Coriander

T:Toxicology&ChemicalFoodSafety

JFS T: Toxicology and Chemical Food Safety

Mutagenicity and Safety Evaluation of WaterExtract of Coriander sativum LeavesMARIANA RAMIREZ REYES, JORGE REYES-ESPARZA, OSCAR TORRES ANGELES, AND LOURDES RODRIGUEZ-FRAGOSO

ABSTRACT: Coriander has been used as a spice and medicinal plant for centuries. Several studies have described itsbiological properties and some reports have indicated its pharmacological actions in some human pathology. How-ever, data on its toxicity and metabolism are limited or null, and no research has been conducted with mammaliancells. The purpose of this study was to evaluate the mutagenicity and safety of Coriandrum sativum extract. Themutagenic effects of C. sativum extract were evaluated by Ames test. Mutagenicity was present when the C. sativumextract was used in high concentrations in both tested strains (Salmonella typhimurium TA97 and TA102). Our re-search showed that C. sativum extract reduced the cell survival of human cell lines (WRL-68 and 293Q cells) byinducing apoptosis and necrosis in the cases where extract concentration was the highest. The C. sativum extractaltered the cell cycle; it increased the G1 phase of hepatic cells and reduced the G2+M phase in both cell lines ina dose-response manner. These results showed correlation with a reduction in the mitotic index. The extract alsoinduced severe malformations during embryonic development. Exposure of chicken embryos to the C. sativum ex-tract resulted in a dose-dependent increase of anomalies. Present results show that C. sativum extract reduced theaxial skeleton and affected the neural tube, the somites, the cardiovascular structures, and the eye. According to thepresent results, the C. sativum aqueous extract cannot be considered safe. These results indicate that some signifi-cant adverse effects of C. sativum extract could be observed in vivo.

Keywords: apoptosis, Coriandrum sativum, cytotoxicity, embryotoxicity, mutagenicity

Introduction

Coriander is an annual herb native to Mediterranean Europeand Western Asia, naturalized in North America, and now ex-

tensively cultivated in many temperate countries (Wichtl and Bis-set 1994; BHP 1996; Leung and Foster 1996). Fresh leaves are usedas a flavoring agent and dried coriander seeds are used as spicesin food preparation. Both an annual and a perennial herb, corian-der is rich in various food elements (Grieve 1971). It contains about1% volatile oil, of which 55% to 74% is linalool; monoterpene hy-drocarbons (a- and b-pinene and limonene), anethole, and cam-phor comprises 20%; oleic, petroselinic, and linolenic fatty acidsmake 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 re-mainder (approximately 1%) contains coumarins, mucilage, andstarch (Lister and Hrhamme 1973; Hansel and others 1992; Wichtland 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 prepara-tions of coriander seed have been used in traditional Chinese, In-dian, Greco-European, and Latin American indigenous medicine.The British Herbal Pharmacopoeia (BHP 1996) and The Merck Indexalso report its therapeutic qualities as a carminative and aromatic(Budavari 1996).

In traditional medicine, Coriandrum sativum has been used totreat a number of medical problems such as dyspepsia, loss of ap-petite, convulsions, insomnia, and anxiety (Breevort 1996; De Smet2002). C. sativum is empirically used in different doses and forms

MS 20090347 Submitted 4/17/2009, Accepted 8/29/2009. Authors are withFacultad de Farmacia, Univ. Autonoma del Estado de Morelos. AvenidaUniv. 1001 Col. Chamilpa 62210, Cuernavaca, Morelos. Mexico. Direct in-quiries to author Rodrıguez-Fragoso (E-mail: [email protected]).

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 expecteach of these preparations to have a different proportion of each ofthe previously mentioned components; the tinctures and extractswill have more nonpolar components, while hydrophilic compo-nents will predominate in the teas and decoctions. The way thesepreparations are made must also be considered (cooking time, tem-perature, amount of water, time of rest, amount of plant used, andso on). Tinctures are more concentrated than infusions and decoc-tions, while the preparation of extracts could lead to loss of volatileoils. Having taken all this into account, the efficacy or toxicity of C.sativum could vary depending on the composition and proportionof constituents in the preparations made for human use.

Recent research involving in vivo pharmacological use of ex-tracts from this plant in experimental models has shown their hightherapeutic effectiveness (Medhin and others 1986; Gray and Flatt1999; Kubo and others 2004; Lal and others 2004; Ramadan andothers 2003; Emamghoreishi and others 2005). But very few (ifany) modern clinical studies have been conducted on coriander.Its approved modern therapeutic applications are based on its longhistory of use in well-established systems of traditional medicine,pharmacological studies conducted on animals, nutrient compo-sition, dietary value studies, and phytochemical research (Burdockand Carabin 2009).

In spite of the wide-ranging, extant research on the therapeu-tic effects of C. sativum, little is known about its toxicologicaleffects, since no extensive studies have been conducted on invitro and in vivo models (Burdock and Carabin 2009). An impor-tant aspect of natural products, especially those that are read-ily available to the public, is safety. Many people assume naturalproducts are safe, but there is recent, abundant evidence involvingserious adverse effects and deaths associated with the use of dietary

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Toxicity of Coriandrum sativum . . .

supplements and nutriments (Marcus and Grollman 2002; Wooltor-ton 2002; Wooltorton and Sibbald 2002; Morrow and others 2005).The purpose of this study was, therefore, to evaluate the toxicologi-cal effect of C. sativum extract on in vitro models and chick embryodevelopment.

Materials and Methods

Preparation of C. sativum extractLeaves of C. sativum were purchased from the Oaxaca Sierra,

Mexico, and transported to the state of Morelos (Mexico) in October2006 (Figure 1). These were then identified by a taxonomist and avoucher sample representing Herbarium nr FL6006 was depositedat the Herbarium of the Autonomous Univ. of the State of Morelos.The leaves were air dried at room temperature, ground, and kept inamber colored bottles until processed. Aqueous extraction was per-formed by soaking a weighed amount of the dry powder in distilledwater and shaking it for 3 h with an electric shaker. The suspen-sion was filtered through muslin gauze and the filtrate kept in deepfreeze for 24 h, then lyophilized. The lyophilized dry powder wascollected in a stoppered sample vial, weighed, and kept in a desic-

Figure 1 --- Coriander (Coriander sativum) (Source: OaxacaSierra, Mexico, 2006).

cator to avoid water absorption until used. Hydroalcoholic extrac-tion using 80% methanol was conducted by percolating 200 to 300 gof the dried and powdered plant material for 5 d, which was thenfiltered through Whatman filter paper nr 1. The solvent was evap-orated using a Rotavapor and the extract was kept in a stopperedsample vial at 4 ◦C until used.

Determination of content andessential oil composition

Coriander leaf oils were analyzed as 1% solutions in hexane,using a Perkin–Elmer autosystem gas chromatograph under thecontrol of Perkin–Elmer Omega (version 5.2) software. GC analy-sis was performed on a Hewlett-Packard 5890 series II gas chro-matograph equipped with 2 flame ionization detectors (255 ◦C), 2fused 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 simultane-ously, and a split injector at 255 ◦C (split ratio) 1 : 150). The tem-perature 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 chro-matograph Q-mass 910 quadrupole mass spectrometer, equippedwith 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 thesame as those mentioned previously, but helium was used as thecarrier gas. Mass units were monitored from 45 to 350 at 70 eV.The oil components were identified by (1) determining their reten-tion indexes (RI) in relation to a homologous series of fatty acidsmethyl esters (C4-C18) and (2) comparing their mass spectra withpublished values (Adams 1995; Baratta and others 1998; Gil andothers 2002) (Table 1).

Cell cultureTwo cell lines were used for this study: 293Q cells derived from

normal epithelial cells of human fetal kidney (CRL-1573 ATCC) andWRL-68 cells derived from epithelial cells of human fetal liver (CRL-48 ATCC). Cell lines were cultured in minimal essential medium(MEM, GIBCO BRL Inc., Grand Island, N.Y., U.S.A.), supplementedwith nonessential amino acids (GIBCO BRL Inc), 10% fetal calfserum (GIBCO BRL Inc), L-glutamine (2 mol/L), and antibiotics.Cells were plated in 100-mm culture dishes (106 cells/dish), andmaintained at 37 ◦C under an atmosphere of 5% CO2 in humid-ified air. The medium was replaced every 2 d and the cells wereharvested and diluted 5-fold every 7 d. Subcultures were obtainedby trypsinization (0.025% trypsin solution containing 0.01% N ,N-diethyldithiocarbamic acid sodium salt, EDTA).

Table 1 --- Identification of C. sativum components.

Identification method

Peak Identity GC RI GC-MS Component level (%)

1 α-pinene + + + 4.82 camphene + + + 0.73 β-pinene + + + 0.44 myrcene + + + 0.95 p-cymene + + + 0.76 limonene + + + 2.77 γ -terpinene + + + 4.98 linalool + + + 75.49 camphor + + + 5.110 decanal + + + 0.311 geraniol + + + 2.812 decanol + + − 0.313 geranyl-acetate + + + 3.0

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Assessment of cell viabilityCells were plated at 10000 cells/well on 96-well plates. After 24 h,

the culture medium was replaced by a fresh one supplemented withdifferent 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 andprocessed. Cell viability was measured by a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay (Wang andothers 1996). Briefly, 20 μL MTT (5 g/L) was added to each welland incubated with the culture for an additional 4 h at 37 ◦C, 5%CO2. Culture media were then discarded, followed by the additionof 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 dis-solved, the optical density was determined on a microplate reader(Bio-Rad) at 450 nm.

Survival of cells and detectionof percentage of apoptosis and necrosis

Survival of cells was evaluated by a flow cytometric method(Nicoletti and others 1991). Annexin V is a Ca2+-dependentphospholipid-binding protein that has a high affinity forphospholipid-like phosphatidylserine (PS) and is useful foridentifying apoptotic cells with exposed PS. Cells (105) werewashed twice with cold PBS and then re-suspended in 1× bindingbuffer at a concentration of 1 × 106 cells/mL. A total of 100 μL of thesolution (1 × 105 cells) were transferred to a 5 mL culture tube. Fivemilliliters of Annexin V–FITC and 10 μL of PI were added. Cells wereincubated for 15 min at room temperature (20 to 25 ◦C) in the darkand then 400 μL of 1× binding buffer were added to each tube. Theresults were analyzed with a CELLQuest program in a FACSCaliburflow cytometer (Becton Dickinson, Calif., U.S.A.). Staining cellssimultaneously with FITC-Annexin V (green fluorescence) and thenonvital dye propidium iodide (red fluorescence) enables bivariateanalysis of the discrimination of intact cells the discriminationof intact cells (FITC-PI-), early apoptotic (FITC+PI-), and lateapoptotic or necrotic cells (FITC+PI+).

Bactericidal toxicity testSamples (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 ofthe WFTS, 0.2 mL of phosphate buffer (0.2 M, pH 7.4), and 0.5 mLof S-9 mix (metabolic activator) or, instead, phosphate buffer. Themixture was diluted sequentially with phosphate buffer and 1 mLof diluted solution was mixed with 12 mL of nutrient agar. After in-cubation at 37 ◦C for 48 h, the number of colonies was counted.A bactericidal toxicity effect was confirmed if the standard platecount of tested compound was lower than that of the control (with-out adding tested compound).

Salmonella mutagenicity test/Ames testThe Ames test was performed as a standard plate incorporation

assay with Salmonella typhimurium strains TA97a and TA102 withmetabolic activation (Maron and Ames 1983). Strain-specific ge-netic markers were verified prior to use. The selection of the strainswas based on the testing and strain selection strategies of Mortel-mans and Zeiger (2000). For each tested strain, a specific positivecontrol was always used to assess the experimental flaws, if any.Nitro phenylenediamine and sodium azide were used as positivecontrols for TA97 and TA102, respectively. To ensure sterility, thedifferent concentrations of the extract were exposed for 15 minto UV-C light (TUV 30W G30T8 Philips, Holland). The absence ofcontaminant growth was checked in a blank set containing the ex-tract but without the addition of bacteria. For each concentration,100 mL of the test solution was used per plate. Positive controls

(20 mg/plate nitrophenylenediamine for TA97a and 1.5 mg/platesodium azide for TA102) and negative control (distilled water) wereconcurrently maintained. Samples were tested on triplicate platesin 2 independent experiments. Following 48 h incubation at 37 ◦C,genotoxic activities were expressed as induction factors (inductionfactors of reversions), that is, as multiples of the background lev-els. The interpretation of the Ames test results followed the U.S. En-vironmental Protection Agency (1996) guidelines for genotoxicitytesting of chemicals. According to the guidelines, a mutagenic po-tential is assumed if the revertant frequency is 2 or higher over thesolvent control (Mortelmans and Zeiger 2000).

Analysis of the cell cycleCells were scraped and washed with PBS. Cells (105) were fixed in

75% ethanol for 24 h and then washed in PBS and resuspendedin 0.1% NP40 (Nonidet P40, Biochimica Fluka, St. Louis Mo.,U.S.A.) and 10 μg/mL RNAse (Ribonuclease A, DNAse-free prepa-ration) for 20 min at room temperature (Darzynkiewicz and others2001). Propidium iodide (PI) was then added (final concentration5 μg/mL) for 12 h at 4 ◦C in darkness. Samples were analyzed us-ing a FACSCalibur flow cytometer. The results were analyzed usingCELLQuest program.

Mitotic activityCells were fixed in 3% paraformaldehyde with 0.25M manni-

tol (45.54 g/L) for 2 h, rinsed in PBS and stained with DAPI (4′,6-diamidino-2-phenylindol, 480 nm). After rinsing in PBS, the cellswere embedded in Citifluor mounting medium. The mitotic indexwas counted with a fluorescent microscope (Optiphot 2 Nikon). Foreach experiment, the indices were determined per 1000 cells andwith 4 replicates.

Embryotoxicity studiesFertile White Leghorn chicken eggs were obtained from A.L.P.E.

S.A. (Puebla, Mexico) and were stored at 6 ◦C. Total of 72 fertilizedeggs were weighed, sterilized, and divided into 9 groups. First groupserved 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 groupreceived caffeine (10 mg/mL) and was considered the positive con-trol. A teratogenicity assay was carried out as described by Jelinekand Marthan (Jelinek and Marhan 1994). Test solutions (1 mL) wereadded to the air sac under sterile conditions. Each solution was in-jected after drilling into the shell at the blunt end of the egg; afterinjection, the holes were immediately sealed with melted paraffinwax. The eggs were then transferred to and maintained in a forceddraft incubator at 37.5 ◦C with a relative humidity of 55% until thedesired stage of development was reached.

To determine the concentration dependency of C. sativum ex-tract teratogenicity, a histological analysis was carried out. Em-bryos in each group were fixed in buffered formal saline (pH 7.4),dehydrated, and embedded in paraffin blocks. Paraffin tissue sec-tions of 6 μm were stained with acetocarmine for routine histolog-ical examination. The embryo was examined and staged accordingto morphological criteria previously outlined by Hamburger andHamilton (1951). Embryonic stages at the time of the C. sativumextract application varied from 14 to 16, which correspond approx-imately to developed somites numbered 22 to 28.

Statistical analysis of resultsIn vitro data were reported as mean ± SD of 3 independent

experiments conducted in quadruplicate. Mean values were com-pared using Student’s t-test or analysis of variance (ANOVA) usingSPSS 10.0 software (SPSS Inc., Chicago, Ill., U.S.A.). Significant dif-ferences were established at P < 0.001.

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Results and Discussion

C. sativum extract caused a marked reduction in the survival of2 human cell lines after 24 h incubation (Figure 2). Concentra-

tions higher than 1.6 μg/mL produced different effects on cell lines.A significant 60% survival reduction was observed in WRL-68 (hep-atic) 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. Higherconcentrations (3.2 to 8 μg/mL) reduced cell survival by 60% or

Figure 2 --- Changes in the survival of cells after treatmentwith different concentrations of C. sativum extract duringa 24-h incubation period (control = 100%). The results arepresented 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 accom-panied by an increase in the amount of apoptotic and necrotic cells(Table 2), P < 0.001. It was found that apoptotic cells outnumberednecrotic cells, especially after treatment with the highest concen-trations of the extract (1.6 to 8 μg/mL). The increase in apoptoticcells took place in a dose-dependent manner. Some reports have in-dicated that low concentrations of some compounds induce apop-tosis, while high concentrations induce necrosis (Del Bino andothers 1991). The appearance of necrotic cells may also be a resultof incomplete apoptosis (Leist and Nicotera 1998). Recent studiesby Bakkali and others (Bakkali and others 2005) have shown that anextract of C. sativum induced cytotoxic effects on the yeast Saccha-romyces cerevisiae, and that the effects were stronger in exponentialrather than stationary phase cells. The results of the present studyshowed that C. sativum is cytotoxic for both human cell lines.

The results of previous comet assays show that C. sativum doesnot induce toxicity in cultured fibroblasts of rat embryo (Heibat-ullah and others 2008). The present study demonstrated that C.sativum extract is mutagenic in the tested range of 1.6 to 8 μg/mLaccording to the Ames test. The results of mutagenicity assays, pre-sented as mean revertants per plate, are shown in Table 3. Themutagenic potential of the extracts showed a positive dose-relatedincrease in the number of revertant colonies in both strains of S.typhimurium. The number of revertant colonies ranged between437 ± 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 differentconcentrations of C. sativum extract.

Concentration ofCell line C. sativum (μg/mL) Normal cells (%) Apoptotic cells (%) Necrotic cells (%)

WRL-68 0 90 ± 6 6 ± 2 4 ± 10.4 86 ± 4 8 ± 1.9 5 ± 30.8 85 ± 3 9 ± 3 6 ± 31.6 63 ± 6 28 ± 5∗ 9 ± 2∗

3.2 48 ± 4 35 ± 3∗ 13 ± 5∗

4.8 42 ± 7 39 ± 4∗ 18 ± 6∗

6.4 40 ± 8 42 ± 1∗ 18 ± 4∗

8 42 ± 3 47 ± 0.5∗ 11 ± 2∗

293Q 0 91 ± 5 5 ± 2 4 ± 10.4 86 ± 3 11 ± 3 3 ± 0.50.8 83 ± 2 8 ± 2 9 ± 31.6 70 ± 6 21 ± 4∗ 9 ± 1∗

3.2 58 ± 7 27 ± 3∗ 15 ± 3∗

4.8 40 ± 4 37 ± 5∗ 23 ± 4∗

6.4 38 ± 5 38 ± 4∗ 24 ± 5∗

8 37 ± 5 41 ± 3∗ 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.

Revertant colonies (UFC/plate)e

TA97a, mean ± SD T102, mean ± SD

Concentration of C. sativum (μg/mL) -S9d S9 -S9 S9

Distilled watera 98 ± 3 89 ± 4 149 ± 21 490 ± 60.4 106 ± 15 188 ± 7 198 ± 34 630 ± 140.8 134 ± 28 296 ± 12 265 ± 42 698 ± 241.6 437 ± 16c 990 ± 9 420 ± 38c 830 ± 143.2 538 ± 22c 1030 ± 14 680 ± 27c 3143 ± 254.8 610 ± 36c 2345 ± 17 945 ± 34c 4423 ± 316.4 690 ± 21c 3423 ± 24 1230 ± 43c 4987 ± 418 734 ± 34c 3567 ± 27 1742 ± 58c 5423 ± 34NDPb 790 ± 45c 3974 ± 34SAb 1985 ± 124c 6423 ± 29aNegative control.bPositive control; NDP = nitrophenylenediamine (20μg/plate) and SA = sodium azide (1.5 μg/plate).cNumber of revertant colonies more than twice that of corresponding control (P < 0.001).dS9 is a metabolic activation system consisting of the postmitochondrial fraction of the livers of rats. The results are presented as means ± SD of 3 independentexperiments. SD = standard deviation.eThe number represent the number of mutants/plate.




TA102, with concentrations between 1.6 and 8 μg/mL of C. sativumextract (P < 0.001). According to the 2-fold rule, the doubling ofspontaneous reversion rate at 1 or 2 test chemical concentrationsconstitutes a positive response (Mortelmans and Zeiger 2000). Themutagenic activity was associated to a metabolic activation by S9mix. Bakkali and others (Bakkali and others 2005) demonstratedthat C. sativum induced cytoplasmic petit mutations on the yeast S.cerevisiae. The results of the present study showed that C. sativumextract is mutagenic toward both tester strains, so that the presenceof apoptosis in human cell lines suggests it might also induce mu-tations in those cells.

The results also showed differences in the effects of C. sativumextract 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 wasobserved in WRL-68 cells with all used concentrations; therewas also a 50% to 80% reduction in the G2/M phase with concen-trations of 3.2 to 8 μg/mL (P < 0.001). No changes in S phase wereobserved with any concentration. On the other hand, 293Q cellsshowed a reduction of 60 to 80% in the G2+M phase with con-centrations higher that 0.8 μg/mL (P < 0.001), while no changesin G1 and S phases were observed. C. sativum extract also reducedthe percentage of cellular divisions. A reduction of 20% to 25%with lower concentrations and 35% to 40% with concentrationshigher 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 293Qcells was observed when they were treated with C. sativum extract(P < 0.001). For many cells, G1 phase is the major period of cellgrowth. During this stage, new organelles are synthesized and thecell requires both structural and functional proteins, resulting inactive protein synthesis and, therefore, a high metabolic rate cell.The presence of cell cycle arrest and apoptosis suggests that cellsare suffering DNA damage. Previous studies have suggested that cy-cle regulation-mediated apoptosis is a mechanism of cell growthinhibition (Ahmad and others 1997; Deigner and Kinscherf 1999;Ahmad and others 2001). The apoptosis is a physiological processthat functions as an essential mechanism of tissue homeostasis andis regarded as the preferred way to eliminate unwanted cells. Previ-ous studies by Cortes-Eslava and others (2004) demonstrated thatan aqueous crude coriander juice significantly decreased the muta-genicity of some metabolized aromatic amines and that the corian-der juice (50 to 1000 μL per coincubation flask) was neither toxicnor mutagenic. Previous studies have also shown that C. sativumextract possesses antioxidative properties (Chithra and Leelamma1999; 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 damageand others are protecting the cell.

Hepatic and renal cells play an important role in drugmetabolism because they have drug-metabolizing enzymes. Mu-tagenic activity in the presence of S9 suggested that C. sativumextract might be metabolized in vivo and cause several biologicalactivities. Metabolic products are often less active than the par-ent drug and may even be inactive. However, some biotransforma-tion products have enhanced activity or toxic properties. Certainherbal medicines have been identified as a cause of acute andchronic hepatitis, cholestasis, drug-induced autoimmunity, vascu-lar lesions, and even hepatic failure and cirrhosis. Several factorsmay contribute to the hepatotoxic effects of herbal preparations.Herbal medicines are usually a mixture of several ingredients orplants harvested during different seasons and extracted by vari-able procedures, which make the identification of both pharma-cologically active and toxic compounds difficult. This is the caseof C. sativum (Smallfield and others 2001; Gil and others 2002;

Ramadan and others 2003). Also, contamination of herbalmedicines with microorganisms, fungal toxins (such as aflatoxin),pesticides, heavy metals, and synthetic drugs could confuse anal-ysis. Moreover, it has been reported that herbal extracts may alsoexert renal toxicity through inherent properties (Wojcikowski andothers 2004). There is currently not enough information regardingthe metabolism of C. sativum in human cells. However, becausethis plant contains a mixture of essential oils and the C. sativumextract here studied induced necrosis, it is possible that C. sativumextract could be metabolized into toxic metabolites in hepatocytesand renal cells (Smallfield and others 2001; Gil and others 2002). Ithas been reported that ingestion of coriander oil led to the incor-poration of petroselinoyl (triacylglycerols of coriander) into heart,

Figure 3 --- Percentage of cells in G1, S, G2/M phases of thecell cycle after treatment with different concentrationsof C. sativum extract during a 24-h incubation period. (A)WRL68 cells, (B) 293Q cells. The results are presented asmeans ± SD of 3 independent experiments. ∗P < 0.001 incomparison with control values.

Figure 4 --- Changes in the mitotic index of cells af-ter incubation for a 24-h period with different con-centrations of C. sativum extract. The results arepresented as means ± SD of 3 independent experiments.∗P < 0.001 as compared with all groups; #P < 0.001 ascompared with 0.4, 0.8, and 1.6 μg/mL.


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Figure 5 --- Morphological appearanceof chick embryos treated withdifferent concentrations of C.sativum. Arrows indicatemorphological alterations in theheart, central nervous system (CNS),placodes (optical and otic), neuraltube, and somites.

Table 4 --- Teratogenic effects of C. sativum.

C. sativum extract (μg/mL)

Embryonic region affected Caffeinea 10 mg/mL Controlb 0.4 0.8 1.6 3.2 4.8 6.4 8

CNS 3/9 0/9 0/9 1/9 3/9 5/9 7/9 8/9 9/9Neural tube 6/9 0/9 0/9 0/9 3/9 5/9 7/9 8/9 9/9Somites 7/9 0/9 0/9 0/9 2/9 4/9 5/9 8/9 9/9Vasculature 7/9 0/9 0/9 0/9 3/9 5/9 7/9 8/9 9/9Heart 7/9 0/9 0/9 1/9 3/9 5/9 7/9 8/9 9/9Eye 5/9 0/9 0/9 0/9 1/9 2/9 4/9 6/9 8/9Axial skeleton 9/9 0/9 3/9 4/9 7/9 8/9 9/9 9/9 9/9aPositive control.bNegative control.The fractions represent the number of abnormal embryos and the total examined for each developmental region of the embryo.

liver, and blood lipids in rats. The consumption of coriander triacyl-glycerols led to significantly greater liver weights (Weber and others1995).

The effect of an aqueous extract of fresh C. sativum seeds onfemale fertility in rats was studied in the 1980s (Al-Said and oth-ers 1987). The results showed that the treatment of animals withfresh coriander seed extract did not produce any significant aborti-facient activity. There was no significant change in the weight andlength 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 ex-tract on a chicken embryo model. Exposures of chicken embryos toC. sativum extract resulted in a dose-dependent increase of anoma-lies with concentrations higher than 1.6 μg/mL (Figure 5 andTable 4). Present results show that C. sativum extract reduced theaxial skeleton and affected the neural tube, the somites, the car-diovascular structures, and the eye. These results indicate that themechanism for the teratogenicity of C. sativum extract includes adirect effect on developing tissue. The nature of the registered ab-normalities implies that this effect might be mediated by the dis-ruption of genes that regulate pattern formation and that its effectscould be directly associated with concentration amount.

Conclusions

The toxicological evaluation of C. sativum aqueous extractshowed that said extract is toxic for human cell lines and

chicken embryos. Study results reveal that C. sativum extract in-duced cytotoxic effects on renal and liver cells in a dose-dependentmanner (P < 0.001). C. sativum extract was able to produce apop-tosis, necrosis, and alterations in the cell cycle, and changes inthe mitotic index in renal and hepatic cell lines. The extract also

presents mutagenicity according to the Ames test and the resultsshowed it induced severe malformations during embryonic devel-opment. The C. sativum aqueous extract cannot be considered safeand these results indicate that some significant adverse effects of C.sativum extract could be observed in vivo.

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