- Email: [email protected]
Inhibitory effects of Zataria multiflora essential oil and its main components on nitric oxide and hydrogen peroxide production in glucose-stimulated human monocyte
Food and Chemical Toxicology 50 (2012) 3079–3085
Contents lists available at SciVerse ScienceDirect
Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Inhibitory effects of Zataria multiflora essential oil and its main components on nitric oxide and hydrogen peroxide production in glucose-stimulated human monocyte Gholamreza Kavoosi a,⇑, Jaime A. Teixeira da Silva b a b
Institute of Biotechnology, Faculty of Agriculture, University of Shiraz, Shiraz 71441-65186, Iran Faculty of Agriculture and Graduate School of Agriculture, Kagawa University, Ikenobe 761-0795, Japan
a r t i c l e
i n f o
Article history: Received 2 November 2011 Accepted 4 June 2012 Available online 13 June 2012 Keywords: Zataria multiflora Essential oil Monocytes Glucose Nitric oxide Hydrogen peroxide
a b s t r a c t The inhibitory effects of Zataria multiflora essential oil on nitric oxide (NO) and hydrogen peroxide (H2O2) production were examined in human monocytes cultured in the presence of 20 mM glucose. Z. multiflora essential oil was extracted by water-distillation and then analyzed by GC–MS. Carvacrol (29.2%), thymol (25.4%), p-cymene (11.2%), linalool (9.6%) and c-terpinene (8%) were the main components detected in the essential oil. Cells cultured in the presence of 20 mM glucose showed an increase in NO and H2O2 production as well as NO synthase (NOS) and NADH oxidase (NOX) activities compared to cells cultured in the presence of 5 mM glucose. Pretreatment with Z. multiflora essential oil, carvacrol and thymol reduced NO and H2O2 production as well as NOS and NOX activities in those cells cultured in the presence of 20 mM glucose. However, p-cymene, linalool and c-terpinene did not show any such activities. Accordingly, it was concluded that Z. multiflora can reduce oxidative stress and can be used in the therapy of oxidative damage accompanying hyperglycemia and some inflammatory conditions. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Extensive research within the last decade has revealed that most diseases such as cancer, cardiovascular, neurological and autoimmune diseases, as well as diabetes are linked to the overproduction of reactive oxygen species (ROS) and reactive nitrogen species (RNS) (Ma, 2010). Although herbal medicines are regarded to have therapeutic potential against these diseases, neither their active components nor their mechanisms of action are fully understood. There is growing interest in the possible therapeutic potential of natural products against these diseases. These compounds exert potent antioxidant actions and have the inherent capacity to scavenge ROS and RNS, thus counteracting conditions of oxidative stress that accompany disorders such as coronary and vascular and inflammatory diseases, diabetes and cancer (Johansen et al., 2005; Pacher et al., 2007; Sze et al., 2010). One of the most important natural products in herbal medicine is the essential oil of a plant. Essential oil, which contains a variety of volatile molecules such as terpenes, terpenoids, phenol-derived aromatic compounds and aliphatic components, is obtained from various natural plant materials including leaves, fruits, roots, flowers and wood. The biological activity of essential oil is complex and depends on its overall composition. Essential oils have been widely used as ⇑ Corresponding author. Tel./fax: 098(711)2272805. E-mail address: [email protected] (G. Kavoosi). 0278-6915/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2012.06.002
bactericidal, virucidal, insecticidal, fungicidal, antiparasitic, analgesic, sedative, anti-inflammatory, spasmolytic and locally anesthetic remedies, and in both medicinal and cosmetic applications, especially in the pharmaceutical, sanitary, cosmetic, and agricultural and food industries (Bakkali et al., 2008; Reichling et al., 2009). Zataria multiflora Boiss, with the Persian name Avishan Shirazi, grows only in warm parts of Iran, Afghanistan and Pakistan. This aromatic plant belongs to the Lamiaceae family. In traditional medicine in the Middle East, the plant is used in flavoring and preserving food and drinks and also used as an antispasmodic, anesthetic and antinociceptive agent (Fazeli et al., 2007; Ramezani et al., 2004). Moreover, antibacterial (Mahmoudabadi et al., 2007), antifungal (Saei-Dehkordi et al., 2008) and acaricidal (Pirali-Kheirabadi and Teixeira da Silva, 2011) activities have also been demonstrated. Even though the composition, antiviral, antifungal and antimicrobial properties of the essential oil of many other medicinal and aromatic species have been previously studied, there is very little information about the anti-oxidative stress of this plant. Thus, in the present study, the chemical composition, reactive oxygen scavenging and reactive nitrogen scavenging activities of Z. multiflora essential oil and its main components were determined. In addition, the effect of Z. multiflora essential oil and its main components on nitric oxide (NO) and hydrogen peroxide (H2O2) production as well as on NO synthase and NADH oxidase activity in glucose-stimulated human monocytes was investigated. Our results clearly demonstrated that Z. multiflora
3080
G. Kavoosi, J.A. Teixeira da Silva / Food and Chemical Toxicology 50 (2012) 3079–3085
essential oil significantly reduced oxidative stress stimulated by high glucose concentration.
of MTT solution (5 mg/mL) was added to each well. After 4 h at 37 °C, 100 lL of lysis buffer (10% SDS in 0.01 M HCl) was added and incubated for 12 h. The absorbance of the solution in the wells was read at 540 nm (Green et al., 1984).
2. Materials and methods
2.7. Cell culture and NO and H2O2 assay
2.1. Chemicals
Human THP-1 monocytes (2 106) were treated with 10 mg/mL phorbol myristate acetate (PMA, Sigma, Germany) for 3 days until cells adhered to the bottom of the culture plate and differentiated into macrophages. The differentiated cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 2 mM L-glutamine, 100 U/mL penicillin, 100 lg/mL streptomycin and 10% heat-inactivated fetal calf serum. The cells incubated in DMEM were supplemented with 5 mM D-glucose or 20 mM D-glucose at 37 °C, 5% CO2 for 48 h. Mannitol was used to control the effects of osmotic pressure. Some wells were pretreated with different concentrations of Z. multiflora essential oil and its main components solubilized in DMSO (1, 10, 100 ng/mL, final concentration). The essential oil was dissolved in DMSO, and the final DMSO concentration was 0.1% in all cultures containing this agent; the same amount of DMSO was added to the control cultures. Finally, nitrite (stable end product of NO) was determined using the Griess reagent (Miranda et al., 2001). H2O2 (diffusible end product of superoxide) was detected by a two-step oxidation of TMB in the presence of H2O2 (Rhee et al., 2010).
Potassium persulfate, 2, 20 -azino-di (3-ethylbenzthiazoline sulfate) (ABTS), 3,5,30 ,50 -tetramethylbenzidine (TMB), H2O2, sodium nitrite, naphthylethylenediamine, sulfanilamide, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), thymol, carvacrol, p-cymene, linalool, c-terpinene were purchased from Sigma and Fluka. All other chemicals and reagents used were of the highest purity commercially available.
2.2. Plant materials and essential oil preparation The aerial parts of Z. multiflora were obtained from wild plants in Arsenjan Mountain, Fars province, Iran. The taxonomic identification of the plant was confirmed by a senior plant taxonomist, Prof. Ahmad Reza Khosravi at the Department of Biology, Shiraz University, Shiraz, Fars Province, Iran. A voucher specimen (24985) has been deposited at the Herbarium of the Department of Biology, Shiraz University. The leaves of plant materials were separated from the stem and dried in the shade for 72 h. The air-dried leaves (100 g) were hydro-distilled for 3 h using an all-glass Clevenger-type apparatus. The essential oil thus obtained was dried over anhydrous sodium sulfate and stored at 20 °C until use.
2.3. Essential oil analysis and identification Analysis of the essential oil was carried out on a Thermoquest-Finnigan trace GC–MS instrument equipped with a DB-5 fused silica column (60 m 0.25 mm i.d., film thickness 0.25 mm). The oven temperature was programmed to increase from 60 to 250 °C at a rate of 4 °C/min and finally held for 10 min. The transfer line temperature was 250 °C. Helium was used as the carrier gas at a flow rate of 1.1 mL/ min with a split ratio of 1/50. The quadruple mass spectrometer was scanned over 35–465 amu with an ionizing voltage of 70 eV and an ionization current of 150 mA. GC–flame ionization detector (FID) analyses of the oil were conducted using a Thermoquest-Finnigan instrument equipped with a DB-5 fused silica column (60 m 0.25 mm i.d., film thickness 0.25 mm). Nitrogen was used as the carrier gas at a constant flow of 1.1 mL/min; the split ratio was the same as for GC–MS. The oven temperature was raised from 60 to 250 °C at a rate of 4 °C/min and was held for 10 min. The injector and detector (FID) temperatures were kept at 250 and 280 °C, respectively. Semi-quantitative data were obtained from FID area percentages without the use of correction factors. For essence identification, retention indices (RI) were calculated by using retention times of n-alkanes (C6–C24) which were injected after the oil at the same temperature and conditions. Compounds were identified by comparison of their RIs reported in the literature (Adams, 2007).
2.4. Total antioxidant determination For total antioxidant capacity, to 1.0 mL of diluted ABTS radical solution (7 mM ABTS and 2.54 mM potassium persulfate) 10 lL of essential oil, thymol, carvacrol, pcymene, linalool, c-terpinene (0, 2, 4, 6, 8, 10 lg/mL in DMSO) was added. After mixing, the absorbance was read at 734 nm (Re et al., 1999). The percentage of oxygen radical scavenging was obtained by the equation: (A734blank A734sample)/ A734blank 100 (Katalinic et al., 2006). The concentration providing 50% inhibition (IC50) was calculated from a graph plotting inhibition percentage against different essential oil concentrations.
2.5. Nitric oxide radical scavenging activity For the NO-scavenging assay, 10 lL of essential oil, thymol, carvacrol, p-cymene, linalool, c-terpinene (0, 2, 4, 6, 8, 10 lg/mL in DMSO) was incubated with 0.5 mL of sodium nitrite (10 lg/mL in 100 mM sodium citrate pH 5) at 37 °C for 2 h. After incubation, 0.5 mL of Griess reagent was added and the absorbance was read at 540 nm (Marcocci et al., 1994). The percentage of nitrogen radical scavenging was obtained by the equation: (A540blank A540sample)/A540blank 100(Katalinic et al., 2006). IC50 was calculated from the graph plotting inhibition percentage against different essential oil concentrations.
2.6. Cell viability assay The human THP-1 monocytes cell line was obtained from the cell bank of the Pasteur Institute of Iran. The monocytes (1 104 cells/well) were incubated in 96-well plates (Tissue, Culture Plate, Jet Biofil, Japan) with different concentrations of the essential oil, thymol, carvacrol, p-cymene, linalool, c-terpinene (final concentration = 1, 10, 100, 1000 ng/mL) at 37 °C in 5% CO2 for 24 h. After incubation, 10 lL
2.8. Nitric oxide synthase assay Monocytes were washed with fresh culture medium and lysed with 1% SDS in water. 0.5 mL of cell lysate was added to a vial containing 0.5 mL of 0.1 M sodium phosphate buffer, pH 7.5 containing protease inhibitors (protease inhibitor mix, 806501-23, Healthcare, Germany). NOS activity was determined using a NOS assay kit (Calbiochem, 482702, UK) according to the manufacturer’s protocol. The values were expressed as U/mL. 2.9. NADH oxidase assay Monocytes were washed with fresh culture medium and lysed with 1% SDS in water. 0.5 mL of cell lysate was added to a vial containing 0.5 mL of 0.1 M sodium phosphate buffer, pH 7.5 containing protease inhibitors. NADH oxidase activity was determined at 340 nm using 0.1 mM NADH, 0.1 M potassium phosphate buffer, pH 7.5 and 1 mM dithiotreitol. The reaction was initiated after adding 0.5 mL of cell homogenate to 0.5 mL of the above mixture (Hummel and Riebel, 2003). The values were expressed as U/mL. 2.10. Statistical analysis All data are representative of at least three independent experiments. Data are expressed as the means plus standard deviations. The significant differences between treatments were analyzed by one-way analysis of variance (ANOVA) and the Kruskal–Wallis test at P < 0.05 using statistical package for the social sciences (SPSS, Abaus Concepts, Berkeley, CA) and Prism 5 (Graph Phad, San Diego, USA) software.
3. Results 3.1. Chemical composition and radical scavenging The essential oil was prepared by water-distillation and its chemical composition was analyzed by GC–MS. According to the GC–MS chromatograph, 26 compounds detected. As shown in Table 1, GC–MS analysis of the oil indicated the main components to be carvacrol (29.2%), thymol (25.4%), p-cymene (11.2%), linalool (9.6%) and c-terpinene (8%). The yield of essential oil was 2% (w/w) from leaves. Reactive oxygen and reactive nitrogen scavenging were assessed and concentrations providing 50% inhibition (IC50) were calculated. The IC50 for reactive oxygen scavenging was estimated to be 5.7, 4.2 and 3 lg/mL for the essential oil, carvacrol and thymol, respectively. The IC50 for reactive nitrogen scavenging was estimated to be 8.6, 6.6 and 4.7 lg/mL for the essential oil, carvacrol and thymol, respectively. Thus the potency of radical scavenging, when ranked, was: thymol > carvacrol > Z. multiflora essential oil > p-cymene = linalool = c-terpinene. Z. multiflora essential oil, thymol and carvacrol displayed a concentration-dependent reactive oxygen and nitrogen scavenging activity, whilst p-cymene, linalool and c-terpinene did not show any such radical scavenging activity.
3081
G. Kavoosi, J.A. Teixeira da Silva / Food and Chemical Toxicology 50 (2012) 3079–3085 Table 1 Chemical composition of essential oil from Z. multiflora. Components
RI
Rel.%
Components
RI
Rel.%
a-Thujene a-Pinene
924 931 945 974 983 988 1015 1025 1027 1029 1059 1064 1070 1086 1104
0.499 1.234 0.269 0.575 0.267 0.729 0.562 11.247 0.448 0.447 8.054 0.451 0.56 0.602 9.363
Borneol Terpinene-4-ol A-Terpineol Thymol methyl ether Carvacrol methyl ether Linalyl acetate Thymol Carvacrol Thymol acetate Carvacrol acetate (E)-Caryophyllenecaryophyllene Aromadendrene Viridiflorene Spathulenol Caryophyllene oxide
1163 1175 1195 1232 1242 1264 1297 1310 1353 1371 1417 1436 1491 1574 1580
0.846 0.486 0.326 0.533 2.4 0.188 25.701 29.489 0.219 0.735 1.331 0.258 0.223 0.585 1.369
Camphene b-Pinene 3-Octanone Myrcene A-Terpinene p-Cymene Limonene 1,8-Cineol c-Terpinene cis-Sabinene hydrate trans-Linalool oxide cis-Linalool oxide Linalool
3.2. MTT assay The MTT assay indicated that Z. multiflora essential oil, thymol, carvacrol, p-cymene, linalool and c-terpinene had no effects on the cell viability at low concentrations (1, 10, 100 ng/mL). However, at a high concentration (1000 ng/mL) all of them reduced cell viability by 40%. 3.3. Effects of Z. multiflora essential oil, carvacrol, thymol, p-cymene, linalool and c-terpinene on NO production in glucose-stimulated monocytes As shown in Fig. 1 the concentration of NO in the culture medium of un-stimulated cells was 78 ± 14.8 nM and after incubation with 20 mM glucose, it was 263 ± 17 nM. Pretreatment with Z. multiflora essential oil at 1, 10 and 100 ng/mL, the concentration of NO was 240.5 ± 17, 180 ± 17.6 and 133 ± 20 nM, respectively. Pretreatment with carvacrol at 1, 10 and 100 ng/mL caused the concentration of NO to increase to 229 ± 17, 194 ± 12.4 and 152 ± 8.5 nM, respectively. Pretreatment with thymol at 1, 10 and 100 ng/mL, the concentration of NO was 214 ± 21, 161 ± 24 and 131 ± 18.5 nM, respectively. Pretreatment with p-cymene, linalool and c-terpinene at 1, 10 and 100 ng/mL caused no change in
the concentration of NO. Thus, essential oil, carvacrol and thymol at 10 and 100 ng/mL significantly (P < 0.01) reduced NO production in glucose-stimulated monocytes in a similar manner.
3.4. Effects of Z. multiflora essential oil, carvacrol, thymol, p-cymene, linalool and c-terpinene on NOS activity in glucose-stimulated monocytes As shown in Fig. 2 the activity of NOS in the un-stimulated cells was 12 ± 2.4 U/mL and after incubation with 20 mM glucose, it was 39.50 ± 2.3 U/mL. Pretreatment with Z. multiflora essential oil at 1, 10 and 100 ng/mL, the concentration of NO was 33 ± 1.7, 20.5 ± 1.8 and 17 ± 1.6 U/mL, respectively. Pretreatment with carvacrol at 1, 10 and 100 ng/mL, caused the concentrations of NO to be 32.6 ± 3.9, 27 ± 2.3 and 19.5 ± 1.6 U/mL, respectively. Pretreatment with thymol at 1, 10 and 100 ng/mL, the concentration of NO was 37 ± 2.6, 22.7 ± 2.27 and 15.3 ± 2.25 U/mL, respectively. Pretreatment with p-cymene, linalool and c-terpinene at 1, 10 and 100 ng/mL caused no change in NOS activity. Thus, essential oil, carvacrol and thymol at 10 and 100 ng/mL significantly (P < 0.01) reduced NOS activity in glucose-stimulated monocytes in a similar manner.
Fig. 1. Effect of Z. multiflora essential oil, carvacrol, thymol, p-cymene, linalool and c-terpinene on NO production. Human monocytes were pretreated with essential oil, carvacrol, thymol, p-cymene, linalool and c-terpinene and then cultured in 5 and 20 mM glucose for 48 h. Finally, NO production was measured in the culture medium. Data are expressed as means ± standard deviation of triplicate experiments. mMG = milli molar glucose; C1, C10 and C100 = 1, 10 and 100 ng/mL of each test compound (essential oil, carvacrol, thymol, p-cymene, linalool and c-terpinene). = compared with 20 mM glucose-treated cells at P < 0.01. Essential oil, carvacrol and thymol at 10 and 100 ng/mL significantly (P < 0.01) reduced NO production in monocytes stimulated by a high concentration of glucose.
3082
G. Kavoosi, J.A. Teixeira da Silva / Food and Chemical Toxicology 50 (2012) 3079–3085
Fig. 2. Effect of Z. multiflora essential oil, carvacrol, thymol, p-cymene, linalool and c-terpinene on NOS activity. Human monocytes were pretreated with essential oil, carvacrol, thymol, p-cymene, linalool and c-terpinene and then cultured in 5 and 20 mM glucose for 48 h. Finally, NOS activity was measured in the culture medium. Data are expressed as means ± standard deviation of triplicate experiments. mMG = milli molar glucose; C1, C10 and C100 = 1, 10 and 100 ng/mL of each test compound (essential oil, carvacrol, thymol, p-cymene, linalool and c-terpinene). = compared with 20 mM glucose-treated cells at P < 0.01. Essential oil, carvacrol and thymol at 10 and 100 ng/mL significantly (P < 0.01) reduced NOS activity in monocytes stimulated by a high concentration of glucose.
3.5. Effects of Z. multiflora essential oil, carvacrol, thymol, p-cymene, linalool and c-terpinene on H2O2 production in glucose-stimulated monocytes As shown in Fig. 3 the concentration of H2O2 in the culture medium of un-stimulated cells was 55.6 ± 4.1 nM and after incubation with 20 mM glucose, it was 136 ± 12 nM. Pretreatment with Z. multiflora essential oil at 1, 10 and 100 ng/mL, the concentration of H2O2 was 123 ± 6.7, 79 ± 6.3 and 62 ± 10.3 nM, respectively. Pretreatment with carvacrol at 1, 10 and 100 ng/ mL caused the concentrations of H2O2 to increase slightly to 99.5 ± 15.7, 80.3 ± 8 and 59.4 ± 4.2 nM, respectively. Pretreatment with thymol at 1, 10 and 100 ng/mL, the concentration of H2O2
was 119.5 ± 12, 79 ± 16 and 63 ± 5.5 nM, respectively. Pretreatment with p-cymene, linalool and c-terpinene at 1, 10 and 100 ng/mL caused no change in the H2O2 concentration. Thus, essential oil, carvacrol and thymol at 10 and 100 ng/mL significantly (P < 0.01) reduced H2O2 production in glucose-stimulated monocytes in a similar manner. 3.6. Effects of Z. multiflora essential oil, carvacrol, thymol, p-cymene, linalool and c-terpinene on NOX production in glucose-stimulated monocytes As shown in Fig. 4, the activity of NOX in the un-stimulated cells was 9 ± 0.65 U/mL and after incubation with 20 mM glucose, it was
Fig. 3. Effect of Z. multiflora essential oil, carvacrol, thymol, p-cymene, linalool and c-terpinene on H2O2 production. Human monocytes were pretreated with essential oil, carvacrol, thymol, p-cymene, linalool and c-terpinene and then cultured in 5 and 20 mM glucose for 48 h. Finally, H2O2 production was measured in the culture medium. Data are expressed as means ± standard deviation of triplicate experiments. mMG = milli molar glucose; C1, C10 and C100 = 1, 10 and 100 ng/mL of each test compound (essential oil, carvacrol, thymol, p-cymene, linalool and c-terpinene). = compared with 20 mM glucose-treated cells at P < 0.01. Essential oil, carvacrol and thymol at 10 and 100 ng/mL significantly (P < 0.01) reduced H2O2 production in monocytes stimulated by a high concentration of glucose.
G. Kavoosi, J.A. Teixeira da Silva / Food and Chemical Toxicology 50 (2012) 3079–3085
3083
Fig. 4. Effect of Z. multiflora essential oil, carvacrol, thymol, p-cymene, linalool and c-terpinene on NOX activity. Human monocytes were pretreated with essential oil, carvacrol, thymol, p-cymene, linalool and c-terpinene and then cultured in 5 and 20 mM glucose for 48 h. Finally, NOX activity was measured in the culture medium. Data are expressed as means ± standard deviation of triplicate experiments. mMG = milli molar glucose; C1, C10 and C100 = 1, 10 and 100 ng/mL of each test compound (essential oil, carvacrol, thymol, p-cymene, linalool and c-terpinene). = compared with 20 mM glucose-treated cells at P < 0.01. Essential oil, carvacrol and thymol at 10 and 100 ng/mL significantly (P < 0.01) reduced NOX activity in monocytes stimulated by a high concentration of glucose.
21.6 ± 2 U/mL. After pretreatment with Z. multiflora essential oil at 1, 10 and 100 ng/mL, NOX activity was 20 ± 3.3, 14.2 ± 1.5 and 9.2 ± 1.1 U/mL, respectively; after pretreatment with carvacrol at 1, 10 and 100 ng/mL, NOX activity became 20.6 ± 1, 12.6 ± 2 and 9.4 ± 1.4 U/mL, respectively, while pretreatment with thymol at 1, 10 and 100 ng/mL resulted in 17 ± 1.4, 10.6 ± 1.6 and 8.7 ± 1.4 U/mL of NOX activity, respectively. Pretreatment with p-cymene, linalool and c-terpinene at 1, 10 and 100 ng/mL caused no change in NOX activity. Thus, essential oil, carvacrol and thymol at 10 and 100 ng/mL significantly (P < 0.01) reduced NOX activity in glucose-stimulated monocytes in a similar manner. 4. Discussion About 80% of the world’s population currently relies on traditional medicines and most of this therapy involves the use of the aqueous extract of a plant in the form of an herbal tea. Until now, thousands of traditional herbs and active components have been tested for antioxidant, antibacterial, antifungal, antiviral, anti-diabetes, anti-cancer and anti-inflammation activities (Harikumar and Aggarwal, 2008; Zick et al., 2009). One of these useful plants is Z. multiflora, which is used in traditional medicine for its antiseptic, analgesic and carminative properties. The plant’s essential oil is mainly composed of monoterpene and aromatic compounds and has antibacterial, antiviral, and antifungal activities (Fazeli et al., 2007; Ramezani et al., 2004). Despite its remarkable array of medical applications, to our knowledge, little research has been carried out on the biological activity of the essential oil constituents of this plant. Our results indicate that the main components of the essential oil are carvacrol (29.2%), thymol (25.4%), pcymene (11.2%), linalool (9.6%) and c-terpinene (8%). The Z. multiflora essential oil has potent radical scavenging activity. The antioxidant activity of compounds is mainly due to their redox properties, which can play an important role in neutralizing free radicals, quenching singlet and triplet oxygen, or decomposing peroxides, and is related to phenolic hydroxyl groups (Buyukbalci and Ei, 2008; Katalinic et al., 2006; Wei and Shibamoto, 2010; Zheng and Wang, 2001). According to GC–MS analysis, Z. multiflora contains high levels of phenolic compounds (thymol and carvacrol).
Thus, potent antioxidant activity is related to the levels of thymol and carvacrol (Zheng and Wang, 2001; Baser, 2008). The side effects of Z. multiflora essential oil and its main components on cell viability were examined by the MTT assay. The essential oil, thymol, carvacrol, p-cymene, linalool and c-terpinene had no effects on cell viability at low concentrations although at a high concentration all of them reduced cell viability. Even though essential oil acts as an antioxidant, in eukaryotic cells essential oils can act as prooxidants affecting inner cell membranes and organelles such as mitochondria but, depending on the type and concentration, they also exhibit cytotoxic effects on living cells (Bakkali et al., 2008; Reichling et al., 2010). The effect of Z. multiflora essential oil and its main components on the modulation of NO and H2O2 production as induced by a high glucose concentration in human monocytes, was investigated. The results of this study also showed that a high glucose concentration induced large amounts of NO and H2O2 production in human monocytes. Previous studies have indicated that a high glucose concentration directly induces superoxide production. Superoxide ion leads to the activation of NF-jB transcription factor, which in turn causes the expression of several immunological mediators, including NO. Thus, the production of NO by a high glucose concentration is indirectly mediated by NF-jB activation (Guha et al., 2000; Shanmugan et al., 2003). However, our results indicate that Z. multiflora essential oil, carvacrol and thymol significantly reduced NO and H2O2 production in a dose-dependent manner, while p-cymene, linalool and c-terpinene did not show any effects on NO and H2O2 production in human monocytes treated with a high glucose concentration. The reduction of NO and H2O2 production by Z. multiflora essential oil, carvacrol and thymol occurs through several mechanisms. The first and simplest way is the scavenging of reactive oxygen and nitrogen radicals. Our study indicates that Z. multiflora essential oil, carvacrol and thymol, which contain a phenol group, have strong radical scavenging activities, which reduce NO and H2O2 concentration in the medium, while p-cymene, linalool and c-terpinene, which lack a phenol group, did not show such effects (Baser, 2008; Bakkali et al., 2008; Wei and Shibamoto, 2010). The second possible mechanism may be that Z. multiflora essential oil, thymol and carvacrol function as NOS and NOX inhibitors
3084
G. Kavoosi, J.A. Teixeira da Silva / Food and Chemical Toxicology 50 (2012) 3079–3085
and thus reduce NOS and NOX activities. The most common pathway for NO and H2O2 production is NOS and NOX activation by stimuli such as lipopolysaccharide (LPS). In response to such stimuli, NO and superoxide production are markedly increased. NO is converted to a stable end product nitrite and then to nitrate while superoxide is easily converted to a stable and diffusible product, H2O2, by superoxide dismutase (Babior et al. 2002). Thus, in this research, the production of nitrate and H2O2 as an indicator of oxidative stress generation was analyzed. Our results indicate that Z. multiflora essential oil, carvacrol and thymol reduced NOS and NOX activities. The mechanism of this inhibitory activity is not clear; however, this inhibition may be due to a redox cycling property of the phenol antioxidant. NOS and NOX require NADH or NADPH as the reducing agent for activity. Thus the ratio of NADH/NAD+ or NADPH/NADP+ affects the activity of the enzymes. Redox cycling of phenol antioxidant disturbs the NADH/NAD+ or NADPH/NADP+ ratio and affects NOS and NOX activities (Choi et al., 2008; Yang et al., 2008). To our knowledge, there is no research on the inhibitory effects of Z. multiflora essential oil, carvacrol and thymol on NO and H2O2 production in human monocytes cultured in the presence of high glucose concentration. Besides high glucose concentration, other stimuli such as LPS may also induce NO and superoxide production. Previous studies on the aqueous and ethanolic extracts as well as the essential oils of other herbal medicines confirm our findings. Extracts from Eucalyptus globulus Labill. and Thymus vulgaris L. (Vigo et al., 2004), Saposhnikovia divaricata (Wang et al., 1999), Pinus sylvestris L. and Plantago lanceolata L. (Vigo et al., 2005), Acanthopanax senticosus (Lin et al., 2008), Cimicifuga racemosa (aqueous) (Schemid et al., 2009), and Actinodaphne lancifolia (methanolic) (Kim et al., 2004) confirmed the inhibitory activity on NO production in LPS-activated macrophage cells during translational and post-translational levels. In addition, the two main compounds in thyme (i.e., thymol and carvacrol) displayed concentrationdependent antioxidant capacity and showed a protective effect against oxidative damage in human lymphocytes and V97 Chinese hamster lung fibroblast cells while c-terpinene, which lacks a phenolic group, did not show any antioxidant capacity (Aydin et al., 2005; Undeger et al., 2009). Furthermore, Vernonia cinerea, Cardiospermum halicacabum, T. vulgaris, Eucalyptus bridgesiana and Cymbopogon martini showed antioxidant and anti-inflammatory activity by modulating proinflammatory cytokines, NO synthase and cylooxygenase-2 (El-Nekeety et al., 2011; Pratheshkumar and Kuttan 2011a,b; Tsai et al., 2011).
5. Conclusions Accordingly, it was concluded that essential oil from Z. multiflora has antioxidant properties, can reduce oxidative stress and can be used in the therapy of oxidative damage accompanying some inflammatory conditions. High glucose concentration can produce large amounts of NO and H2O2 in human monocytes. Pretreatment of monocytes cultured on medium containing a high glucose concentration with Z. multiflora essential oil, thymol and carvacrol leads to a significant reduction in NO and H2O2 production. Although more studies are needed for understanding the major strategy for these reductions, they are probably mediated by two mechanisms: (1) by the radical scavenging activity of phenolic antioxidants; (2) through inhibition of the related enzyme by phenolic antioxidants. Overall it can be concluded that Z. multiflora can be used in the therapy of oxidative damage that accompanies hyperglycemia and some inflammatory conditions. However, further studies are necessary to confirm the antioxidant activities of the essential oil in vivo. In addition, the present study provides a stronger case for supporting the use of Z. multiflora as a tea or addi-
tive and traditional remedy for the treatment of inflammatory diseases. Conflict of Interest The authors declare that there are no conflicts of interest. Acknowledgements This work was financially supported by Shiraz University(Grant No. 88-GR-AGRST-108). The authors thank their colleagues at the Plants and Drug Research Institute, University of Shahid Beheshti, for assistance with GC–MS analysis. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.fct.2012.06.002. References Adams, R.P., 2007. Identification of essential oil components by gas chromatography/mass spectrometery, Fourth ed. Allured Publishing Corporation, Illinois, USA, pp. 456. Aydin, S., Basaran, A.A., Basaran, N., 2005. Modulating effects of thyme and its major ingredients on oxidative DNA damage in human lymphocytes. J. Agr. Food Chem. 53, 1299–1305. Babior, B.M., Lamaeth, J.D., Nauseef, W., 2002. The neutrophil NADPH oxidase. Arch. Biochem. Biophys. 397, 342–344. Bakkali, F., Averbeck, S., Averbeck, D., Idaomar, M., 2008. Biological effects of essential oils. Food Chem. Toxicol. 46, 447–475. Baser, K.H., 2008. Biological and pharmacological activities of carvacrol and carvacrol bearing essential oils. Curr. Pharm. Dis. 14, 3106–3119. Buyukbalci, A., Ei, S.N., 2008. Determination of in vitro antidiabetic effects, antioxidant activities and phenol contents of some herbal teas. Plant Foods Human Nutr. 63, 27–33. Choi, S.W., Bensie, I.F.F., Ma, S.W., Strain, J.J., Hannigan, B.M., 2008. Acute hyperglycemia and oxidative stress: Direct cause and effect. Free Radical Biol. Med. 44, 1217–1231. El-Nekeety, A.A., Mohamed, S.R., Hathout, A.S., Hassan, N.S., Aly, S.E., AbdelWahhab, M.A., 2011. Antioxidant properties of Thymus vulgaris oil against aflatoxin-induced oxidative stress in male rats. Toxicon 57, 984–991. Fazeli, M.R., Amin, G., Attari, M.M.A., Ashtiani, H., Jamalifar, H., Samadi, N., 2007. Antimicrobial activities of Iranian sumac and avishan-e shirazi (Zataria multiflora) against some food-borne bacteria. Food Control 18, 646–649. Green, L.M., Reade, J.L., Ware, C.F., 1984. Rapid colorimetric assay for cell viability: Application to the quantitation of cytotoxic and growth inhibitory lymphokines. J. Immunol. Methods 70, 257–268. Guha, M., Bai, W., Nadler, J.L., Natarajan, R., 2000. Molecular mechanisms of tumor necrosis factor gene expression in monocytic cells via hyperglycemia-induced oxidant stress-dependent and independent pathways. J. Biol. Chem. 275, 17728–17739. Harikumar, K.B., Aggarwal, B.B., 2008. Resveratrol: A multitargeted agent for ageassociated chronic diseases. Cell Cycle 7, 1020–1035. Hummel, W., Riebel, B., 2003. Isolation and biochemical characterization of a new NADH oxidase from Lactobacillus brevis. Biotechnol. Lett. 25, 51–54. Johansen, J.S., Harris, A.K., Rychly, D.J., Ergul, A., 2005. Oxidative stress and the use of antioxidants in diabetes: Linking basic science to clinical practice. Cardiovasc. Diabetol. 4, 5–16. Katalinic, V., Milos, M., Kulisic, T., Jukic, M., 2006. Screening of 70 medicinal plant extracts for antioxidant capacity and total phenols. Food Chem. 94, 550–557. Kim, Y., Min, H.Y., Park, H.J., Lee, E.J., Park, E.J., Wang, H.J., Jin, C., Lee, Y.S., Lee, S.K., 2004. Suppressive effect of inducible nitric oxide synthase expression by the methanol extract of Actinodaphene lancifolia. Phytother. Res. 18, 853–856. Lin, Q.Y., Jin, L.J., Cao, Z.H., Xu, Y.P., 2008. Inhibition of inducible nitric oxide synthase by Acanthopanax senticosus extract in RAW264.7 macrophages. J. Ethnopharmacol. 118, 231–236. Ma, Q., 2010. Transcriptional responses to oxidative stress: Pathological and toxicological implications. Pharmacol. Ther. 125, 376–393. Mahmoudabadi, A.Z., Dabbagh, M.A., Fouladi, Z., 2007. In vitro anti-Candida activity of Zataria multiflora Boiss. Evidence Based Complem. Alter. Med. 4, 351–353. Marcocci, L., Maguire, J.J., Droylefaix, M.T., Packer, L., 1994. The nitric oxide scavenging properties of Ginkgo biloba extract EGB 761. Biochem. Biophys. Res. Commun. 201, 748–755. Miranda, M.K., Espey, M.G., Wink, D.A., 2001. A rapid, simple spectrophotometric method for simultaneous detection of nitrate and nitrite. Nitric Oxide 5, 62–71. Pacher, P., Beckman, J.S., Liaudet, L., 2007. Nitric oxide and peroxynitrite in health and disease. Physiol. Rev. 87, 315–324.
G. Kavoosi, J.A. Teixeira da Silva / Food and Chemical Toxicology 50 (2012) 3079–3085 Pirali-Kheirabadi, K., Teixeira da Silva, J.A., 2011. In-vitro assessment of the acaricidal properties of Artemisia annua and Zataria multiflora essential oils to control cattle ticks. Iranian J. Parasitol. 6, 58–65. Pratheshkumar, P., Kuttan, G., 2011a. Cardiospermum halicacabum inhibits cyclophosphamide induced immunosupression and oxidative stress in mice and also regulates iNOS and COX-2 gene expression in LPS-stimulated macrophages. Asian Pacific J. Cancer Prev. 11, 1245–1252. Pratheshkumar, P., Kuttan, G., 2011b. Modulation of immune response by Vernonia cinerea L. inhibits the proinflammatory cytokine profile, iNOS, and COX-2 expression in LPS-stimulated macrophages. Immunopharmacol. Immunotoxicol. 33, 73–83. Ramezani, M., Hosseinzadeh, H., Samizadeh, S., 2004. Antinociceptive effects of Zataria multiflora Boiss. fractions in mice. J. Ethnopharmacol. 91, 167–170. Re, R., Pellegrini, N., Protegenete, A., Pannala, A., Yang, M., Rice-Evans, C., 1999. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biol. Med. 26, 1231–1237. Reichling, J., Schnitzler, P., suschke, U., Saller, R., 2009. Essential oils of aromatic plants with antibacterial, antifungal, antiviral and cytotoxic properties. Forch. Komplement. Med. 16, 79–90. Rhee, S.G., Chang, T.S., Jeong, W., Kang, D., 2010. Methods for detection and measurement of hydrogen peroxide inside and outside of cells. Mol. Cells 29, 539–549. Saei-Dehkordi, S.S., Razavi Rohani, S.M., Tajik, H., Moradi, M., Aliakbarlou, J., 2008. Antimicrobial effects of lysozyme in combination with Zataria multiflora Boiss. essential oil at different pH and NaCl concentrations on E. coli O157:H7 and Staphylococcus aureus. J. Animal Vet. Adv. 7, 1458–1463. Schemid, D., Gruber, M., Woehs, F., Prinz, Z., Etzlstorfer, B., Prucker, C., Fuzzati, N., Kopp, B., Moeslinger, T., 2009. Inhibition of inducible nitric oxide synthesis by Cimicifuga racemosa extracts in LPS-stimulated RAW264.7 macrophages. J. Pharm. Pharmacol. 61, 1089–1096.
3085
Shanmugan, N., Reddy, M.A., Guha, M., Natarajan, R., 2003. High-glucose expression of proinflammatory cytokines and chemokine genes in monocytic cells. Diabetes 52, 1256–1264. Sze, S.C., Tong, Y., Ng, T.B., Cheng, C.L., Cheung, H.P., 2010. Herba epimedii: Antioxidative properties and its medical implications. Molecules 15, 7861–7870. Tsai, M.L., Lin, C.C., Lin, W.C., Yang, C.H., 2011. Antimicrobial, antioxidant and antiinflammatory activities of essential oils from five selected herbs. Biosci. Biotechnol. Biochem. 75, 110377-1–110377-7. Undeger, U., Basaran, A., Degen, G.H., Basaran, N., 2009. Antioxidant activities of major thyme ingredients and lack of oxidative DNA damages in V97 Chinese hamster lung cells al low levels of carvacrol and thymol. Food Chem. Toxicol. 47, 2037–2043. Vigo, E., Cepeda, A., Gualillo, O., Perez-Fernandez, R., 2004. In vitro antiinflammatory effect of Eucalyptus globulus and Thymus vulgaris: Nitric oxide inhibition in J774A.1 murine macrophages. J. Pharm. Pharmacol. 56, 257–263. Vigo, E., Cepeda, A., Gualillo, O., Perez-Fernandez, R., 2005. In vitro antiinflammatory activity of Pinus sylvestris and Plantago lanceolata extracts: Effects on inducible NOS, COX-1, COX-2 and their products in J774A.1 murine macrophages. J. Pharm. Pharmacol. 57, 383–391. Wang, C.C., Chen, L.G., Yang, L.L., 1999. Inducible nitric oxide synthse inhibitor of the Chinese herbs I Saposhnikovia divaricata. Cancer Lett. 145, 151–157. Wei, A., Shibamoto, T., 2010. Antioxidant/lipoixygenase inhibitory activities and chemical compositions of selected essential oil. J. Agri. Food Chem. 58, 7218– 7225. Yang, Z., Laubach, V.E., French, B.A., Kron, I.L., 2008. Acute hyperglycemia enhances oxidative stress and exacerbates myocardial infarction by activating NADH oxidase during reperfusion. J. Thorac. Cardiovas. Surg. 137, 723–729. Zheng, W., Wang, S.Y., 2001. Antioxidant activity and phenolic compounds in selected herbs. J. Agri. Food Chem. 49, 5165–5170. Zick, S.M., Schwabl, H., Flower, A., Charkraborty, B., Hirschkon, K., 2009. Unique aspects of herbal system research. Explore 5, 97–103.
Contents lists available at SciVerse ScienceDirect
Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Inhibitory effects of Zataria multiflora essential oil and its main components on nitric oxide and hydrogen peroxide production in glucose-stimulated human monocyte Gholamreza Kavoosi a,⇑, Jaime A. Teixeira da Silva b a b
Institute of Biotechnology, Faculty of Agriculture, University of Shiraz, Shiraz 71441-65186, Iran Faculty of Agriculture and Graduate School of Agriculture, Kagawa University, Ikenobe 761-0795, Japan
a r t i c l e
i n f o
Article history: Received 2 November 2011 Accepted 4 June 2012 Available online 13 June 2012 Keywords: Zataria multiflora Essential oil Monocytes Glucose Nitric oxide Hydrogen peroxide
a b s t r a c t The inhibitory effects of Zataria multiflora essential oil on nitric oxide (NO) and hydrogen peroxide (H2O2) production were examined in human monocytes cultured in the presence of 20 mM glucose. Z. multiflora essential oil was extracted by water-distillation and then analyzed by GC–MS. Carvacrol (29.2%), thymol (25.4%), p-cymene (11.2%), linalool (9.6%) and c-terpinene (8%) were the main components detected in the essential oil. Cells cultured in the presence of 20 mM glucose showed an increase in NO and H2O2 production as well as NO synthase (NOS) and NADH oxidase (NOX) activities compared to cells cultured in the presence of 5 mM glucose. Pretreatment with Z. multiflora essential oil, carvacrol and thymol reduced NO and H2O2 production as well as NOS and NOX activities in those cells cultured in the presence of 20 mM glucose. However, p-cymene, linalool and c-terpinene did not show any such activities. Accordingly, it was concluded that Z. multiflora can reduce oxidative stress and can be used in the therapy of oxidative damage accompanying hyperglycemia and some inflammatory conditions. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Extensive research within the last decade has revealed that most diseases such as cancer, cardiovascular, neurological and autoimmune diseases, as well as diabetes are linked to the overproduction of reactive oxygen species (ROS) and reactive nitrogen species (RNS) (Ma, 2010). Although herbal medicines are regarded to have therapeutic potential against these diseases, neither their active components nor their mechanisms of action are fully understood. There is growing interest in the possible therapeutic potential of natural products against these diseases. These compounds exert potent antioxidant actions and have the inherent capacity to scavenge ROS and RNS, thus counteracting conditions of oxidative stress that accompany disorders such as coronary and vascular and inflammatory diseases, diabetes and cancer (Johansen et al., 2005; Pacher et al., 2007; Sze et al., 2010). One of the most important natural products in herbal medicine is the essential oil of a plant. Essential oil, which contains a variety of volatile molecules such as terpenes, terpenoids, phenol-derived aromatic compounds and aliphatic components, is obtained from various natural plant materials including leaves, fruits, roots, flowers and wood. The biological activity of essential oil is complex and depends on its overall composition. Essential oils have been widely used as ⇑ Corresponding author. Tel./fax: 098(711)2272805. E-mail address: [email protected] (G. Kavoosi). 0278-6915/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2012.06.002
bactericidal, virucidal, insecticidal, fungicidal, antiparasitic, analgesic, sedative, anti-inflammatory, spasmolytic and locally anesthetic remedies, and in both medicinal and cosmetic applications, especially in the pharmaceutical, sanitary, cosmetic, and agricultural and food industries (Bakkali et al., 2008; Reichling et al., 2009). Zataria multiflora Boiss, with the Persian name Avishan Shirazi, grows only in warm parts of Iran, Afghanistan and Pakistan. This aromatic plant belongs to the Lamiaceae family. In traditional medicine in the Middle East, the plant is used in flavoring and preserving food and drinks and also used as an antispasmodic, anesthetic and antinociceptive agent (Fazeli et al., 2007; Ramezani et al., 2004). Moreover, antibacterial (Mahmoudabadi et al., 2007), antifungal (Saei-Dehkordi et al., 2008) and acaricidal (Pirali-Kheirabadi and Teixeira da Silva, 2011) activities have also been demonstrated. Even though the composition, antiviral, antifungal and antimicrobial properties of the essential oil of many other medicinal and aromatic species have been previously studied, there is very little information about the anti-oxidative stress of this plant. Thus, in the present study, the chemical composition, reactive oxygen scavenging and reactive nitrogen scavenging activities of Z. multiflora essential oil and its main components were determined. In addition, the effect of Z. multiflora essential oil and its main components on nitric oxide (NO) and hydrogen peroxide (H2O2) production as well as on NO synthase and NADH oxidase activity in glucose-stimulated human monocytes was investigated. Our results clearly demonstrated that Z. multiflora
3080
G. Kavoosi, J.A. Teixeira da Silva / Food and Chemical Toxicology 50 (2012) 3079–3085
essential oil significantly reduced oxidative stress stimulated by high glucose concentration.
of MTT solution (5 mg/mL) was added to each well. After 4 h at 37 °C, 100 lL of lysis buffer (10% SDS in 0.01 M HCl) was added and incubated for 12 h. The absorbance of the solution in the wells was read at 540 nm (Green et al., 1984).
2. Materials and methods
2.7. Cell culture and NO and H2O2 assay
2.1. Chemicals
Human THP-1 monocytes (2 106) were treated with 10 mg/mL phorbol myristate acetate (PMA, Sigma, Germany) for 3 days until cells adhered to the bottom of the culture plate and differentiated into macrophages. The differentiated cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 2 mM L-glutamine, 100 U/mL penicillin, 100 lg/mL streptomycin and 10% heat-inactivated fetal calf serum. The cells incubated in DMEM were supplemented with 5 mM D-glucose or 20 mM D-glucose at 37 °C, 5% CO2 for 48 h. Mannitol was used to control the effects of osmotic pressure. Some wells were pretreated with different concentrations of Z. multiflora essential oil and its main components solubilized in DMSO (1, 10, 100 ng/mL, final concentration). The essential oil was dissolved in DMSO, and the final DMSO concentration was 0.1% in all cultures containing this agent; the same amount of DMSO was added to the control cultures. Finally, nitrite (stable end product of NO) was determined using the Griess reagent (Miranda et al., 2001). H2O2 (diffusible end product of superoxide) was detected by a two-step oxidation of TMB in the presence of H2O2 (Rhee et al., 2010).
Potassium persulfate, 2, 20 -azino-di (3-ethylbenzthiazoline sulfate) (ABTS), 3,5,30 ,50 -tetramethylbenzidine (TMB), H2O2, sodium nitrite, naphthylethylenediamine, sulfanilamide, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), thymol, carvacrol, p-cymene, linalool, c-terpinene were purchased from Sigma and Fluka. All other chemicals and reagents used were of the highest purity commercially available.
2.2. Plant materials and essential oil preparation The aerial parts of Z. multiflora were obtained from wild plants in Arsenjan Mountain, Fars province, Iran. The taxonomic identification of the plant was confirmed by a senior plant taxonomist, Prof. Ahmad Reza Khosravi at the Department of Biology, Shiraz University, Shiraz, Fars Province, Iran. A voucher specimen (24985) has been deposited at the Herbarium of the Department of Biology, Shiraz University. The leaves of plant materials were separated from the stem and dried in the shade for 72 h. The air-dried leaves (100 g) were hydro-distilled for 3 h using an all-glass Clevenger-type apparatus. The essential oil thus obtained was dried over anhydrous sodium sulfate and stored at 20 °C until use.
2.3. Essential oil analysis and identification Analysis of the essential oil was carried out on a Thermoquest-Finnigan trace GC–MS instrument equipped with a DB-5 fused silica column (60 m 0.25 mm i.d., film thickness 0.25 mm). The oven temperature was programmed to increase from 60 to 250 °C at a rate of 4 °C/min and finally held for 10 min. The transfer line temperature was 250 °C. Helium was used as the carrier gas at a flow rate of 1.1 mL/ min with a split ratio of 1/50. The quadruple mass spectrometer was scanned over 35–465 amu with an ionizing voltage of 70 eV and an ionization current of 150 mA. GC–flame ionization detector (FID) analyses of the oil were conducted using a Thermoquest-Finnigan instrument equipped with a DB-5 fused silica column (60 m 0.25 mm i.d., film thickness 0.25 mm). Nitrogen was used as the carrier gas at a constant flow of 1.1 mL/min; the split ratio was the same as for GC–MS. The oven temperature was raised from 60 to 250 °C at a rate of 4 °C/min and was held for 10 min. The injector and detector (FID) temperatures were kept at 250 and 280 °C, respectively. Semi-quantitative data were obtained from FID area percentages without the use of correction factors. For essence identification, retention indices (RI) were calculated by using retention times of n-alkanes (C6–C24) which were injected after the oil at the same temperature and conditions. Compounds were identified by comparison of their RIs reported in the literature (Adams, 2007).
2.4. Total antioxidant determination For total antioxidant capacity, to 1.0 mL of diluted ABTS radical solution (7 mM ABTS and 2.54 mM potassium persulfate) 10 lL of essential oil, thymol, carvacrol, pcymene, linalool, c-terpinene (0, 2, 4, 6, 8, 10 lg/mL in DMSO) was added. After mixing, the absorbance was read at 734 nm (Re et al., 1999). The percentage of oxygen radical scavenging was obtained by the equation: (A734blank A734sample)/ A734blank 100 (Katalinic et al., 2006). The concentration providing 50% inhibition (IC50) was calculated from a graph plotting inhibition percentage against different essential oil concentrations.
2.5. Nitric oxide radical scavenging activity For the NO-scavenging assay, 10 lL of essential oil, thymol, carvacrol, p-cymene, linalool, c-terpinene (0, 2, 4, 6, 8, 10 lg/mL in DMSO) was incubated with 0.5 mL of sodium nitrite (10 lg/mL in 100 mM sodium citrate pH 5) at 37 °C for 2 h. After incubation, 0.5 mL of Griess reagent was added and the absorbance was read at 540 nm (Marcocci et al., 1994). The percentage of nitrogen radical scavenging was obtained by the equation: (A540blank A540sample)/A540blank 100(Katalinic et al., 2006). IC50 was calculated from the graph plotting inhibition percentage against different essential oil concentrations.
2.6. Cell viability assay The human THP-1 monocytes cell line was obtained from the cell bank of the Pasteur Institute of Iran. The monocytes (1 104 cells/well) were incubated in 96-well plates (Tissue, Culture Plate, Jet Biofil, Japan) with different concentrations of the essential oil, thymol, carvacrol, p-cymene, linalool, c-terpinene (final concentration = 1, 10, 100, 1000 ng/mL) at 37 °C in 5% CO2 for 24 h. After incubation, 10 lL
2.8. Nitric oxide synthase assay Monocytes were washed with fresh culture medium and lysed with 1% SDS in water. 0.5 mL of cell lysate was added to a vial containing 0.5 mL of 0.1 M sodium phosphate buffer, pH 7.5 containing protease inhibitors (protease inhibitor mix, 806501-23, Healthcare, Germany). NOS activity was determined using a NOS assay kit (Calbiochem, 482702, UK) according to the manufacturer’s protocol. The values were expressed as U/mL. 2.9. NADH oxidase assay Monocytes were washed with fresh culture medium and lysed with 1% SDS in water. 0.5 mL of cell lysate was added to a vial containing 0.5 mL of 0.1 M sodium phosphate buffer, pH 7.5 containing protease inhibitors. NADH oxidase activity was determined at 340 nm using 0.1 mM NADH, 0.1 M potassium phosphate buffer, pH 7.5 and 1 mM dithiotreitol. The reaction was initiated after adding 0.5 mL of cell homogenate to 0.5 mL of the above mixture (Hummel and Riebel, 2003). The values were expressed as U/mL. 2.10. Statistical analysis All data are representative of at least three independent experiments. Data are expressed as the means plus standard deviations. The significant differences between treatments were analyzed by one-way analysis of variance (ANOVA) and the Kruskal–Wallis test at P < 0.05 using statistical package for the social sciences (SPSS, Abaus Concepts, Berkeley, CA) and Prism 5 (Graph Phad, San Diego, USA) software.
3. Results 3.1. Chemical composition and radical scavenging The essential oil was prepared by water-distillation and its chemical composition was analyzed by GC–MS. According to the GC–MS chromatograph, 26 compounds detected. As shown in Table 1, GC–MS analysis of the oil indicated the main components to be carvacrol (29.2%), thymol (25.4%), p-cymene (11.2%), linalool (9.6%) and c-terpinene (8%). The yield of essential oil was 2% (w/w) from leaves. Reactive oxygen and reactive nitrogen scavenging were assessed and concentrations providing 50% inhibition (IC50) were calculated. The IC50 for reactive oxygen scavenging was estimated to be 5.7, 4.2 and 3 lg/mL for the essential oil, carvacrol and thymol, respectively. The IC50 for reactive nitrogen scavenging was estimated to be 8.6, 6.6 and 4.7 lg/mL for the essential oil, carvacrol and thymol, respectively. Thus the potency of radical scavenging, when ranked, was: thymol > carvacrol > Z. multiflora essential oil > p-cymene = linalool = c-terpinene. Z. multiflora essential oil, thymol and carvacrol displayed a concentration-dependent reactive oxygen and nitrogen scavenging activity, whilst p-cymene, linalool and c-terpinene did not show any such radical scavenging activity.
3081
G. Kavoosi, J.A. Teixeira da Silva / Food and Chemical Toxicology 50 (2012) 3079–3085 Table 1 Chemical composition of essential oil from Z. multiflora. Components
RI
Rel.%
Components
RI
Rel.%
a-Thujene a-Pinene
924 931 945 974 983 988 1015 1025 1027 1029 1059 1064 1070 1086 1104
0.499 1.234 0.269 0.575 0.267 0.729 0.562 11.247 0.448 0.447 8.054 0.451 0.56 0.602 9.363
Borneol Terpinene-4-ol A-Terpineol Thymol methyl ether Carvacrol methyl ether Linalyl acetate Thymol Carvacrol Thymol acetate Carvacrol acetate (E)-Caryophyllenecaryophyllene Aromadendrene Viridiflorene Spathulenol Caryophyllene oxide
1163 1175 1195 1232 1242 1264 1297 1310 1353 1371 1417 1436 1491 1574 1580
0.846 0.486 0.326 0.533 2.4 0.188 25.701 29.489 0.219 0.735 1.331 0.258 0.223 0.585 1.369
Camphene b-Pinene 3-Octanone Myrcene A-Terpinene p-Cymene Limonene 1,8-Cineol c-Terpinene cis-Sabinene hydrate trans-Linalool oxide cis-Linalool oxide Linalool
3.2. MTT assay The MTT assay indicated that Z. multiflora essential oil, thymol, carvacrol, p-cymene, linalool and c-terpinene had no effects on the cell viability at low concentrations (1, 10, 100 ng/mL). However, at a high concentration (1000 ng/mL) all of them reduced cell viability by 40%. 3.3. Effects of Z. multiflora essential oil, carvacrol, thymol, p-cymene, linalool and c-terpinene on NO production in glucose-stimulated monocytes As shown in Fig. 1 the concentration of NO in the culture medium of un-stimulated cells was 78 ± 14.8 nM and after incubation with 20 mM glucose, it was 263 ± 17 nM. Pretreatment with Z. multiflora essential oil at 1, 10 and 100 ng/mL, the concentration of NO was 240.5 ± 17, 180 ± 17.6 and 133 ± 20 nM, respectively. Pretreatment with carvacrol at 1, 10 and 100 ng/mL caused the concentration of NO to increase to 229 ± 17, 194 ± 12.4 and 152 ± 8.5 nM, respectively. Pretreatment with thymol at 1, 10 and 100 ng/mL, the concentration of NO was 214 ± 21, 161 ± 24 and 131 ± 18.5 nM, respectively. Pretreatment with p-cymene, linalool and c-terpinene at 1, 10 and 100 ng/mL caused no change in
the concentration of NO. Thus, essential oil, carvacrol and thymol at 10 and 100 ng/mL significantly (P < 0.01) reduced NO production in glucose-stimulated monocytes in a similar manner.
3.4. Effects of Z. multiflora essential oil, carvacrol, thymol, p-cymene, linalool and c-terpinene on NOS activity in glucose-stimulated monocytes As shown in Fig. 2 the activity of NOS in the un-stimulated cells was 12 ± 2.4 U/mL and after incubation with 20 mM glucose, it was 39.50 ± 2.3 U/mL. Pretreatment with Z. multiflora essential oil at 1, 10 and 100 ng/mL, the concentration of NO was 33 ± 1.7, 20.5 ± 1.8 and 17 ± 1.6 U/mL, respectively. Pretreatment with carvacrol at 1, 10 and 100 ng/mL, caused the concentrations of NO to be 32.6 ± 3.9, 27 ± 2.3 and 19.5 ± 1.6 U/mL, respectively. Pretreatment with thymol at 1, 10 and 100 ng/mL, the concentration of NO was 37 ± 2.6, 22.7 ± 2.27 and 15.3 ± 2.25 U/mL, respectively. Pretreatment with p-cymene, linalool and c-terpinene at 1, 10 and 100 ng/mL caused no change in NOS activity. Thus, essential oil, carvacrol and thymol at 10 and 100 ng/mL significantly (P < 0.01) reduced NOS activity in glucose-stimulated monocytes in a similar manner.
Fig. 1. Effect of Z. multiflora essential oil, carvacrol, thymol, p-cymene, linalool and c-terpinene on NO production. Human monocytes were pretreated with essential oil, carvacrol, thymol, p-cymene, linalool and c-terpinene and then cultured in 5 and 20 mM glucose for 48 h. Finally, NO production was measured in the culture medium. Data are expressed as means ± standard deviation of triplicate experiments. mMG = milli molar glucose; C1, C10 and C100 = 1, 10 and 100 ng/mL of each test compound (essential oil, carvacrol, thymol, p-cymene, linalool and c-terpinene). = compared with 20 mM glucose-treated cells at P < 0.01. Essential oil, carvacrol and thymol at 10 and 100 ng/mL significantly (P < 0.01) reduced NO production in monocytes stimulated by a high concentration of glucose.
3082
G. Kavoosi, J.A. Teixeira da Silva / Food and Chemical Toxicology 50 (2012) 3079–3085
Fig. 2. Effect of Z. multiflora essential oil, carvacrol, thymol, p-cymene, linalool and c-terpinene on NOS activity. Human monocytes were pretreated with essential oil, carvacrol, thymol, p-cymene, linalool and c-terpinene and then cultured in 5 and 20 mM glucose for 48 h. Finally, NOS activity was measured in the culture medium. Data are expressed as means ± standard deviation of triplicate experiments. mMG = milli molar glucose; C1, C10 and C100 = 1, 10 and 100 ng/mL of each test compound (essential oil, carvacrol, thymol, p-cymene, linalool and c-terpinene). = compared with 20 mM glucose-treated cells at P < 0.01. Essential oil, carvacrol and thymol at 10 and 100 ng/mL significantly (P < 0.01) reduced NOS activity in monocytes stimulated by a high concentration of glucose.
3.5. Effects of Z. multiflora essential oil, carvacrol, thymol, p-cymene, linalool and c-terpinene on H2O2 production in glucose-stimulated monocytes As shown in Fig. 3 the concentration of H2O2 in the culture medium of un-stimulated cells was 55.6 ± 4.1 nM and after incubation with 20 mM glucose, it was 136 ± 12 nM. Pretreatment with Z. multiflora essential oil at 1, 10 and 100 ng/mL, the concentration of H2O2 was 123 ± 6.7, 79 ± 6.3 and 62 ± 10.3 nM, respectively. Pretreatment with carvacrol at 1, 10 and 100 ng/ mL caused the concentrations of H2O2 to increase slightly to 99.5 ± 15.7, 80.3 ± 8 and 59.4 ± 4.2 nM, respectively. Pretreatment with thymol at 1, 10 and 100 ng/mL, the concentration of H2O2
was 119.5 ± 12, 79 ± 16 and 63 ± 5.5 nM, respectively. Pretreatment with p-cymene, linalool and c-terpinene at 1, 10 and 100 ng/mL caused no change in the H2O2 concentration. Thus, essential oil, carvacrol and thymol at 10 and 100 ng/mL significantly (P < 0.01) reduced H2O2 production in glucose-stimulated monocytes in a similar manner. 3.6. Effects of Z. multiflora essential oil, carvacrol, thymol, p-cymene, linalool and c-terpinene on NOX production in glucose-stimulated monocytes As shown in Fig. 4, the activity of NOX in the un-stimulated cells was 9 ± 0.65 U/mL and after incubation with 20 mM glucose, it was
Fig. 3. Effect of Z. multiflora essential oil, carvacrol, thymol, p-cymene, linalool and c-terpinene on H2O2 production. Human monocytes were pretreated with essential oil, carvacrol, thymol, p-cymene, linalool and c-terpinene and then cultured in 5 and 20 mM glucose for 48 h. Finally, H2O2 production was measured in the culture medium. Data are expressed as means ± standard deviation of triplicate experiments. mMG = milli molar glucose; C1, C10 and C100 = 1, 10 and 100 ng/mL of each test compound (essential oil, carvacrol, thymol, p-cymene, linalool and c-terpinene). = compared with 20 mM glucose-treated cells at P < 0.01. Essential oil, carvacrol and thymol at 10 and 100 ng/mL significantly (P < 0.01) reduced H2O2 production in monocytes stimulated by a high concentration of glucose.
G. Kavoosi, J.A. Teixeira da Silva / Food and Chemical Toxicology 50 (2012) 3079–3085
3083
Fig. 4. Effect of Z. multiflora essential oil, carvacrol, thymol, p-cymene, linalool and c-terpinene on NOX activity. Human monocytes were pretreated with essential oil, carvacrol, thymol, p-cymene, linalool and c-terpinene and then cultured in 5 and 20 mM glucose for 48 h. Finally, NOX activity was measured in the culture medium. Data are expressed as means ± standard deviation of triplicate experiments. mMG = milli molar glucose; C1, C10 and C100 = 1, 10 and 100 ng/mL of each test compound (essential oil, carvacrol, thymol, p-cymene, linalool and c-terpinene). = compared with 20 mM glucose-treated cells at P < 0.01. Essential oil, carvacrol and thymol at 10 and 100 ng/mL significantly (P < 0.01) reduced NOX activity in monocytes stimulated by a high concentration of glucose.
21.6 ± 2 U/mL. After pretreatment with Z. multiflora essential oil at 1, 10 and 100 ng/mL, NOX activity was 20 ± 3.3, 14.2 ± 1.5 and 9.2 ± 1.1 U/mL, respectively; after pretreatment with carvacrol at 1, 10 and 100 ng/mL, NOX activity became 20.6 ± 1, 12.6 ± 2 and 9.4 ± 1.4 U/mL, respectively, while pretreatment with thymol at 1, 10 and 100 ng/mL resulted in 17 ± 1.4, 10.6 ± 1.6 and 8.7 ± 1.4 U/mL of NOX activity, respectively. Pretreatment with p-cymene, linalool and c-terpinene at 1, 10 and 100 ng/mL caused no change in NOX activity. Thus, essential oil, carvacrol and thymol at 10 and 100 ng/mL significantly (P < 0.01) reduced NOX activity in glucose-stimulated monocytes in a similar manner. 4. Discussion About 80% of the world’s population currently relies on traditional medicines and most of this therapy involves the use of the aqueous extract of a plant in the form of an herbal tea. Until now, thousands of traditional herbs and active components have been tested for antioxidant, antibacterial, antifungal, antiviral, anti-diabetes, anti-cancer and anti-inflammation activities (Harikumar and Aggarwal, 2008; Zick et al., 2009). One of these useful plants is Z. multiflora, which is used in traditional medicine for its antiseptic, analgesic and carminative properties. The plant’s essential oil is mainly composed of monoterpene and aromatic compounds and has antibacterial, antiviral, and antifungal activities (Fazeli et al., 2007; Ramezani et al., 2004). Despite its remarkable array of medical applications, to our knowledge, little research has been carried out on the biological activity of the essential oil constituents of this plant. Our results indicate that the main components of the essential oil are carvacrol (29.2%), thymol (25.4%), pcymene (11.2%), linalool (9.6%) and c-terpinene (8%). The Z. multiflora essential oil has potent radical scavenging activity. The antioxidant activity of compounds is mainly due to their redox properties, which can play an important role in neutralizing free radicals, quenching singlet and triplet oxygen, or decomposing peroxides, and is related to phenolic hydroxyl groups (Buyukbalci and Ei, 2008; Katalinic et al., 2006; Wei and Shibamoto, 2010; Zheng and Wang, 2001). According to GC–MS analysis, Z. multiflora contains high levels of phenolic compounds (thymol and carvacrol).
Thus, potent antioxidant activity is related to the levels of thymol and carvacrol (Zheng and Wang, 2001; Baser, 2008). The side effects of Z. multiflora essential oil and its main components on cell viability were examined by the MTT assay. The essential oil, thymol, carvacrol, p-cymene, linalool and c-terpinene had no effects on cell viability at low concentrations although at a high concentration all of them reduced cell viability. Even though essential oil acts as an antioxidant, in eukaryotic cells essential oils can act as prooxidants affecting inner cell membranes and organelles such as mitochondria but, depending on the type and concentration, they also exhibit cytotoxic effects on living cells (Bakkali et al., 2008; Reichling et al., 2010). The effect of Z. multiflora essential oil and its main components on the modulation of NO and H2O2 production as induced by a high glucose concentration in human monocytes, was investigated. The results of this study also showed that a high glucose concentration induced large amounts of NO and H2O2 production in human monocytes. Previous studies have indicated that a high glucose concentration directly induces superoxide production. Superoxide ion leads to the activation of NF-jB transcription factor, which in turn causes the expression of several immunological mediators, including NO. Thus, the production of NO by a high glucose concentration is indirectly mediated by NF-jB activation (Guha et al., 2000; Shanmugan et al., 2003). However, our results indicate that Z. multiflora essential oil, carvacrol and thymol significantly reduced NO and H2O2 production in a dose-dependent manner, while p-cymene, linalool and c-terpinene did not show any effects on NO and H2O2 production in human monocytes treated with a high glucose concentration. The reduction of NO and H2O2 production by Z. multiflora essential oil, carvacrol and thymol occurs through several mechanisms. The first and simplest way is the scavenging of reactive oxygen and nitrogen radicals. Our study indicates that Z. multiflora essential oil, carvacrol and thymol, which contain a phenol group, have strong radical scavenging activities, which reduce NO and H2O2 concentration in the medium, while p-cymene, linalool and c-terpinene, which lack a phenol group, did not show such effects (Baser, 2008; Bakkali et al., 2008; Wei and Shibamoto, 2010). The second possible mechanism may be that Z. multiflora essential oil, thymol and carvacrol function as NOS and NOX inhibitors
3084
G. Kavoosi, J.A. Teixeira da Silva / Food and Chemical Toxicology 50 (2012) 3079–3085
and thus reduce NOS and NOX activities. The most common pathway for NO and H2O2 production is NOS and NOX activation by stimuli such as lipopolysaccharide (LPS). In response to such stimuli, NO and superoxide production are markedly increased. NO is converted to a stable end product nitrite and then to nitrate while superoxide is easily converted to a stable and diffusible product, H2O2, by superoxide dismutase (Babior et al. 2002). Thus, in this research, the production of nitrate and H2O2 as an indicator of oxidative stress generation was analyzed. Our results indicate that Z. multiflora essential oil, carvacrol and thymol reduced NOS and NOX activities. The mechanism of this inhibitory activity is not clear; however, this inhibition may be due to a redox cycling property of the phenol antioxidant. NOS and NOX require NADH or NADPH as the reducing agent for activity. Thus the ratio of NADH/NAD+ or NADPH/NADP+ affects the activity of the enzymes. Redox cycling of phenol antioxidant disturbs the NADH/NAD+ or NADPH/NADP+ ratio and affects NOS and NOX activities (Choi et al., 2008; Yang et al., 2008). To our knowledge, there is no research on the inhibitory effects of Z. multiflora essential oil, carvacrol and thymol on NO and H2O2 production in human monocytes cultured in the presence of high glucose concentration. Besides high glucose concentration, other stimuli such as LPS may also induce NO and superoxide production. Previous studies on the aqueous and ethanolic extracts as well as the essential oils of other herbal medicines confirm our findings. Extracts from Eucalyptus globulus Labill. and Thymus vulgaris L. (Vigo et al., 2004), Saposhnikovia divaricata (Wang et al., 1999), Pinus sylvestris L. and Plantago lanceolata L. (Vigo et al., 2005), Acanthopanax senticosus (Lin et al., 2008), Cimicifuga racemosa (aqueous) (Schemid et al., 2009), and Actinodaphne lancifolia (methanolic) (Kim et al., 2004) confirmed the inhibitory activity on NO production in LPS-activated macrophage cells during translational and post-translational levels. In addition, the two main compounds in thyme (i.e., thymol and carvacrol) displayed concentrationdependent antioxidant capacity and showed a protective effect against oxidative damage in human lymphocytes and V97 Chinese hamster lung fibroblast cells while c-terpinene, which lacks a phenolic group, did not show any antioxidant capacity (Aydin et al., 2005; Undeger et al., 2009). Furthermore, Vernonia cinerea, Cardiospermum halicacabum, T. vulgaris, Eucalyptus bridgesiana and Cymbopogon martini showed antioxidant and anti-inflammatory activity by modulating proinflammatory cytokines, NO synthase and cylooxygenase-2 (El-Nekeety et al., 2011; Pratheshkumar and Kuttan 2011a,b; Tsai et al., 2011).
5. Conclusions Accordingly, it was concluded that essential oil from Z. multiflora has antioxidant properties, can reduce oxidative stress and can be used in the therapy of oxidative damage accompanying some inflammatory conditions. High glucose concentration can produce large amounts of NO and H2O2 in human monocytes. Pretreatment of monocytes cultured on medium containing a high glucose concentration with Z. multiflora essential oil, thymol and carvacrol leads to a significant reduction in NO and H2O2 production. Although more studies are needed for understanding the major strategy for these reductions, they are probably mediated by two mechanisms: (1) by the radical scavenging activity of phenolic antioxidants; (2) through inhibition of the related enzyme by phenolic antioxidants. Overall it can be concluded that Z. multiflora can be used in the therapy of oxidative damage that accompanies hyperglycemia and some inflammatory conditions. However, further studies are necessary to confirm the antioxidant activities of the essential oil in vivo. In addition, the present study provides a stronger case for supporting the use of Z. multiflora as a tea or addi-
tive and traditional remedy for the treatment of inflammatory diseases. Conflict of Interest The authors declare that there are no conflicts of interest. Acknowledgements This work was financially supported by Shiraz University(Grant No. 88-GR-AGRST-108). The authors thank their colleagues at the Plants and Drug Research Institute, University of Shahid Beheshti, for assistance with GC–MS analysis. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.fct.2012.06.002. References Adams, R.P., 2007. Identification of essential oil components by gas chromatography/mass spectrometery, Fourth ed. Allured Publishing Corporation, Illinois, USA, pp. 456. Aydin, S., Basaran, A.A., Basaran, N., 2005. Modulating effects of thyme and its major ingredients on oxidative DNA damage in human lymphocytes. J. Agr. Food Chem. 53, 1299–1305. Babior, B.M., Lamaeth, J.D., Nauseef, W., 2002. The neutrophil NADPH oxidase. Arch. Biochem. Biophys. 397, 342–344. Bakkali, F., Averbeck, S., Averbeck, D., Idaomar, M., 2008. Biological effects of essential oils. Food Chem. Toxicol. 46, 447–475. Baser, K.H., 2008. Biological and pharmacological activities of carvacrol and carvacrol bearing essential oils. Curr. Pharm. Dis. 14, 3106–3119. Buyukbalci, A., Ei, S.N., 2008. Determination of in vitro antidiabetic effects, antioxidant activities and phenol contents of some herbal teas. Plant Foods Human Nutr. 63, 27–33. Choi, S.W., Bensie, I.F.F., Ma, S.W., Strain, J.J., Hannigan, B.M., 2008. Acute hyperglycemia and oxidative stress: Direct cause and effect. Free Radical Biol. Med. 44, 1217–1231. El-Nekeety, A.A., Mohamed, S.R., Hathout, A.S., Hassan, N.S., Aly, S.E., AbdelWahhab, M.A., 2011. Antioxidant properties of Thymus vulgaris oil against aflatoxin-induced oxidative stress in male rats. Toxicon 57, 984–991. Fazeli, M.R., Amin, G., Attari, M.M.A., Ashtiani, H., Jamalifar, H., Samadi, N., 2007. Antimicrobial activities of Iranian sumac and avishan-e shirazi (Zataria multiflora) against some food-borne bacteria. Food Control 18, 646–649. Green, L.M., Reade, J.L., Ware, C.F., 1984. Rapid colorimetric assay for cell viability: Application to the quantitation of cytotoxic and growth inhibitory lymphokines. J. Immunol. Methods 70, 257–268. Guha, M., Bai, W., Nadler, J.L., Natarajan, R., 2000. Molecular mechanisms of tumor necrosis factor gene expression in monocytic cells via hyperglycemia-induced oxidant stress-dependent and independent pathways. J. Biol. Chem. 275, 17728–17739. Harikumar, K.B., Aggarwal, B.B., 2008. Resveratrol: A multitargeted agent for ageassociated chronic diseases. Cell Cycle 7, 1020–1035. Hummel, W., Riebel, B., 2003. Isolation and biochemical characterization of a new NADH oxidase from Lactobacillus brevis. Biotechnol. Lett. 25, 51–54. Johansen, J.S., Harris, A.K., Rychly, D.J., Ergul, A., 2005. Oxidative stress and the use of antioxidants in diabetes: Linking basic science to clinical practice. Cardiovasc. Diabetol. 4, 5–16. Katalinic, V., Milos, M., Kulisic, T., Jukic, M., 2006. Screening of 70 medicinal plant extracts for antioxidant capacity and total phenols. Food Chem. 94, 550–557. Kim, Y., Min, H.Y., Park, H.J., Lee, E.J., Park, E.J., Wang, H.J., Jin, C., Lee, Y.S., Lee, S.K., 2004. Suppressive effect of inducible nitric oxide synthase expression by the methanol extract of Actinodaphene lancifolia. Phytother. Res. 18, 853–856. Lin, Q.Y., Jin, L.J., Cao, Z.H., Xu, Y.P., 2008. Inhibition of inducible nitric oxide synthase by Acanthopanax senticosus extract in RAW264.7 macrophages. J. Ethnopharmacol. 118, 231–236. Ma, Q., 2010. Transcriptional responses to oxidative stress: Pathological and toxicological implications. Pharmacol. Ther. 125, 376–393. Mahmoudabadi, A.Z., Dabbagh, M.A., Fouladi, Z., 2007. In vitro anti-Candida activity of Zataria multiflora Boiss. Evidence Based Complem. Alter. Med. 4, 351–353. Marcocci, L., Maguire, J.J., Droylefaix, M.T., Packer, L., 1994. The nitric oxide scavenging properties of Ginkgo biloba extract EGB 761. Biochem. Biophys. Res. Commun. 201, 748–755. Miranda, M.K., Espey, M.G., Wink, D.A., 2001. A rapid, simple spectrophotometric method for simultaneous detection of nitrate and nitrite. Nitric Oxide 5, 62–71. Pacher, P., Beckman, J.S., Liaudet, L., 2007. Nitric oxide and peroxynitrite in health and disease. Physiol. Rev. 87, 315–324.
G. Kavoosi, J.A. Teixeira da Silva / Food and Chemical Toxicology 50 (2012) 3079–3085 Pirali-Kheirabadi, K., Teixeira da Silva, J.A., 2011. In-vitro assessment of the acaricidal properties of Artemisia annua and Zataria multiflora essential oils to control cattle ticks. Iranian J. Parasitol. 6, 58–65. Pratheshkumar, P., Kuttan, G., 2011a. Cardiospermum halicacabum inhibits cyclophosphamide induced immunosupression and oxidative stress in mice and also regulates iNOS and COX-2 gene expression in LPS-stimulated macrophages. Asian Pacific J. Cancer Prev. 11, 1245–1252. Pratheshkumar, P., Kuttan, G., 2011b. Modulation of immune response by Vernonia cinerea L. inhibits the proinflammatory cytokine profile, iNOS, and COX-2 expression in LPS-stimulated macrophages. Immunopharmacol. Immunotoxicol. 33, 73–83. Ramezani, M., Hosseinzadeh, H., Samizadeh, S., 2004. Antinociceptive effects of Zataria multiflora Boiss. fractions in mice. J. Ethnopharmacol. 91, 167–170. Re, R., Pellegrini, N., Protegenete, A., Pannala, A., Yang, M., Rice-Evans, C., 1999. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biol. Med. 26, 1231–1237. Reichling, J., Schnitzler, P., suschke, U., Saller, R., 2009. Essential oils of aromatic plants with antibacterial, antifungal, antiviral and cytotoxic properties. Forch. Komplement. Med. 16, 79–90. Rhee, S.G., Chang, T.S., Jeong, W., Kang, D., 2010. Methods for detection and measurement of hydrogen peroxide inside and outside of cells. Mol. Cells 29, 539–549. Saei-Dehkordi, S.S., Razavi Rohani, S.M., Tajik, H., Moradi, M., Aliakbarlou, J., 2008. Antimicrobial effects of lysozyme in combination with Zataria multiflora Boiss. essential oil at different pH and NaCl concentrations on E. coli O157:H7 and Staphylococcus aureus. J. Animal Vet. Adv. 7, 1458–1463. Schemid, D., Gruber, M., Woehs, F., Prinz, Z., Etzlstorfer, B., Prucker, C., Fuzzati, N., Kopp, B., Moeslinger, T., 2009. Inhibition of inducible nitric oxide synthesis by Cimicifuga racemosa extracts in LPS-stimulated RAW264.7 macrophages. J. Pharm. Pharmacol. 61, 1089–1096.
3085
Shanmugan, N., Reddy, M.A., Guha, M., Natarajan, R., 2003. High-glucose expression of proinflammatory cytokines and chemokine genes in monocytic cells. Diabetes 52, 1256–1264. Sze, S.C., Tong, Y., Ng, T.B., Cheng, C.L., Cheung, H.P., 2010. Herba epimedii: Antioxidative properties and its medical implications. Molecules 15, 7861–7870. Tsai, M.L., Lin, C.C., Lin, W.C., Yang, C.H., 2011. Antimicrobial, antioxidant and antiinflammatory activities of essential oils from five selected herbs. Biosci. Biotechnol. Biochem. 75, 110377-1–110377-7. Undeger, U., Basaran, A., Degen, G.H., Basaran, N., 2009. Antioxidant activities of major thyme ingredients and lack of oxidative DNA damages in V97 Chinese hamster lung cells al low levels of carvacrol and thymol. Food Chem. Toxicol. 47, 2037–2043. Vigo, E., Cepeda, A., Gualillo, O., Perez-Fernandez, R., 2004. In vitro antiinflammatory effect of Eucalyptus globulus and Thymus vulgaris: Nitric oxide inhibition in J774A.1 murine macrophages. J. Pharm. Pharmacol. 56, 257–263. Vigo, E., Cepeda, A., Gualillo, O., Perez-Fernandez, R., 2005. In vitro antiinflammatory activity of Pinus sylvestris and Plantago lanceolata extracts: Effects on inducible NOS, COX-1, COX-2 and their products in J774A.1 murine macrophages. J. Pharm. Pharmacol. 57, 383–391. Wang, C.C., Chen, L.G., Yang, L.L., 1999. Inducible nitric oxide synthse inhibitor of the Chinese herbs I Saposhnikovia divaricata. Cancer Lett. 145, 151–157. Wei, A., Shibamoto, T., 2010. Antioxidant/lipoixygenase inhibitory activities and chemical compositions of selected essential oil. J. Agri. Food Chem. 58, 7218– 7225. Yang, Z., Laubach, V.E., French, B.A., Kron, I.L., 2008. Acute hyperglycemia enhances oxidative stress and exacerbates myocardial infarction by activating NADH oxidase during reperfusion. J. Thorac. Cardiovas. Surg. 137, 723–729. Zheng, W., Wang, S.Y., 2001. Antioxidant activity and phenolic compounds in selected herbs. J. Agri. Food Chem. 49, 5165–5170. Zick, S.M., Schwabl, H., Flower, A., Charkraborty, B., Hirschkon, K., 2009. Unique aspects of herbal system research. Explore 5, 97–103.