isolated compounds, and their calculated IC50 values (experiments run in triplicates). For isoorientin and chryseriol-7-O-glucoside, due to the low amount of the isolated pure compounds, the single-point test was performed twice for ADP and collagen at concentrations of 0.037 mg mLÀ1. At this concentration, chryseriol-inhibited aggregation of ADP by 63.8% and that of collagen by 39.5%. The least inhibition was observed with isoorientin (12.2%, 8.2% for ADP and collagen, respectively). Luteolin was the most potent compound in the inhibition of ADP-induced platelet aggregation while vitexin exhibited the highest potency in the inhibition of the collagen-induced platelet aggregation. Natural Product Research 3 Table 1. Biological activities of E. spiculatum crude extract and some of the isolated compounds. Platelet aggregation inhibition Antiproliferative activity À1 À1 À1 IC50 (mg mL )IC50 (mgmL ) Æ standard deviation (mmol L ) Collagen ADP MCF-7 T47D Crude extract (E. spiculatum) 0.133 0.108 138 Æ 26.8 190 Æ 5.21 Luteolin 0.034 0.015 5.33 Æ 0.379, (18.49) 4.30 Æ 0.08, (14.92) Luteolin-7-O-glucoside 0.046 0.067 Non-toxic (0.1 and Non-toxic (0.1 and 100 mgmLÀ1) 100 mgmLÀ1) Vitexin 0.021 0.030 Non-toxic (0.1 and Non-toxic (0.1 and 100 mgmLÀ1) 100 mgmLÀ1) Isoorientin # # 135 Æ 15.3, (301.08) 49.8 Æ 3.17, (111.06) Chrysoeriol-7-O-glucoside # # 77.86 Æ 4.58, (168.53) 67.61 Æ 7.90, (146.34) -Sitosterol NT NT 61.85 Æ 4.7, (149.74) 39.02 Æ 10.3, (94.09) Cisplatin (mmol LÀ1) 7.34 Æ 1.89 21.29 Æ 9.72 Doxorubicin (mmol LÀ1) 0.16 Æ 0.02 0.17 Æ 0.03 À1 Notes: The calculated IC50 values (mg mL ) in inhibiting the ADP and collagen-induced platelet aggregation and in vitro cytotoxicity activity (IC50 Æ standard deviation) against MCF-7 and T47D cell lines after 72 h incubation. Results express average percentage Æ standard deviation of six replicates done on at least two passages. # refers to text for single-point determination. NT, not determined.