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Mutagenicity and Safety Evaluation of Water Extract of Coriander sativum Leaves ´ NGELES, AND LOURDES RODR´IGUEZ-FRAGOSO MARIANA RAM´IREZ REYES, JORGE REYES-ESPARZA, OSCAR TORRES A
ABSTRACT: Coriander has been used as a spice and medicinal plant for centuries. Several studies have described its biological properties and some reports have indicated its pharmacological actions in some human pathology. However, data on its toxicity and metabolism are limited or null, and no research has been conducted with mammalian cells. The purpose of this study was to evaluate the mutagenicity and safety of Coriandrum sativum extract. The mutagenic effects of C. sativum extract were evaluated by Ames test. Mutagenicity was present when the C. sativum extract was used in high concentrations in both tested strains (Salmonella typhimurium TA97 and TA102). Our research showed that C. sativum extract reduced the cell survival of human cell lines (WRL-68 and 293Q cells) by inducing apoptosis and necrosis in the cases where extract concentration was the highest. The C. sativum extract altered the cell cycle; it increased the G1 phase of hepatic cells and reduced the G2+M phase in both cell lines in a dose-response manner. These results showed correlation with a reduction in the mitotic index. The extract also induced severe malformations during embryonic development. Exposure of chicken embryos to the C. sativum extract resulted in a dose-dependent increase of anomalies. Present results show that C. sativum extract reduced the axial skeleton and affected the neural tube, the somites, the cardiovascular structures, and the eye. According to the present results, the C. sativum aqueous extract cannot be considered safe. These results indicate that some significant adverse effects of C. sativum extract could be observed in vivo. Keywords: apoptosis, Coriandrum sativum, cytotoxicity, embryotoxicity, mutagenicity
Introduction
for medicinal purposes. These include powered seeds or dry extract (2 to 5 g/d), tea (4 to 8 g/100 mL), tinctures (1.8 g/mL), decoctions, and infusions ( Breevort 1996; De Smet 2002). One would expect each of these preparations to have a different proportion of each of the previously mentioned components; the tinctures and extracts will have more nonpolar components, while hydrophilic components will predominate in the teas and decoctions. The way these preparations are made must also be considered (cooking time, temperature, amount of water, time of rest, amount of plant used, and so on). Tinctures are more concentrated than infusions and decoctions, while the preparation of extracts could lead to loss of volatile oils. Having taken all this into account, the efficacy or toxicity of C. sativum could vary depending on the composition and proportion of constituents in the preparations made for human use. Recent research involving in vivo pharmacological use of extracts from this plant in experimental models has shown their high therapeutic effectiveness (Medhin and others 1986; Gray and Flatt 1999; Kubo and others 2004; Lal and others 2004; Ramadan and others 2003; Emamghoreishi and others 2005). But very few (if any) modern clinical studies have been conducted on coriander. Its approved modern therapeutic applications are based on its long history of use in well-established systems of traditional medicine, pharmacological studies conducted on animals, nutrient composition, dietary value studies, and phytochemical research (Burdock and Carabin 2009). In spite of the wide-ranging, extant research on the therapeutic effects of C. sativum, little is known about its toxicological effects, since no extensive studies have been conducted on in vitro and in vivo models (Burdock and Carabin 2009). An important aspect of natural products, especially those that are readMS 20090347 Submitted 4/17/2009, Accepted 8/29/2009. Authors are with Facultad de Farmacia, Univ. Aut´onoma del Estado de Morelos. Avenida ily available to the public, is safety. Many people assume natural Univ. 1001 Col. Chamilpa 62210, Cuernavaca, Morelos. Mexico. Direct in- products are safe, but there is recent, abundant evidence involving quiries to author Rodr´ıguez-Fragoso (E-mail:
[email protected]). serious adverse effects and deaths associated with the use of dietary
C
oriander is an annual herb native to Mediterranean Europe and Western Asia, naturalized in North America, and now extensively cultivated in many temperate countries (Wichtl and Bisset 1994; BHP 1996; Leung and Foster 1996). Fresh leaves are used as a flavoring agent and dried coriander seeds are used as spices in food preparation. Both an annual and a perennial herb, coriander is rich in various food elements (Grieve 1971). It contains about 1% volatile oil, of which 55% to 74% is linalool; monoterpene hydrocarbons (a- and b-pinene and limonene), anethole, and camphor comprises 20%; oleic, petroselinic, and linolenic fatty acids make up 26%; approximately 20% is comprises flavonoid glycosides (quercetin, isoquercitrin, and rutin), chlorogenic and caffeic acids, tannins, and sugars while proteins comprise 11% to 17%. The remainder (approximately 1%) contains coumarins, mucilage, and starch (Lister and Hˆrhamme 1973; Hansel and others 1992; Wichtl and Bisset 1994; Budavari 1996; Leung and Foster 1996). Coriander essential oil has a long history in traditional medicine (Uma and others 1993; Kiple and Ornelas 2000). Galenical preparations of coriander seed have been used in traditional Chinese, Indian, Greco-European, and Latin American indigenous medicine. The British Herbal Pharmacopoeia (BHP 1996) and The Merck Index also report its therapeutic qualities as a carminative and aromatic (Budavari 1996). In traditional medicine, Coriandrum sativum has been used to treat a number of medical problems such as dyspepsia, loss of appetite, convulsions, insomnia, and anxiety (Breevort 1996; De Smet 2002). C. sativum is empirically used in different doses and forms
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R 2009 Institute of Food Technologists! doi: 10.1111/j.1750-3841.2009.01403.x
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Further reproduction without permission is prohibited
Toxicity of Coriandrum sativum . . . supplements and nutriments (Marcus and Grollman 2002; Wooltorton 2002; Wooltorton and Sibbald 2002; Morrow and others 2005). The purpose of this study was, therefore, to evaluate the toxicological effect of C. sativum extract on in vitro models and chick embryo development.
Materials and Methods
cator to avoid water absorption until used. Hydroalcoholic extraction using 80% methanol was conducted by percolating 200 to 300 g of the dried and powdered plant material for 5 d, which was then filtered through Whatman filter paper nr 1. The solvent was evaporated using a Rotavapor and the extract was kept in a stoppered sample vial at 4 ◦ C until used.
Determination of content and essential oil composition
Preparation of C. sativum extract Leaves of C. sativum were purchased from the Oaxaca Sierra, Mexico, and transported to the state of Morelos (Mexico) in October 2006 (Figure 1). These were then identified by a taxonomist and a voucher sample representing Herbarium nr FL6006 was deposited at the Herbarium of the Autonomous Univ. of the State of Morelos. The leaves were air dried at room temperature, ground, and kept in amber colored bottles until processed. Aqueous extraction was performed by soaking a weighed amount of the dry powder in distilled water and shaking it for 3 h with an electric shaker. The suspension was filtered through muslin gauze and the filtrate kept in deep freeze for 24 h, then lyophilized. The lyophilized dry powder was collected in a stoppered sample vial, weighed, and kept in a desic-
Coriander leaf oils were analyzed as 1% solutions in hexane, using a Perkin–Elmer autosystem gas chromatograph under the control of Perkin–Elmer Omega (version 5.2) software. GC analysis was performed on a Hewlett-Packard 5890 series II gas chromatograph equipped with 2 flame ionization detectors (255 ◦ C), 2 fused capillary columns of different polarities, a methyl silicone, and a polyethylene glycol 20000 column (HP-1 and HP-Wax, 50 m × 0.25 mm, film thickness 0.25 mm), which were used simultaneously, and a split injector at 255 ◦ C (split ratio) 1 : 150). The temperature was programmed from 90 to 220 ◦ C at a rate of 3 ◦ C/min. Nitrogen was used as the carrier gas at a flow rate of 0.8 mL/min. GC-MS was performed on a Perkin–Elmer Autosystem gas chromatograph Q-mass 910 quadrupole mass spectrometer, equipped with 2 fused silica columns, a nonpolar J&W DB-1 (30 m × 0.25 mm, 0.25 µm film thickness) and a polar DB-Wax (30 m × 0.25 mm, 0.25 µm film thickness). The GC parameters were the same as those mentioned previously, but helium was used as the carrier gas. Mass units were monitored from 45 to 350 at 70 eV. The oil components were identified by (1) determining their retention indexes (RI) in relation to a homologous series of fatty acids methyl esters (C4-C18) and (2) comparing their mass spectra with published values (Adams 1995; Baratta and others 1998; Gil and others 2002) (Table 1).
Cell culture Two cell lines were used for this study: 293Q cells derived from normal epithelial cells of human fetal kidney (CRL-1573 ATCC) and WRL-68 cells derived from epithelial cells of human fetal liver (CRL48 ATCC). Cell lines were cultured in minimal essential medium (MEM, GIBCO BRL Inc., Grand Island, N.Y., U.S.A.), supplemented with nonessential amino acids (GIBCO BRL Inc), 10% fetal calf serum (GIBCO BRL Inc), L-glutamine (2 mol/L), and antibiotics. Cells were plated in 100-mm culture dishes (106 cells/dish), and maintained at 37 ◦ C under an atmosphere of 5% CO2 in humidified air. The medium was replaced every 2 d and the cells were harvested and diluted 5-fold every 7 d. Subcultures were obtained Figure 1 --- Coriander (Coriander sativum) (Source: Oaxaca by trypsinization (0.025% trypsin solution containing 0.01% N,Ndiethyldithiocarbamic acid sodium salt, EDTA). Sierra, Mexico, 2006). Table 1 --- Identification of C. sativum components. Identity
1 2 3 4 5 6 7 8 9 10 11 12 13
α-pinene camphene β-pinene myrcene p-cymene limonene γ -terpinene linalool camphor decanal geraniol decanol geranyl-acetate
GC
RI
GC-MS
Component level (%)
+ + + + + + + + + + + + +
+ + + + + + + + + + + + +
+ + + + + + + + + + + − +
4.8 0.7 0.4 0.9 0.7 2.7 4.9 75.4 5.1 0.3 2.8 0.3 3.0
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Identification method Peak
Toxicity of Coriandrum sativum . . . Assessment of cell viability Cells were plated at 10000 cells/well on 96-well plates. After 24 h, the culture medium was replaced by a fresh one supplemented with different concentrations of C. sativum extract (0.4, 0.8, 1.6, 3.2, 4.8, 6.4, and 8 µg/mL). After 24 h incubation, cells were collected and processed. Cell viability was measured by a 3-(4, 5-dimethylthiazol2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay (Wang and others 1996). Briefly, 20 µL MTT (5 g/L) was added to each well and incubated with the culture for an additional 4 h at 37 ◦ C, 5% CO2 . Culture media were then discarded, followed by the addition of 200 µL DMSO with 25 µL Sørensen’s glycine buffer (glycine 0.1 M, NaCl 0.1 M, pH 10.5) to each well. When the blue crystals were dissolved, the optical density was determined on a microplate reader (Bio-Rad) at 450 nm.
Survival of cells and detection of percentage of apoptosis and necrosis Survival of cells was evaluated by a flow cytometric method (Nicoletti and others 1991). Annexin V is a Ca2+-dependent phospholipid-binding protein that has a high affinity for phospholipid-like phosphatidylserine (PS) and is useful for identifying apoptotic cells with exposed PS. Cells (105 ) were washed twice with cold PBS and then re-suspended in 1× binding buffer at a concentration of 1 × 106 cells/mL. A total of 100 µL of the solution (1 × 105 cells) were transferred to a 5 mL culture tube. Five milliliters of Annexin V–FITC and 10 µL of PI were added. Cells were incubated for 15 min at room temperature (20 to 25 ◦ C) in the dark and then 400 µL of 1× binding buffer were added to each tube. The results were analyzed with a CELLQuest program in a FACSCalibur flow cytometer (Becton Dickinson, Calif., U.S.A.). Staining cells simultaneously with FITC-Annexin V (green fluorescence) and the nonvital dye propidium iodide (red fluorescence) enables bivariate analysis of the discrimination of intact cells the discrimination of intact cells (FITC-PI-), early apoptotic (FITC+PI-), and late apoptotic or necrotic cells (FITC+PI+).
Bactericidal toxicity test Samples (1 to 5 mg/plate) were prepared with 0.1 mL of fresh culture of the tester strain (approximately 108 cells/mL), 0.1 mL of the WFTS, 0.2 mL of phosphate buffer (0.2 M, pH 7.4), and 0.5 mL of S-9 mix (metabolic activator) or, instead, phosphate buffer. The mixture was diluted sequentially with phosphate buffer and 1 mL of diluted solution was mixed with 12 mL of nutrient agar. After incubation at 37 ◦ C for 48 h, the number of colonies was counted. A bactericidal toxicity effect was confirmed if the standard plate count of tested compound was lower than that of the control (without adding tested compound).
Salmonella mutagenicity test/Ames test
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The Ames test was performed as a standard plate incorporation assay with Salmonella typhimurium strains TA97a and TA102 with metabolic activation (Maron and Ames 1983). Strain-specific genetic markers were verified prior to use. The selection of the strains was based on the testing and strain selection strategies of Mortelmans and Zeiger (2000). For each tested strain, a specific positive control was always used to assess the experimental flaws, if any. Nitro phenylenediamine and sodium azide were used as positive controls for TA97 and TA102, respectively. To ensure sterility, the different concentrations of the extract were exposed for 15 min to UV-C light (TUV 30W G30T8 Philips, Holland). The absence of contaminant growth was checked in a blank set containing the extract but without the addition of bacteria. For each concentration, 100 mL of the test solution was used per plate. Positive controls T8
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(20 mg/plate nitrophenylenediamine for TA97a and 1.5 mg/plate sodium azide for TA102) and negative control (distilled water) were concurrently maintained. Samples were tested on triplicate plates in 2 independent experiments. Following 48 h incubation at 37 ◦ C, genotoxic activities were expressed as induction factors (induction factors of reversions), that is, as multiples of the background levels. The interpretation of the Ames test results followed the U.S. Environmental Protection Agency (1996) guidelines for genotoxicity testing of chemicals. According to the guidelines, a mutagenic potential is assumed if the revertant frequency is 2 or higher over the solvent control (Mortelmans and Zeiger 2000).
Analysis of the cell cycle Cells were scraped and washed with PBS. Cells (105) were fixed in 75% ethanol for 24 h and then washed in PBS and resuspended in 0.1% NP40 (Nonidet P40, Biochimica Fluka, St. Louis Mo., U.S.A.) and 10 µg/mL RNAse (Ribonuclease A, DNAse-free preparation) for 20 min at room temperature (Darzynkiewicz and others 2001). Propidium iodide (PI) was then added (final concentration 5 µg/mL) for 12 h at 4 ◦ C in darkness. Samples were analyzed using a FACSCalibur flow cytometer. The results were analyzed using CELLQuest program.
Mitotic activity Cells were fixed in 3% paraformaldehyde with 0.25M mannitol (45.54 g/L) for 2 h, rinsed in PBS and stained with DAPI (4$ ,6diamidino-2-phenylindol, 480 nm). After rinsing in PBS, the cells were embedded in Citifluor mounting medium. The mitotic index was counted with a fluorescent microscope (Optiphot 2 Nikon). For each experiment, the indices were determined per 1000 cells and with 4 replicates.
Embryotoxicity studies Fertile White Leghorn chicken eggs were obtained from A.L.P.E. S.A. (Puebla, Mexico) and were stored at 6 ◦ C. Total of 72 fertilized eggs were weighed, sterilized, and divided into 9 groups. First group served as a nontreated control. The next 6 groups received the C. sativum extract (0.4, 0.8, 1.6, 3.2, 6.4, and 8 µg/mL). The last group received caffeine (10 mg/mL) and was considered the positive control. A teratogenicity assay was carried out as described by Jelinek and Marthan (Jelinek and Marhan 1994). Test solutions (1 mL) were added to the air sac under sterile conditions. Each solution was injected after drilling into the shell at the blunt end of the egg; after injection, the holes were immediately sealed with melted paraffin wax. The eggs were then transferred to and maintained in a forced draft incubator at 37.5 ◦ C with a relative humidity of 55% until the desired stage of development was reached. To determine the concentration dependency of C. sativum extract teratogenicity, a histological analysis was carried out. Embryos in each group were fixed in buffered formal saline (pH 7.4), dehydrated, and embedded in paraffin blocks. Paraffin tissue sections of 6 µm were stained with acetocarmine for routine histological examination. The embryo was examined and staged according to morphological criteria previously outlined by Hamburger and Hamilton (1951). Embryonic stages at the time of the C. sativum extract application varied from 14 to 16, which correspond approximately to developed somites numbered 22 to 28.
Statistical analysis of results In vitro data were reported as mean ± SD of 3 independent experiments conducted in quadruplicate. Mean values were compared using Student’s t-test or analysis of variance (ANOVA) using SPSS 10.0 software (SPSS Inc., Chicago, Ill., U.S.A.). Significant differences were established at P < 0.001.
Toxicity of Coriandrum sativum . . .
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Results and Discussion
. sativum extract caused a marked reduction in the survival of 2 human cell lines after 24 h incubation (Figure 2). Concentrations higher than 1.6 µg/mL produced different effects on cell lines. A significant 60% survival reduction was observed in WRL-68 (hepatic) cells with concentrations of 1.6 to 8 µg/mL (P < 0.001); 293Q (renal) cells showed a 30% survival reduction at 1.6 µg/mL. Higher concentrations (3.2 to 8 µg/mL) reduced cell survival by 60% or
Figure 2 --- Changes in the survival of cells after treatment with different concentrations of C. sativum extract during a 24-h incubation period (control = 100%). The results are presented as means ± SD of 3 independent experiments. ∗ P < 0.001 in comparison with control value.
more (P < 0.001). The decreased percentage of live cells was accompanied by an increase in the amount of apoptotic and necrotic cells (Table 2), P < 0.001. It was found that apoptotic cells outnumbered necrotic cells, especially after treatment with the highest concentrations of the extract (1.6 to 8 µg/mL). The increase in apoptotic cells took place in a dose-dependent manner. Some reports have indicated that low concentrations of some compounds induce apoptosis, while high concentrations induce necrosis (Del Bino and others 1991). The appearance of necrotic cells may also be a result of incomplete apoptosis (Leist and Nicotera 1998). Recent studies by Bakkali and others (Bakkali and others 2005) have shown that an extract of C. sativum induced cytotoxic effects on the yeast Saccharomyces cerevisiae, and that the effects were stronger in exponential rather than stationary phase cells. The results of the present study showed that C. sativum is cytotoxic for both human cell lines. The results of previous comet assays show that C. sativum does not induce toxicity in cultured fibroblasts of rat embryo (Heibatullah and others 2008). The present study demonstrated that C. sativum extract is mutagenic in the tested range of 1.6 to 8 µg/mL according to the Ames test. The results of mutagenicity assays, presented as mean revertants per plate, are shown in Table 3. The mutagenic potential of the extracts showed a positive dose-related increase in the number of revertant colonies in both strains of S. typhimurium. The number of revertant colonies ranged between 437 ± 16 and 7905 ± 45 in TA97a and 420 ± 38 and 1742 ± 58 in
Table 2 --- Changes in the percentage of normal, apoptotic, and necrotic cells after 24 h incubation with different concentrations of C. sativum extract. Cell line WRL-68
293Q
Concentration of C. sativum (µg/mL)
Normal cells (%)
0 0.4 0.8 1.6
Apoptotic cells (%)
90 ± 6 86 ± 4 85 ± 3 63 ± 6
3.2 4.8 6.4 8 0 0.4 0.8 1.6 3.2 4.8 6.4 8
6±2 8 ± 1.9 9±3 28 ± 5∗
48 ± 4 42 ± 7 40 ± 8 42 ± 3 91 ± 5 86 ± 3 83 ± 2 70 ± 6 58 ± 7 40 ± 4 38 ± 5 37 ± 5
Necrotic cells (%) 4±1 5±3 6±3 9 ± 2∗
35 ± 3∗ 39 ± 4∗ 42 ± 1∗ 47 ± 0.5∗ 5±2 11 ± 3 8±2 21 ± 4∗ 27 ± 3∗ 37 ± 5∗ 38 ± 4∗ 41 ± 3∗
13 ± 5∗ 18 ± 6∗ 18 ± 4∗ 11 ± 2∗ 4±1 3 ± 0.5 9±3 9 ± 1∗ 15 ± 3∗ 23 ± 4∗ 24 ± 5∗ 22 ± 4∗
The results are presented as means ± SD of 3 independent experiments. ∗ P < 0.001 in comparison with control value.
Table 3 --- Mutagenicity of C. sativum in tester strain of S. typhimurium.
Distilled watera 0.4 0.8 1.6 3.2 4.8 6.4 8 NDPb SAb
-S9d
98 ± 3 106 ± 15 134 ± 28 437 ± 16c 538 ± 22c 610 ± 36c 690 ± 21c 734 ± 34c 790 ± 45c
a Negative control. b Positive control; NDP = nitrophenylenediamine (20µg/plate) and SA = sodium azide (1.5 c Number of revertant colonies more than twice that of corresponding control (P < 0.001). d
S9
89 ± 4 188 ± 7 296 ± 12 990 ± 9 1030 ± 14 2345 ± 17 3423 ± 24 3567 ± 27 3974 ± 34
-S9
S9
149 ± 21 198 ± 34 265 ± 42 420 ± 38c 680 ± 27c 945 ± 34c 1230 ± 43c 1742 ± 58c
490 ± 6 630 ± 14 698 ± 24 830 ± 14 3143 ± 25 4423 ± 31 4987 ± 41 5423 ± 34
1985 ± 124c
6423 ± 29
µg/plate).
S9 is a metabolic activation system consisting of the postmitochondrial fraction of the livers of rats. The results are presented as means ± SD of 3 independent experiments. SD = standard deviation. e The number represent the number of mutants/plate.
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Concentration of C. sativum (µg/mL)
Revertant colonies (UFC/plate)e TA97a, mean ± SD T102, mean ± SD
Toxicity of Coriandrum sativum . . .
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TA102, with concentrations between 1.6 and 8 µg/mL of C. sativum extract (P < 0.001). According to the 2-fold rule, the doubling of spontaneous reversion rate at 1 or 2 test chemical concentrations constitutes a positive response (Mortelmans and Zeiger 2000). The mutagenic activity was associated to a metabolic activation by S9 mix. Bakkali and others (Bakkali and others 2005) demonstrated that C. sativum induced cytoplasmic petit mutations on the yeast S. cerevisiae. The results of the present study showed that C. sativum extract is mutagenic toward both tester strains, so that the presence of apoptosis in human cell lines suggests it might also induce mutations in those cells. The results also showed differences in the effects of C. sativum extract on the cell cycle of both cell lines after 24 h incubation (Figure 3). A 30% increase in the number of cells in G1 phase was observed in WRL-68 cells with all used concentrations; there was also a 50% to 80% reduction in the G2/M phase with concentrations of 3.2 to 8 µg/mL (P < 0.001). No changes in S phase were observed with any concentration. On the other hand, 293Q cells showed a reduction of 60 to 80% in the G2+M phase with concentrations higher that 0.8 µg/mL (P < 0.001), while no changes in G1 and S phases were observed. C. sativum extract also reduced the percentage of cellular divisions. A reduction of 20% to 25% with lower concentrations and 35% to 40% with concentrations higher than 3.2 µg/mL was observed in WRL-68 cells, P < 0.001 (Figure 4). A 20% to 34% reduction in the mitotic index of 293Q cells was observed when they were treated with C. sativum extract (P < 0.001). For many cells, G1 phase is the major period of cell growth. During this stage, new organelles are synthesized and the cell requires both structural and functional proteins, resulting in active protein synthesis and, therefore, a high metabolic rate cell. The presence of cell cycle arrest and apoptosis suggests that cells are suffering DNA damage. Previous studies have suggested that cycle regulation-mediated apoptosis is a mechanism of cell growth inhibition (Ahmad and others 1997; Deigner and Kinscherf 1999; Ahmad and others 2001). The apoptosis is a physiological process that functions as an essential mechanism of tissue homeostasis and is regarded as the preferred way to eliminate unwanted cells. Previous studies by Cort´es-Eslava and others (2004) demonstrated that an aqueous crude coriander juice significantly decreased the mutagenicity of some metabolized aromatic amines and that the coriander juice (50 to 1000 µL per coincubation flask) was neither toxic nor mutagenic. Previous studies have also shown that C. sativum extract possesses antioxidative properties (Chithra and Leelamma 1999; Satyanarayana and others 2004; Sreelatha and others 2008). Because we are testing an aqueous extract (a mix of essential oils), it is possible that some toxic constituents are inducing cell damage and others are protecting the cell. Hepatic and renal cells play an important role in drug metabolism because they have drug-metabolizing enzymes. Mutagenic activity in the presence of S9 suggested that C. sativum extract might be metabolized in vivo and cause several biological activities. Metabolic products are often less active than the parent drug and may even be inactive. However, some biotransformation products have enhanced activity or toxic properties. Certain herbal medicines have been identified as a cause of acute and chronic hepatitis, cholestasis, drug-induced autoimmunity, vascular lesions, and even hepatic failure and cirrhosis. Several factors may contribute to the hepatotoxic effects of herbal preparations. Herbal medicines are usually a mixture of several ingredients or plants harvested during different seasons and extracted by variable procedures, which make the identification of both pharmacologically active and toxic compounds difficult. This is the case of C. sativum (Smallfield and others 2001; Gil and others 2002; T10
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Ramadan and others 2003). Also, contamination of herbal medicines with microorganisms, fungal toxins (such as aflatoxin), pesticides, heavy metals, and synthetic drugs could confuse analysis. Moreover, it has been reported that herbal extracts may also exert renal toxicity through inherent properties (Wojcikowski and others 2004). There is currently not enough information regarding the metabolism of C. sativum in human cells. However, because this plant contains a mixture of essential oils and the C. sativum extract here studied induced necrosis, it is possible that C. sativum extract could be metabolized into toxic metabolites in hepatocytes and renal cells (Smallfield and others 2001; Gil and others 2002). It has been reported that ingestion of coriander oil led to the incorporation of petroselinoyl (triacylglycerols of coriander) into heart,
Figure 3 --- Percentage of cells in G1, S, G2/M phases of the cell cycle after treatment with different concentrations of C. sativum extract during a 24-h incubation period. (A) WRL68 cells, (B) 293Q cells. The results are presented as means ± SD of 3 independent experiments. ∗ P < 0.001 in comparison with control values.
Figure 4 --- Changes in the mitotic index of cells after incubation for a 24-h period with different concentrations of C. sativum extract. The results are presented as means ± SD of 3 independent experiments. ∗ P < 0.001 as compared with all groups; #P < 0.001 as compared with 0.4, 0.8, and 1.6 µg/mL.
Toxicity of Coriandrum sativum . . . Figure 5 --- Morphological appearance of chick embryos treated with different concentrations of C. sativum. Arrows indicate morphological alterations in the heart, central nervous system (CNS), placodes (optical and otic), neural tube, and somites.
Table 4 --- Teratogenic effects of C. sativum. Embryonic region affected
Caffeinea 10 mg/mL
Controlb
0.4
0.8
3/9 6/9 7/9 7/9 7/9 5/9 9/9
0/9 0/9 0/9 0/9 0/9 0/9 0/9
0/9 0/9 0/9 0/9 0/9 0/9 3/9
1/9 0/9 0/9 0/9 1/9 0/9 4/9
CNS Neural tube Somites Vasculature Heart Eye Axial skeleton
C. sativum extract (µg/mL) 1.6 3.2 4.8 3/9 3/9 2/9 3/9 3/9 1/9 7/9
5/9 5/9 4/9 5/9 5/9 2/9 8/9
7/9 7/9 5/9 7/9 7/9 4/9 9/9
6.4
8
8/9 8/9 8/9 8/9 8/9 6/9 9/9
9/9 9/9 9/9 9/9 9/9 8/9 9/9
a Positive b
control. Negative control. The fractions represent the number of abnormal embryos and the total examined for each developmental region of the embryo.
T
Conclusions
he toxicological evaluation of C. sativum aqueous extract showed that said extract is toxic for human cell lines and chicken embryos. Study results reveal that C. sativum extract induced cytotoxic effects on renal and liver cells in a dose-dependent manner (P < 0.001). C. sativum extract was able to produce apoptosis, necrosis, and alterations in the cell cycle, and changes in the mitotic index in renal and hepatic cell lines. The extract also
presents mutagenicity according to the Ames test and the results showed it induced severe malformations during embryonic development. The C. sativum aqueous extract cannot be considered safe and these results indicate that some significant adverse effects of C. sativum extract could be observed in vivo.
References Adams RP. 1995. Identification of essential oil components by gas chromatography/ mass spectroscopy. Carol Stream, Ill.: Allured Publishing. Ahmad N, Feyes DK, Nieminen AL, Agarwal R, Mukhtar H. 1997. Green tea constituent epigallocatechin-3-gallate and induction of apoptosis and cell cycle arrest in human carcinoma cells. J Natl Cancer Inst 89:1881–6. Ahmad N, Adhami VM, Afaq F, Feyes DK, Mukhtar H. 2001. Resveratrol causes WAF1/p21-mediated G1 -phase arrest of cell cycle and induction of apoptosis in human epidermoid carcinoma A431 cells. Clin Can Res 7:1466–73. Al-Said MS, Al-Khamis KI, Islam MW, Parmar NS, Tariq M, Ageel AM. 1987. Postcoital antifertility activity of the seeds of Coriandrum sativum in rats. J Ethnopharm 21:165–73. Bakkali F, Averbeck S, Averbeck D, Zhiri A, Idaomar M. 2005. Cytotoxicity and gene induction by some essential oils in the yeast Saccharomyces cerevisiae. Mutation Res 585:1–13. Baratta MT, Dorman HJD, Deans SG, Biondi DM, Ruberto G. 1998. Chemical composition, antimicrobial and antioxidative activity of laurel, sage, rosemary, oregano and coriander essential oils. J Essent Oil Res 10:618–27. Breevort P. 1996. The U.S. botanical market: an overview. Herbalogramm 36:49– 57. [BHP] British Herbal Pharmacopoeia. 1996. Exeter, U.K.: British Herbal Medicine Assoc. 12 p. Budavari S, O’Neil M, Smith A, Heckelman P, Obenchain J. 1999. Oil of coriander. The Merck Index. Boca Raton, Fla.: Chapman and Hall. Burdock GA, Carabin IG. 2009. Safety assessment of coriander (Coriandrum sativum L.) essential oil as a food ingredient. Food Chem Toxicol 47:22–34. Cort´es-Eslava J, G´omez-Arroyo S, Villalobos-Pietrini R, Espinosa-Aguirre JJ. 2004. Antimutagenicity of coriander (Coriandrum sativum) juice on the mutagenesis produced by plant metabolites of aromatic amines. Toxicol Lett 153(2):283–92. Chithra V, Leelamma S. 1999. Coriandrum sativum changes the levels of lipid peroxides and activity of antioxidant enzymes in experimental animals. Indian J Biochem Biophy 36:59–61. Darzynkiewicz Z, Bedner E, Smolewski P. 2001. Flow cytometry in analysis of cell cycle and apoptosis. Sem Hematol 38:179–93.
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liver, and blood lipids in rats. The consumption of coriander triacylglycerols led to significantly greater liver weights (Weber and others 1995). The effect of an aqueous extract of fresh C. sativum seeds on female fertility in rats was studied in the 1980s (Al-Said and others 1987). The results showed that the treatment of animals with fresh coriander seed extract did not produce any significant abortifacient activity. There was no significant change in the weight and length of the fetuses delivered by rats treated with the extract, and no abnormalities were found in the organs of their offspring. The present study evaluated the teratogenic effect of C. sativum extract on a chicken embryo model. Exposures of chicken embryos to C. sativum extract resulted in a dose-dependent increase of anomalies with concentrations higher than 1.6 µg/mL (Figure 5 and Table 4). Present results show that C. sativum extract reduced the axial skeleton and affected the neural tube, the somites, the cardiovascular structures, and the eye. These results indicate that the mechanism for the teratogenicity of C. sativum extract includes a direct effect on developing tissue. The nature of the registered abnormalities implies that this effect might be mediated by the disruption of genes that regulate pattern formation and that its effects could be directly associated with concentration amount.
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