Oxidative stress and reactive oxygen species-mediated cell damage have been implicated in the development of various human chronic diseases such as Crohn's disease, CVD, certain cancers and a number of neurodegenerative diseases (Babbs, Reference Babbs1992; Halliwell & Gutteridge, Reference Halliwell and Gutteridge1999; Parthasarathy et al. Reference Parthasarathy, Santanam, Ramachandran and Meilhac1999; Sohal et al. Reference Sohal, Mockett and Orr2002). At the cellular level, subjecting cells to oxidative stress can result in severe metabolic dysfunction, including lipid peroxidation, protein oxidation, membrane disruption and DNA damage (Halliwell & Gutteridge, Reference Halliwell and Gutteridge1999).
Oxidative damage can also have undesirable effects on foods such as the production of rancid odours and flavours which can reduce the shelf-life, nutritional quality and safety of food products (Zainol et al. Reference Zainol, Abd-Hamid, Yusof and Muse2003; Chanwitheesuk et al. Reference Chanwitheesuk, Teerawutgulrag and Rakariyatham2005). In order to lower the risk of oxidative deterioration, synthetic antioxidants have been added to many food products. However, due to safety issues and consumer demand, there has been considerable interest in replacing synthetic antioxidants with natural plant-based alternatives. Therefore, an increasing number of plant-derived antioxidant preparations are commercially available for use in food applications (Aruoma et al. Reference Aruoma, Spencer, Rossi, Aeschbach, Khan, Mahmood, Munoz, Murcia, Butler and Halliwell1996; Mielnik et al. Reference Mielnik, Aaby and Skrede2003).
Over the past few decades, research has focused on the health effects of phytochemicals and plant-derived extracts. The Lamiaceae family is a large group of plant species that contain substantial amounts of phenolic compounds (Moreno et al. Reference Moreno, Scheyer, Romano and Vojnov2006). Popular varieties from this family include the culinary herbs: rosemary (Rosmarinus officinalis L.), oregano (Origanum vulgare L.) and sage (Salvia officinalis L.). Experimental evidence suggests that most herbs and spices, especially those of the Lamiaceae family, possess a wide range of biological and pharmacological activities that may protect tissues against O2-induced damage and therefore lower the risk of human chronic diseases (Craig, Reference Craig1999; Kris-Etherton et al. Reference Kris-Etherton, Hecker, Bonanome, Coval, Binkoski, Hilpert, Griel and Etherton2002; Bozin et al. Reference Bozin, Mimica-Dukic, Simin and Anackov2006).
Natural phytochemicals present at low levels in fruit, vegetables, herbs and spices offer many health benefits, but these compounds may not be effective or safe when consumed at higher does (Liu, Reference Liu2003). If herbal use is to be extended to applications that require high doses, the increased exposure to human consumers is a matter of concern (Aydin et al. Reference Aydin, Basaran and Basaran2005). The objective of the present study, therefore, was to investigate the toxicity and biological activity of rosemary, oregano, sage and echinacea extracts using a human colon carcinoma cell line, Caco-2 cells, as a model system. The paper presents the effects of rosemary, oregano, sage and echinacea extracts on the viability, membrane integrity, antioxidant status and DNA integrity of Caco-2 cells. In the present study, the four plant extracts were also investigated for their potential cytoprotective and genoprotective effects against H2O2-induced stress in Caco-2 cells.
Materials and methods
Materials
All chemicals and cell culture reagents were obtained from Sigma Chemical Co. (Poole, Dorset, UK) unless otherwise stated. Rosemary (Rosmarinus officinalis), oregano (Origanum vulgare), sage (Salvia officinalis) and echinacea (Echinacea purpurea (L.), Moench–Asteraceae family) extracts were purchased from Guinness Chemicals (Portlaoise, Co. Laois, Republic of Ireland). Suppliers provided the following information based on HPLC analysis: No information was available on the oregano extract. Rosemary extract contained rosmarinic acid (7 %, w/v). The key compounds detected in the sage extract were rosmarinic acid (7·03 %, w/v), flavones (3·21 %, w/v) and flavanones (5·13 %, w/v). Echinacea extract contained various caffeic acid compounds (8·35 % total, w/v).
Total phenol content
Total phenolic content of the extracts was determined by employing the methods given in the literature involving Folin–Ciocalteu reagent and gallic acid as standard (Singleton & Rossi, Reference Singleton and Rossi1965).
Cell culture
Human Caucasian colon adenocarcinoma Caco-2 cells were purchased from the European Collection of Animal Cell Cultures (Salisbury, Wilts, UK). Cells were cultured in an atmosphere of CO2–air (5:95, v/v; O2 partial pressure of 19·998 kPa) at 37°C. Caco-2 cells were grown in Dulbecco's modified Eagle's medium supplemented with fetal bovine serum (10 %, v/v) and non-essential amino acids (1 %, v/v). Cells were maintained in the absence of antibiotics. For all experiments, Caco-2 cells were seeded at a density of 3 × 104 cells per cm2 in cell culture plates.
Incubation of Caco-2 cells with plant extracts or oxidants
After 24 h growth in the appropriate culture plates, growth media was replaced with growth medium containing reduced fetal bovine serum (5 %, v/v) and non-essential amino acids (1 %, v/v) in the presence or absence of the herb extracts, except for the lactate dehydrogenase (LDH) release assay where 2·5 % (v/v) fetal bovine serum was used. Rosemary, oregano, sage and echinacea extracts were dissolved in non-supplemented Dulbecco's modified Eagle's medium. Control cultures were exposed to the equivalent concentration of carrier and were found not to significantly differ from cells grown in media only. H2O2 was dissolved in ice-cold PBS and treatments with the oxidant were in the absence of serum.
For the determination of cell viability, concentration resulting in 50 % cell death (IC50) values and cell membrane integrity, Caco-2 cells were supplemented with increasing concentrations (5–1000 μg/ml) of rosemary, oregano, sage or echinacea extracts for 24 h. For subsequent assays, the concentration of each plant extract that corresponded to greater than 90 % cell viability was selected, namely: 15 μg/ml rosemary, 60 μg/ml oregano, 60 μg/ml sage and 250 μg/ml echinacea. Cell viability was also determined in Caco-2 cells exposed to H2O2 (50 μm for 30 min) and (100–500 μm for 2 h). To determine protection against oxidant-induced DNA damage, cells were supplemented with or without the plant extracts for 24 h followed by exposure to 50 μm-H2O2 for 30 min at 37°C. To investigate protection against oxidant-induced cytotoxicity, Caco-2 cells were supplemented in the presence or absence of each plant extract for 24 h followed with or without exposure to H2O2 (100–500 μm) for 2 h at 37°C.
Cell membrane integrity (lactate dehydrogenase release assay)
Media was collected from all test wells of a ninety-six-well plate. LDH leakage into the growth media and total LDH (LDH leakage plus LDH inside cells) were measured using an in vitro lactate dehydrogenase release assay kit (Biogenesis, Poole, Dorset, UK). LDH release was expressed as a percentage of total LDH.
Cell viability (neutral red uptake assay)
The neutral red uptake assay, by the method of Babich & Borenfreund (Reference Babich, Borenfreund and Watson1992), was used to assess cell viability. Neutral red dye was prepared as a 0·4 % (w/v) stock solution, which was diluted 1/100 using Dulbecco's modified Eagle's medium and pre-incubated overnight at 37°C. After 3 h incubation with the dye (40 μg/ml) at 37°C, cells were quickly washed with a fixative (1 % CaCl2, 0·5 % formaldehyde) and then solubilised. Absorbance was read at 540 nm on a microtitre plate reader. The IC50 value for each plant extract (concentration of compound that reduced cell viablity by 50 %) was calculated using the data obtained from the neutral red uptake assay and GraphPad Prism software version 4.0 (GraphPad Inc., San Diego, CA, USA).
Reduced glutathione content
GSH content was determined by the method of Hissin & Hilf (Reference Hissin and Hilf1976). Perchloric acid (15 %, v/v) was added to the cell sonicates and samples were centrifuged at 14 000 rpm for 30 min at 4°C. The final GSH assay mixture contained 100 μl sample, 1·8 ml phosphate–EDTA buffer (pH 8) and 100 μl σ-phthaldialdehyde (1 mg/ml). Fluorescence was detected at 430 nm following excitation at 360 nm. GSH content was expressed relative to the protein content, as determined by the bicinchoninic acid method (Smith et al. 1985).
Antioxidant enzyme activity assays
Catalase activity was determined using a modification of the method of Baudhuin et al. (Reference Baudhuin, Beaufay, Rahman-Li, Sellinger, Wattiaux, Jacques and deDuve1964), where any remaining H2O2 is determined as a yellow ‘peroxy titanium sulfate’. One unit of catalase activity is defined as the amount of catalase required to decompose 1 μmol H2O2 per min at pH 7·5 and 25°C. Superoxide dismutase activity was measured by the method of McCord & Fridovich (Reference McCord and Fridovich1969). One unit of superoxide dismutase activity is defined as the amount of superoxide dismutase required to inhibit the maximum rate of cytochrome C reduction by 50 %. Enzyme activities were expressed as units of enzyme activity per mg protein (Smith et al. 1985).
Alkaline single cell gel electrophoresis (comet) assay
DNA damage was assessed using the comet assay by the method of Tice et al. (Reference Tice, Andrews, Hirai, Singh and Witmer1990). Briefly, treated and non-treated cells were gently scraped and embedded in low-melting-point agarose on coded microscope slides. Samples were then placed in cold lysis solution (2·5 m-NaCl, 100 mm-EDTA, 10 mm-tri(hydroxymethyl)-aminomethane, 1 % sodium sarcosinate (pH 10), with fresh 1 % Triton® X-100 and 10 % dimethyl sulfoxide) for 1·5 h at 4°C. Slides were aligned in a horizontal gel electrophoresis tank (Horizon® 20·25; GIBCO BRL Life Technologies, Gaithersburg, MD, USA) which was filled with fresh electrophoresis solution (1 mm-EDTA, 300 mm-NaOH; pH 13). Slides were allowed to sit in this buffer for 30 min. Electrophoresis was conducted at 1 V/cm for 25 min at 4°C. After electrophoresis, the slides were washed three times with neutralising buffer and stained with ethidium bromide (20 μg/ml). Fifty nuclei per slide were analysed using computer-assisted image analysis (Komet 4; Kinetic Imaging, Liverpool, UK). The extent of DNA damage was expressed in two ways: (i) the mean percentage of tail DNA, reflecting the proportion of DNA that has migrated from the head and (ii) olive tail moment (OTM), which represents the product of the tail length and the percentage DNA in the tail.
Statistical analysis
Results are presented as mean values with their standard errors. Data were analysed by one-way ANOVA followed by Dunnett's test. The level of statistical significance was taken at P < 0·05 or P < 0·01.
Results
Total phenolic content of plant extracts
The total phenolic content of the four plant extracts was as follows: rosemary, 207 mg gallic acid equivalents (GAE)/g; sage, 192 mg GAE/g; oregano, 78 mg GAE/g; echinacea, 58 mg GAE/g.
Effects of plant extracts on the membrane integrity of Caco-2 cells
Caco-2 cells were grown in the presence or absence of rosemary, oregano, sage or echinacea (5–1000 μg/ml) for a period of 24 h. Following incubation, cell membrane integrity was determined using the LDH release assay. At concentrations ≥ 120 μg/ml, rosemary and sage significantly increased LDH leakage from the cells (Fig. 1). Supplementation of Caco-2 cells with oregano ( ≥ 500 μg/ml) or echinacea (1000 μg/ml) significantly induced LDH release (Fig. 1). The membrane-toxic effects of the plant extracts were in the order of rosemary>sage>oregano>echinacea.
Fig. 1 Cell membrane integrity of Caco-2 cells following supplementation with or without increasing concentrations (5–1000 μg/ml) of rosemary (-○-), oregano (-□-), sage (-▲-) or echinacea (-■-) extracts for 24 h. Cell membrane integrity was assessed using the lactate dehydrogenase release assay (see p. 322). Data are the means of four independent experiments, with standard errors represented by vertical bars.
Effects of plant extracts on cell viability in Caco-2 cells
Caco-2 cells were supplemented with or without increasing concentrations of rosemary, oregano, sage or echinacea (5–1000 μg/ml) for 24 h. Following incubation, the effect of the plant extracts on cell viability was assessed using the neutral red uptake assay. Cell viability in control and carrier control samples was greater than 95 % for all experiments (data not shown). On the addition of rosemary ( ≥ 30 μg/ml), cell viability was significantly reduced (P < 0·01; Fig. 2). The addition of higher concentrations of sage ( ≥ 120 μg/ml) or oregano ( ≥ 250 μg/ml) significantly decreased cell viability compared with control cells (P < 0·01). Echinacea was toxic only at the highest concentration tested (P < 0·01; Fig. 2). The cytotoxic effects of the plant extracts were in the order of rosemary>sage>oregano>echinacea. Using data obtained from the neutral red uptake assay, the IC50 value for each plant extract was determined. Rosemary had an IC50 value of 123 μg/ml, sage had an IC50 value of 214 μg/ml whereas oregano had a value of 318 μg/ml. Because the highest concentration of echinacea resulted in approximately 64 % cell viability (P < 0·01), the predicted IC50 value was 1421 μg/ml. Concentrations below the IC50 value that corresponded to greater than 90 % cell viability were selected for use in subsequent assays. Exposing Caco-2 cells to H2O2 (50 μm for 30 min) in the absence of serum did not affect cell viability (data not shown).
Fig. 2 Viability of Caco-2 cells following supplementation with increasing concentrations (5–1000 μg/ml) of rosemary (-○-), oregano (-□-), sage (-▲-) or echinacea (-■-) extracts for 24 h. Cell viability was determined using the neutral red uptake assay (see p. 322). Data are the means of four independent experiments, with standard errors represented by vertical bars.
Effects of plant extracts on antioxidant status of Caco-2 cells
Caco-2 cells were supplemented with or without rosemary (15 μg/ml), oregano (60 μg/ml), sage (60 μg/ml) or echinacea (250 μg/ml) for a period of 24 h. Following incubation, GSH content, catalase activity and superoxide dismutase activity were determined. Treatment with rosemary, oregano or echinacea had no effect on cellular GSH content whereas incubation of cells with sage for 24 h significantly increased GSH content (P < 0·01) compared with control cells (Fig. 3). There was no significant difference in catalase activity and superoxide dismutase activity between treated and non-treated cells (data not shown).
Fig. 3 GSH content of Caco-2 cells following supplementation with or without rosemary (15 μg/ml), oregano (60 μg/ml), sage (60 μg/ml) or echinacea (250 μg/ml) extracts for 24 h. Data are the means of four independent experiments, with standard errors represented by vertical bars. * Mean value was significantly different from that for control (P < 0·01; one-way ANOVA followed by Dunnett's test).
Effects of plant extracts or hydrogen peroxide on DNA integrity of Caco-2 cells
The potential of the plant extracts to induce single-strand DNA breaks was assessed using the comet assay. Caco-2 cells were supplemented with or without rosemary (15 μg/ml), oregano (60 μg/ml), sage (60 μg/ml) or echinacea (250 μg/ml) for 24 h. None of the four plant extracts investigated significantly increased the formation of DNA single-strand breaks when compared with control cells (data not shown). Caco-2 cells were exposed to 50 μm-H2O2 for 30 min at 37°C. Addition of H2O2 to the culture medium significantly increased DNA damage both in terms of percentage tail DNA and OTM (P < 0·01; Figs. 4 (A) and (B)).
Fig. 4 DNA damage in Caco-2 cells following pre-treatment with or without extracts of rosemary (R; 15 μg/ml), oregano (O; 60 μg/ml), sage (S; 60 μg/ml) or echinacea (E; 250 μg/ml) for 24 h then exposed to 50 μm-H2O2 (H) for 30 min at 37°C. DNA damage was assessed by the comet assay (see pp. 322–323). (A) Percentage tail DNA; (B) olive tail moment (OTM), which represents the product of the tail length and the percentage tail DNA. Data are the means of four independent experiments, with standard errors represented by vertical bars. Statistical analysis was by one-way ANOVA followed by Dunnett's test. * Mean value was significantly different from that for control (P < 0·01). Mean value was significantly different from that for H2O2-treated cells: † P < 0·05, †† P < 0·01.
Effects of plant extracts on hydrogen peroxide-induced DNA damage
Caco-2 cells were pre-incubated with or without rosemary (15 μg/ml), oregano (60 μg/ml), sage (60 μg/ml) or echinacea (250 μg/ml) extracts for 24 h followed by exposure to 50 μm-H2O2 for 30 min at 37°C. Similar trends of genoprotection were seen when DNA damage was expressed as either percentage tail DNA or OTM (Figs. 4 (A) and (B)). Pre-treatment with rosemary or oregano significantly decreased H2O2-induced DNA damage (P < 0·05) when compared with cells treated only with H2O2 (Figs. 4 (A) and (B)). Supplementation with sage produced a more significant reduction in H2O2-induced DNA damage (P < 0·01; Figs. 4 (A) and (B)).
Effects of plant extracts on hydrogen peroxide-induced cell injury
Caco-2 cells were pre-incubated with or without rosemary (15 μg/ml), oregano (60 μg/ml), sage (60 μg/ml) or echinacea (250 μg/ml) extracts for 24 h followed by exposure to increasing concentrations of H2O2 (100–500 μm) for 2 h at 37°C. Although treatment with 100 μm-H2O2 for 2 h reduced cell viability, the effect was not significant (Table 1). None of the plant extracts modulated this oxidant-induced reduction in cell viability. The addition of higher concentrations of H2O2 (250 and 500 μm) significantly decreased (P < 0·01) the viability of cells compared with control (Table 1). Sage was the only extract to significantly protect against H2O2-induced cytotoxicity (P < 0·05). Although not significant, pre-treatment with rosemary and echinacea did show a certain degree of cytoprotection against H2O2 (Table 1).
Table 1 Viability of Caco-2 cells after pre-treatment with or without plant extracts for 24 h followed by exposure to hydrogen peroxide‡ (Mean values with their standard errors; three individual experiments)
*Mean value was significantly different from that for control (P < 0·01; one-way ANOVA, followed by Dunnett's test).
† Mean value was significantly different from that for H2O2-treated cells (P < 0·05; one-way ANOVA, followed by Dunnett's test).
‡ Caco-2 cells were grown in the presence or absence of rosemary (15 μg/ml), oregano (60 μg/ml), sage (60 μg/ml) or echinacea (250 μg/ml) extracts for 24 h followed by exposure to increasing concentrations of H2O2 for 2 h at 37°C.
§ Determined using the neutral red uptake assay (see p. 322). Results are expressed as a percentage of the control (100 %).
Discussion
Fruits, vegetables and herbs contain a wide variety of phytochemicals that possess bioactive properties which may help protect cellular systems from oxidative damage (Liu, Reference Liu2003). In the present study, the biological effects of four plant extracts, namely rosemary, oregano, sage and echinacea were investigated. While there has been interest in the antioxidant activity of rosemary, oregano and sage and immunomodulatory effects of echinacea, limited information exists on their cytoprotective and genoprotective effects on cells in culture.
Many constituents in rosemary, oregano and sage such as phenolic acids (including rosmarinic and caffeic), phenolic compounds and flavonoids have been reported to exhibit a broad range of biological activities in vitro and in vivo including antioxidant, anti-inflammatory, anti-microbial, anti-mutagenic, and anti-tumour effects (Aruoma et al. Reference Aruoma, Spencer, Rossi, Aeschbach, Khan, Mahmood, Munoz, Murcia, Butler and Halliwell1996; Miura et al. Reference Miura, Kikuzaki and Nakatani2002; Petersen & Simmonds, Reference Petersen and Simmonds2003; Chorianopoulos et al. Reference Chorianopoulos, Kalpoutzakis, Aligiannis, Mitaku, Nychas and Haroutounian2004; Osakabe et al. Reference Osakabe, Yasuda, Natsume and Yoshikawa2004; Aydin et al. Reference Aydin, Basaran and Basaran2005; Capecka et al. Reference Capecka, Mareczek and Leja2005). Echinacea purpurea has been reported to act as an anti-inflammatory agent and an immunostimulant (Craig, Reference Craig1999; Linde et al. Reference Linde, Barrett, Wolkart, Bauer and Melchart2006) and is widely consumed for the prevention and treatment of upper respiratory tract infections (Barrett et al. Reference Barrett, Vohmann and Calabrese1999, Reference Barrett, Brown, Locken, Maberry, Bobula and D'Alessio2002; Hwang et al. Reference Hwang, Dasgupta and Actor2004; Linde et al. Reference Linde, Barrett, Wolkart, Bauer and Melchart2006). However, the exact mechanism(s) of action remain unclear and available evidence from clinical trials on the effectiveness of echinacea has been considered inconsistent (Barrett et al. Reference Barrett, Brown, Locken, Maberry, Bobula and D'Alessio2002; Linde et al. Reference Linde, Barrett, Wolkart, Bauer and Melchart2006).
To establish the effects of the four plant extracts on membrane integrity in Caco-2 cells, leakage of the membrane-bound enzyme LDH was determined. The order of ‘membrane potency’ among the four plant extracts correlated well with our cytotoxicity data and IC50 values. Supplementation with increasing concentrations of rosemary for 24 h caused cytotoxicity at concentrations ≥ 30 μg/ml in Caco-2 cells. Slameňová et al. (Reference Slameňová, Kuboskova, Horvathova and Robichova2002) determined the viability of Chinese hamster lung fibroblast V79 cells following supplementation with various concentrations of rosemary for 24 h. Cytotoxicity was reported at concentrations ≥ 15 μg/ml. In the present study both oregano and sage, also members of the Lamiaceae family, affected cell viability in a dose-dependent manner similar to that of rosemary. However, rosemary, having the lowest IC50 value of 123 μg/ml, was most toxic of the extracts to Caco-2 cells and echinacea was the least toxic with a predicted IC50 of 1421 μg/ml. Carpenter et al. (Reference Carpenter, O'Callaghan, O'Grady, Kerry and O'Brien2006) who investigated the effects of various plant extracts on the viability of U937 cells reported that echinacea was the least toxic plant extract of those tested, with the highest IC50 value of 9400 μg/ml.
There are many ways to express DNA damage as assessed by the comet assay, and the two most common endpoints reported are OTM and percentage tail DNA. According to Møller (Reference Møller2006), in order to provide feasible comparisons of DNA damage between studies, it is important that common reference endpoints are agreed upon and that researchers provide the opportunity for comparison. Therefore we expressed DNA damage in terms of OTM and percentage tail DNA (Fig. 4). Similar trends were seen when DNA damage was expressed as either of these two endpoints.
It is known that DNA strand breaks occur when cells are exposed to H2O2 (Meneghini & Martins, Reference Meneghini, Martins, Halliwell and Aruoma1993; Kruszewski et al. Reference Kruszewski, Green, Lowe and Szumiel1994). In the present study, we exposed Caco-2 cells to 50 μm-H2O2 for 30 min at 37°C which resulted in significant DNA single-strand-break formation (Figs. 4 (A) and (B)). These findings are in agreement with previous work carried out in our laboratory (Aherne & O'Brien, Reference Aherne and O'Brien1999, Reference Aherne and O'Brien2000). The plant extracts themselves did not induce DNA damage at the concentrations and exposure times tested. Even though oregano extract had less than half the total phenolic content of rosemary or sage, we found that sage, oregano and rosemary, in that order, protected against H2O2-induced DNA damage in Caco-2 cells. Research has shown that the antioxidant effectiveness of rosemary, oregano and sage extracts in vitro is due to their ability to act as reducing agents and free radical scavengers, as quenchers of singlet O2 formation and to complex with pro-oxidant metal ions (Miura et al. Reference Miura, Kikuzaki and Nakatani2002; Dorman et al. Reference Dorman, Peltoketo, Hiltunen and Tikkanen2003; Amarowicz et al. Reference Amarowicz, Pegg, Rahimi-Moghaddam, Barl and Weil2004; Moreno et al. Reference Moreno, Scheyer, Romano and Vojnov2006; Slameňová et al. 2002). Thus the genoprotective effects of the plant extracts could be due, in part, to their free radical-scavenging efficiency and reducing power, as a result of their phenolic and/or non-phenolic constituents.
Sage was the only plant extract to exert a protective effect against H2O2-induced cytotoxicity. Therefore, sage may afford this protection against H2O2-induced cell injury by mechanisms other than those already mentioned. Chiou & Tzeng (Reference Chiou and Tzeng2000) reported that the resistance of K300 cells to menadione-induced cytotoxicity may be due, in part, to the elevated concentrations of GSH in these cells. Therefore, it is possible that sage protected against H2O2-induced cytotoxicity by improving the antioxidant status of Caco-2 cells. The present results show that rosemary, oregano and echinacea had no effect on the antioxidant status of Caco-2 cells or on H2O2-induced cytotoxicity. However, supplementation with sage enhanced cellular GSH content and protected cells against H2O2-induced cytotoxicity. This mechanism could also explain why sage afforded the greatest protection against H2O2-induced DNA damage.
Prevention is a more effective strategy than treatment of chronic diseases. Incorporation of herbs into everyday meals may be beneficial, as a diet in which culinary herbs are used generously provides a variety of active phytochemicals that could promote good health by protecting tissues against O2-induced damage, and preventing the onset of chronic diseases (Craig, Reference Craig1999). Health benefits resulting from the use of natural plant products rich in bioactive substances has promoted growing interest from pharmaceutical, food and cosmetic industries (Capecka et al. Reference Capecka, Mareczek and Leja2005). The findings of the present study suggest that the proper use of herb products is safe and may provide some beneficial effects; however, our toxicity data justify concerns regarding the consumption of high doses that are unsafe and possibly harmful to human health.
Acknowledgements
The present study was funded under the National Development Plan (2000–6), through the Food Institutional Research Measure, administered by the Irish Department of Agriculture and Food.
