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NPC Natural Product Communications

EDITOR-IN-CHIEF

DR. PAWAN K AGRAWAL Natural Product Inc. 7963, Anderson Park Lane, Westerville, Ohio 43081, USA agrawal@naturalproduct.us EDITORS

PROFESSOR ALESSANDRA BRACA Dipartimento di Chimica Bioorganicae Biofarmacia, Universita di Pisa, via Bonanno 33, 56126 Pisa, Italy braca@farm.unipi.it

PROFESSOR DEAN GUO State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100083, China gda5958@163.com

PROFESSOR J. ALBERTO MARCO Departamento de Quimica Organica, Universidade de Valencia, E-46100 Burjassot, Valencia, Spain alberto.marco@uv.es

PROFESSOR YOSHIHIRO MIMAKI School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Horinouchi 1432-1, Hachioji, Tokyo 192-0392, Japan mimakiy@ps.toyaku.ac.jp

PROFESSOR STEPHEN G. PYNE Department of Chemistry University of Wollongong Wollongong, New South Wales, 2522, Australia spyne@uow.edu.au

PROFESSOR MANFRED G. REINECKE Department of Chemistry, Texas Christian University, Forts Worth, TX 76129, USA m.reinecke@tcu.edu

PROFESSOR WILLIAM N. SETZER Department of Chemistry The University of Alabama in Huntsville Huntsville, AL 35809, USA wsetzer@chemistry.uah.edu

PROFESSOR YASUHIRO TEZUKA Institute of Natural Medicine Institute of Natural Medicine, University of Toyama, 2630-Sugitani, Toyama 930-0194, Japan tezuka@inm.u-toyama.ac.jp

PROFESSOR DAVID E. THURSTON Department of Pharmaceutical and Biological Chemistry, The School of Pharmacy, University of London, 29-39 Brunswick Square, London WC1N 1AX, UK david.thurston@pharmacy.ac.uk

ADVISORY BOARD Prof. Berhanu M. Abegaz Gaborone, Botswana

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Prof. Julie Banerji Kolkata, India

Prof. Anna R. Bilia Florence, Italy

Prof. Maurizio Bruno Palermo, Italy

Prof. Josep Coll Barcelona, Spain

Prof. Geoffrey Cordell Chicago, IL, USA

Prof. Cristina Gracia-Viguera Murcia, Spain

Prof. Duvvuru Gunasekar Tirupati, India

Prof. A.A. Leslie Gunatilaka Tucson, AZ, USA

Prof. Kurt Hostettmann Lausanne, Switzerland

Prof. Martin A. Iglesias Arteaga Mexico, D. F, Mexico

Prof. Jerzy Jaroszewski Copenhagen, Denmark

Prof. Leopold Jirovetz Vienna, Austria

Prof. Teodoro Kaufman Rosario, Argentina

Prof. Norbert De Kimpe Gent, Belgium

Prof. Karsten Krohn Paderborn, Germany

Prof. Hartmut Laatsch Gottingen, Germany

Prof. Marie Lacaille-Dubois Dijon, France

Prof. Shoei-Sheng Lee Taipei, Taiwan

Prof. Francisco Macias Cadiz, Spain

Prof. Imre Mathe Szeged, Hungary

Prof. Joseph Michael Johannesburg, South Africa

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Prof. Luc Pieters Antwerp, Belgium

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Prof. Peter Proksch Düsseldorf, Germany

Prof. Phila Raharivelomanana Tahiti, French Plynesia

Prof. Satyajit Sarker Wolverhampton, UK

Prof. Monique Simmonds Richmond, UK

Prof. Valentin Stonik Vladivostok, Russia

Prof. Winston F. Tinto Barbados, West Indies

Prof. Karen Valant-Vetschera Vienna, Austria

Prof. Peter G. Waterman Lismore, Australia

HONORARY EDITOR

PROFESSOR GERALD BLUNDEN The School of Pharmacy & Biomedical Sciences,

University of Portsmouth, Portsmouth, PO1 2DT U.K.

axuf64@dsl.pipex.com

In Vitro Testing for Genotoxicity of Indigo Naturalis Assessed by Micronucleus Test Luca Dominici, Barbara Cerbone, Milena Villarini, Cristina Fatigoni and Massimo Moretti*

Department of Medical-Surgical Specialties and Public Health (Section of Public Health), University of Perugia, Perugia, Italy massimo.moretti@unipg.it Received: February 19th, 2010; Accepted: May 19th, 2010

In the field of cosmetic dyes, used for coloring the hair and skin, there is a clear tendency to replace the widely used synthetic dyes by natural colorants, such as henna and mixtures of henna with indigo. The aim of this study was to estimate the genotoxicity of water and DMSO solutions of indigo naturalis (prepared from Indigofera tinctoria leaves) using the cytokinesis-blocked micronucleus (CBMN) assay in the human metabolically active HepG2 cell line. The cytotoxic effects of indigo solutions were first assessed by propidium iodide and fluorescein-diacetate simultaneous staining. For both solutions, cytotoxicity was always under 10%. Data obtained in the CBMN assay (for all concentrations tested) indicated that the frequency of MN (micronuclei) in exposed cells was no higher than the control. Both the water and DMSO solutions showed the same behavior. These results indicate that indigo naturalis exhibits neither cytotoxicity, nor genotoxicity for all concentrations tested, which may justify excluding indigofera and its components from the list of carcinogenic agents. Keywords: Indigofera tinctoria, Indigo naturalis, HepG2 cells, Genotoxicity, Micronucleus test. In the field of cosmetic dyes, used for coloring the hair and skin, there is a clear tendency to replace the widely used synthetic dyes by natural colorants, such as henna and mixtures of henna with indigo. Henna gives a red to orange color but if it is combined with indigo (Figure 1), it yields a brown color, so-called henna black. Indigo-related pigments are widely used, not only in cosmetics, but also in textile, pottery and mural paintings [1]. Blue dye indigo is extracted principally from the leaves of Indigofera tinctoria Linn., a perennial shrub, belonging to the family Fabaceae (alt. Leguminosae), cultivated in India, China and other countries. Indigo is also widely used in traditional Chinese and Indian medicine to treat various inflammatory diseases and dermatosis [2], epilepsy, nervous disorders, bronchitis and liver ailments [3,4]. Indigo is said to be good for mouth ulcers and, externally, in ointment form, is used for infected ulcers, psoriasis, hemorrhoids and sore nipples [5-8]. Although much work has been done on the pharmacological properties of indigo, very few studies have evaluated its genotoxic potential [9,10] or that of extracts from species belonging to the genus Indigofera (I. truxillensis and I. suffruticosa) [11].

Figure 1: Chemical structure of indigo, 2,2’-Bis(2,3-dihydro-3- oxoindolyliden (C16H10N2O2) [CAS Reg. # 482-89-3]. With regard to indigo mutagenicity, the data are controversial. Herbert et al. [9] observed no statistically significant increase in micronucleus frequency in the bone marrow of mice treated with natural indigo by oral gavage or an increase of mutagenicity towards strains TA1538 and TA98 in the Salmonella/microsome test with metabolic activation. On the other hand, Jongen and Alink [10] investigated the mutagenic potential of two natural and seven synthetic commercial indigo dye products and observed that the natural products showed no mutagenicity in S. typhimurium strains TA98 and TA100. As regards I. truxillensis and I. suffruticosa extracts, mutagenic activity was observed in the Salmonella/microsome mutagenicity test for methanol extracts, and the authors suggested that the alkaloid indigo was the main compound responsible for this effect [11].

NPC Natural Product Communications 2010 Vol. 5 No. 7

1039 - 1042

1040 Natural Product Communications Vol. 5 (7) 2010 Dominici et al.

The aim of the present study was to evaluate, in an in vitro laboratory approach, the possible genotoxicity of Indigofera tinctoria leaf extracts (indigo) by evaluating chromosomal damage (i.e. micronucleus test) in the human liver HepG2 cell line. Micronuclei (MN) appear in the cytoplasm of interphasic cells as small additional nuclei, smaller than the main nuclei (1/20 to 1/3), when the daughter cells separate. MN generate during the anaphase from acentric chromosome fragments or whole chromosomes that are left behind during mitotic cellular division and, consequently, are excluded from both of the daughter nuclei. The formation of MN in dividing cells is the result of chromosome breakage or chromosome malsegregation due to spindle dysfunction produced by either clastogen or aneugen agents, respectively. Thus, MN provide a measure of both chromosome damage and chromosome loss and this biomarker is considered to be at least as sensitive an indicator of chromosome damage as classical metaphase chromosome aberrations analysis [12]. Genotoxicity testing has been performed on the HepG2 cell line (ATCC HB 8065). The HepG2 cell line is an excellent model to investigate toxicity of drugs, since it retains many of the morphological characteristics of the liver parenchymal cells from which they originate [13] and present endogenous bioactivation capacity expressing phase I and phase II enzymes involved in the activation and/or detoxification of xenobiotics [14,15]. The use of metabolically competent cells, such as HepG2, in genotoxicity testing is considered to be closer to the in vivo situation as the addition of an exogenous metabolic activation system (i.e. S9-mix) is not needed [16]. At the end of the experiments cell viability, as evaluated with the fluorochrome-mediated viability test by simultaneous staining with fluorescein diacetate and propidium iodide, was always found to be over 90% (data not shown). The MN frequencies in HepG2 cells, following 24 h in vitro treatment with various amounts of indigo naturalis prepared from Indigofera tinctoria leaves, are shown in Tables 1 and 2. Neither aqueous nor DMSO solutions induced an increase in the MN frequency relative to negative controls (untreated cells). Statistical analysis did not show any statistically significant difference, for either solution, compared to the respective negative control. The positive controls showed the expected significant increase (p<0.001). To investigate the possible influence of in vitro treatment with indigo naturalis solutions on cell cycle

Table 1: Frequency of micronuclei (MN) in binucleated cells (MN/1,000 cells), mitotic index (MI) and nuclear division index (NDI) in HepG2 cells treated in vitro (24 h) with different concentrations of indigo naturalis (from Indigofera tinctoria leaves), in aqueous solution. Data reported as the experimental mean values ± standard deviation.

Indigo naturalis aqueous solutions

(µg/mL)

MN MI NDI

25 12.5 ± 0.7 0.47±0.09 1.49±0.01 50 13.7 ± 4.4 0.47±0.02 1.48±0.01 100 12.0 ± 4.2 0.49±0.02 1.51±0.02 250 12.3 ± 1.7 0.47±0.03 1.48±0.03 500 13.5 ± 4.1 0.45±0.01 1.46±0.01 800 13.0 ± 1.4 0.35±0.05 1.42±0.04

1,000 15.0 ± 4.2 0.48±0.02 1.47±0.03 Controls (–)a 14.8 ± 2.9 0.48±0.02 1.49±0.02 (+)a 36.7 ± 2.9 0.47±0.01 1.45±0.02

a Negative control: MEM; positive control: B(a)P 1.6 µg/mL.

Table 2: Frequency of micronuclei (MN) in binucleated cells (MN/1,000 cells), mitotic index (MI) and nuclear division index (NDI) in HepG2 cells treated in vitro (24 h) with different concentrations of indigo naturalis (from Indigofera tinctoria leaves), in DMSO. Data reported as the experimental mean values ± standard deviation.

Indigo naturalis DMSO solutions

(µg/mL)

MN MI NDI

25 9.8 ± 0.9 0.47±0.05 1.49±0.06 50 11.5 ± 3.3 0.47±0.01 1.49±0.06 100 12.7 ± 2.5 0.42±0.01 1.42±0.02 250 14.8 ± 3.5 0.40±0.01 1.42±0.01 500 10.3 ± 2.5 0.43±0.01 1.45±0.01 800 14.0 ± 3.2 0.43±0.05 1.45±0.06

1,000 16.3 ± 3.4 0.46±0.02 1.48±0.01 Controls (–)a 12.5 ± 2.1 0.55±0.06 1.56±0.06 (+)a 37.5 ± 3.5 0.45±0.02 1.46±0.01

a Negative (vehicle) control: DMSO 5 µl/mL; positive control: B(a)P 1.6 µg/mL.

and speed of replication of HepG2 cells, the mitotic index (MI) and the nuclear division index (NDI) were evaluated (Tables 1 and 2). Neither of the two considered indices showed a possible interference (negative or positive) on the proliferation properties of HepG2 cells in the range of the doses tested. Studies on the mutagenic/genotoxic effects of indigo naturalis have rarely been found in the literature and to our knowledge there are no studies that have tested indigo genotoxicity in human cell lines. The negative results obtained in this in vitro approach are in agreement with the results of the only in vivo study that evaluated the clastogenic potential of indigo naturalis by the MN test in the bone marrow of male mice [9]. In conclusion, even though the results of this study are largely negative in terms of genotoxicity, for a final decision it seems to be necessary to conduct further research, particularly in vivo, in animal models, to exclude any genotoxic risk to the consumer.

Genotoxicity of Indigo Naturalis Natural Product Communications Vol. 5 (7) 2010 1041

Experimental

Chemicals and Media: Indigo naturalis powder (from leaves) was of commercial quality. All reagents used were of analytical grade. Acetic acid, dimethyl sulfoxide (DMSO), Giemsa stain solution, methanol, potassium chloride (KCl), disodium phosphate (Na2HPO4), monobasic potassium phosphate (KH2PO4) and sodium hydroxide (NaOH) were purchased from Carlo Erba Reagenti Srl, Milan, Italy. Benzo[a]pyrene (B[a]P), cytochalasin-B, fluorescein-diacetate (FDA) and propidium iodide (PI), were obtained from Sigma-Aldrich Srl, Milan, Italy. Gibco Eagle’s Minimum Essential Medium (MEM), fetal bovine serum (FBS), L-glutamine, antibiotics, sodium pyruvate, and Dulbecco’s phosphate-buffered saline, pH 7.4 (PBS), were purchased from Invitrogen Srl, Milan, Italy. Conventional microscope slides and coverslips were supplied by Knittel-Glaser, Braunschweig, Germany. Eukitt was from O. Kindler GmbH, Freiburg, Germany. Distilled water was used throughout the experiments. Sample preparation: Indigo naturalis powder (produced by shredding the leaves of Indigofera tinctoria) was purchased from an herbalist's shop. The solutions used in this experimental approach were obtained by dissolving commercial powder in MEM at concentrations of 5 mg/mL (aqueous solution) and in DMSO at concentrations of 100 mg/mL (DMSO solution). For the procedure, to ensure optimal solubilization of the Indigofera tinctoria powder, the solutions were subjected to sonication. Prior to use, the solutions were filtered to remove the coarse woody debris. Cell cultures: Human HepG2 cells were obtained from Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna “Bruno Ubertini”, Brescia, Italy. The cells were grown as monolayer cultures, in 25 cm2 tissue flasks, in MEM with the addition of 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/mL penicillin and 0.1 mg/mL streptomycin. HepG2 subcultures were incubated at 37°C in a humidified atmosphere containing 5% CO2. Cell stocks were routinely frozen and stored in liquid nitrogen (N2). Cell treatment and micronucleus test: HepG2 cells were cultured in 6-well tissue culture plates (Orange Scientific, Belgium) inoculated with 5 mL of complete MEM containing 1×106 cells per well. The overall culture time was 72 h throughout the experiments. After 24 h the cells were maintained in complete MEM, the culture medium was replaced by fresh complete growth medium containing different concentrations of the natural indigo solutions (25 to 1,000 µg/mL in MEM

and in DMSO), and the cells were incubated further for 24 h. Positive and negative controls were included in each experiment. The model mutagen B[a]P (1.6 µg/mL) was used as positive control. The cytokinesis-block micronucleus (CBMN) test was performed following the original method [17], with minor modifications. After the in vitro treatment was performed the medium was replaced by fresh MEM containing cytochalasin B (final concentration: 3 µg/mL) to inhibit cell division after mitosis. The cells were then incubated for the final 24 h period. After completing the treatments, the cells were washed twice with 2 mL PBS, harvested by trypsinization (300 µl of 0.05% trypsin-EDTA, 5 min) and centrifuged for 5 min at 105×g. The pellets were then resuspended in hypotonic solution (3 mL of 0.56% KCl) at room temperature and fixed with 3 mL of Carnoy's reagent (methanol:glacial acetic acid - 5:1 v/v). The cell suspensions were centrifuged again for 5 min at 105×g and resuspended in 6 mL of fixative. Next, the tubes were centrifuged for 5 min, the supernatant discarded and the cell suspensions poured onto pre-cleaned frosted microscope slides. After drying, the slides were stained with 2% Giemsa in phosphate buffer (0.06 M Na2HPO4 and 0.06 M KH2PO4, pH 6.8) for 8 min, washed with distilled water, air-dried and finally mounted with Eukitt. Cells were examined for MN at 400× magnification according to established criteria [18]. MN were scored in 1,000 binucleated cells (BNC) for any concentration of each repeated experiment. Two independent experiments were performed. Furthermore, to investigate the impact of the tested extracts on cell proliferation, the mitotic index (MI) and nuclear division index (NDI) were determined. The MI was calculated by the ratio of binucleated and polynucleated cells on a total number of 1,000 cells. The NDI was calculated for each experimental point using the formula [19]:

[1×N1]+[2× N2]+[3×N3]+[4×N4] NDI = N

where N1−N4 represent the numbers of cells with 1–4 nuclei, respectively, and N is the total number of cells scored. The results for MN, MI and NDI were expressed as the mean ± standard deviation of duplicate determinations from independent experiments. Fluorochrome-mediated viability test: At the end of the treatments, cytotoxic effects were evaluated in culture aliquots by simultaneous staining with FDA and PI [20]. Dye working solutions were prepared immediately prior to use by adding 20 µl of 5 mg/mL

1042 Natural Product Communications Vol. 5 (7) 2010 Dominici et al.

FDA (in acetone) to 5 mL of PBS, or 20 µl of 20 µg/mL PI to 1 mL of PBS. Subsequently, 100 µl of 20 µg/mL FDA and 30 µl of 0.4 µg/mL PI were added directly to 200 µl of each cell suspension. The cells were then stained for 3 min at room temperature. Cell counts were performed using ten-chamber disposable microscope slides provided with a haemocytometer-like counting grid (Kova, Hycor Biomedical, USA). After the

counting chamber was loaded, the cells were examined with a standard fluorescence microscope (Dialux 20, Leitz, Germany) equipped with epi-illumination provided by a 50 W high-pressure mercury lamp (HBO 50, Osram, Germany). Viable cells fluoresced green, whereas dead cells were indicated by orange-stained nuclei.

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New Metabolite from Viburnum dilatatum Bin Wu, Xing Zeng and Yufeng Zhang 1097

Two New Glycosides from Conyza bonariensis Aqib Zahoor, Imran Nafees Siddiqui, Afsar Khan, Viqar Uddin Ahmad, Amir Ahmed, Zahid Hassan, Saleha Suleman Khan and Shazia Iqbal 1099

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Chemical Composition of the Essential Oil of Leaves and Roots of Ottoa oenanthoides (Apiaceae) from Mérida,Venezuela Janne Rojas, Alexis Buitrago, Luis B. Rojas, Antonio Morales and Shirley Baldovino 1115

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Volatile Constituents of Two Rare Subspecies of Thymus praecox Danijela Vidic, Sanja Ćavar, Marija Edita Šolić and Milka Maksimović 1123

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Essential Oil Composition and Insecticidal Activity of Blumea perrottetiana Growing in Southwestern Nigeria Moses S. Owolabi, Labunmi Lajide, Heather E. Villanueva and William N. Setzer 1135

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Natural Product Communications 2010

Volume 5, Number 7

Contents

Original Paper Page

A DFT Analysis of Thermal Decomposition Reactions Important to Natural Products William N. Setzer 993

Novel Terpenoids from the New Zealand Liverworts Jamesoniella colorata and Bazzania novae-zelandiae Masao Toyota, Ikuko Omatsu, Fumi Sakata, John Braggins and Yoshinori Asakawa 999

Two New C20-Diterpenoid Alkaloids from Delphinium anthriscifolium var. savatieri Xiao-Yu Liu, Lei Song, Qiao-Hong Chen, and Feng-Peng Wang 1005

Cytotoxic Activity of Quassinoids from Eurycoma longifolia Katsunori Miyake, Feng Li, Yasuhiro Tezuka, Suresh Awale and Shigetoshi Kadota 1009

Antifungal Activity of Saponin-rich Extracts of Phytolacca dioica and of the Sapogenins Obtained through Hydrolysis Melina Di Liberto, Laura Svetaz, Ricardo L. E. Furlán, Susana A. Zacchino, Carla Delporte, Marco A. Novoa, Marcelo Asencio and Bruce K. Cassels 1013

New Lupane-type Triterpenoid Saponins from Leaves of Oplopanax horridus (Devil’s Club) Pei-Pei Liu, Mo Li, Ting-Guo Kang, De-Qiang Dou and David C Smith 1019

Triterpene Saponins from Cyclamen persicum Ghezala Mihci-Gaidi, David Pertuit, Tomofumi Miyamoto, Jean-François Mirjolet, Olivier Duchamp, Anne-Claire Mitaine-Offer and Marie-Aleth Lacaille-Dubois 1023

Cytotoxic Pentacyclic Triterpenoids from Combretum oliviforme Xiao-Peng Wu, Chang-Ri Han, Guang-Ying Chen, Yuan Yuan and Jian-Ying Xie 1027

Preparative Separation of Four Major Bufadienolides from the Chinese Traditional Medicine, Chansu, Using High-Speed Counter-Current Chromatography Xiu Lan Xin, Junying Liu, Xiao Chi Ma, Qing Wei, Li Lv, Chang Yuan Wang, Ji Hong Yao and Jian Cui 1031

Acetylcholinesterase and Butyrylcholinesterase Inhibitory Compounds from Eschscholzia californica (Papaveraceae) Lucie Cahlíková, Kateřina Macáková, Jiří Kuneš, Milan Kurfürst, Lubomír Opletal, Josef Cvačka, Jakub Chlebek and Gerald Blunden 1035

In Vitro Testing for Genotoxicity of Indigo Naturalis Assessed by Micronucleus Test Luca Dominici, Barbara Cerbone, Milena Villarini, Cristina Fatigoni and Massimo Moretti 1039

Metabolites from Withania aristata with Potential Phytotoxic Activity Gabriel G. Llanos, Rosa M. Varela, Ignacio A. Jiménez, José M. G. Molinillo, Francisco A. Macías and Isabel L. Bazzocchi 1043

Bioassay-guided Isolation and Quantification of the α-Glucosidase Inhibitory Compound, Glycyrrhisoflavone, from Glycyrrhiza uralensis Wei Li, Songpei Li , Lin Lin, Hong Bai, YingHua Wang, Hiroyoshi Kato, Yoshihisa Asada, Qingbo Zhang and Kazuo Koike 1049

Xanthones, Biflavanones and Triterpenes from Pentadesma grandifolia (Clusiaceae): Structural Determination and Bioactivity Grace Leontine Nwabouloun Djoufack, Karin M. Valant-Vetschera, Johann Schinnerl, Lothar Brecker, Eberhard Lorbeer and Wolfgang Robien 1055

Gmelinoside I, a New Flavonol Glycoside from Limonium gmelinii Zhanar A. Kozhamkulova, Mohamed M. Radwan, Galiya E. Zhusupova, Zharilkasin Zh. Abilov, Saniya N. Rahadilova and Samir A. Ross 1061

2-Arylbenzofuran Neolignans from the Bark of Nectandra purpurascens (Lauraceae) Jaime Rios-Motta and Eliseo Avella 1063

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