PHYTOTHERAPY RESEARCH Phytother. Res. 28: 1739–1748 (2014) Published online 20 July 2014 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ptr.5200
REVIEW
Antiinflammatory, Antioxidant, and Immunological Effects of Carum copticum L. and Some of Its Constituents Azam Alavinezhad and Mohammad Hossein Boskabady* Neurogenic Inflammation Research Centre, Department of Physiology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
Carum copticum L. has been used traditionally for its various therapeutic effects. The plant contains various components such as thymol and carvacrol. Different therapeutic effects such as antifungal, antioxidant, antibacterial, antiparasitic, and antilipidemic were described for the plant and its constituents. Therefore, antiinflammatory, antioxidant, and immunological effects of C. copticum and its constituents, thymol and carvacrol, were discussed in the present review. Previous studies have shown potent antiinflammatory, antioxidant, and immunological effects for C. copticum and its constituents, thymol and carvacrol. Therefore, the plant and its constituents have therapeutic values in several inflammatory and immunological disorders as well as in the oxidative stress conditions. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: Carum copticum; thymol; carvacrol; antiinflammatory; antioxidant; immunological effect.
INTRODUCTION Herbal medicines have been used for the prevention and the treatment of chronic disorders such as inflammatory diseases, diabetes, allergy, arthritis, and other similar conditions for a long time. Carum copticum is a grassy plant that belongs to the Apiaceae (Umbelliferae) family with white flowers and grayish seeds (Fig. 1) (Dwivedi et al., 2012). C. copticum is known as Ajowan, with other names in different languages including Trachyspermum copticum, Ammi copticum L., bishop’s weed, Ethiopian caraway, kummel, ajawa, ajwain, and ajamoda (Bairwa et al., 2012). This plant may increase body resistance and has antiinflammatory and antispasmodic properties (Thangam and Dhananjayan, 2003). The relaxant effect of C. copticum and its constituent, carvacrol on tracheal smooth muscle (Boskabady et al., 2003; Boskabady et al., 1998; Boskabady and Jandaghi, 2003), and its possible mechanism including anticholinergic effect, stimulatory effect on the β-adrenergic, and inhibitory effect on histamine receptors were previously demonstrated (Boskabady et al., 2011a, 2011b; Boskabady and Shaikhi, 2000; Boskabady et al., 2011a, 2011b; Boskabady and Moemeni., 2000; Boskabady and Krachian, 2000; Boskabady et al., 2012). Furthermore, bronchodilatory effect of the plant extract on asthmatic patients was also shown (Boskabady et al., 2007). The antitussive effect of C. copticum was also evaluated, which was even greater than codeine at concentrations used (Boskabady et al., 2005).
Carum copticum oil has antifungal, antimicrobial, and antiparasitic effects (Kavoosi et al., 2013), and extracts of this plant showed the highest antioxidant activity among 125 herbs (Huang et al., 2011). Its fruit has stimulant, antispasmodic, and carminative properties, and it is an important therapeutic agent in indigestion and diarrhea (Bentely and Trimen, 1983). Thymol is an aromatic substance with antimicrobial (Singh and Singh, 2000), antispasmodic (cramps), and antiparasitic (Sivropoulou et al., 1996) properties. It is used for the treatment of gastrointestinal ailments, loss of appetite, and respiratory problems (Cortés-Arroyo et al., 2011). With regard to the wide usage of C. copticum in Iranian traditional medicine and because of several studies indicating antiinflammatory, antioxidant, and immunological effects of the plant and its main constituents, thymol and carvacrol, these effects of C. copticum and the two constituents of the plant were reviewed in the present article. These effects of the plant and its constituents are therapeutically important and scientifically valuable to review. The other plants containing thymol and carvacrol, which have antiinflammatory, antioxidant, and immunological effects, were also included in this review, which supports the mentioned effect for thymol and carvacrol.
CHEMICAL COMPOSITION * Correspondence to: Mohammad Hossein Boskabady, Neurogenic Inflammation Research Centre, Department of Physiology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran. E-mail: [email protected]
Copyright © 2014 John Wiley & Sons, Ltd.
The main components of C. copticum oil are thymol (known as 2-isopropyl-5-methylphenol, C10H14O) (Fig. 2a) and glycosyl constituents such as carvacrol or isothymol (know as 5-isopropyl-2-methyl phenol, Received 12 March 2014 Revised 25 June 2014 Accepted 25 June 2014
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Figure 1. Flowers and seeds of Carum copticum.
C10H14O), (Fig. 2b), 2-O-β-D-glucopyranoside, 3,5dihydroxytoluene, and 3-O-β-D-galactopyranoside (Ishikawa et al., 2001; Zahin et al., 2010). The other constituents of C. copticum oil are γ-terpinene, β-pinene, o-cymene, and p-cymene (Zarshenas et al., 2014; Kazemi et al., 2011; Khajeh et al., 2004; Singh et al., 2004). However, acetone extract of C. copticum had different percentages of some constituents from the total extract (68.8%) such as β-pinene (0.2%), p-cymene (1.6%), γ-terpinene (2.6%), thymol (39. 1%), and carvacrol (0.3%), (Singh et al., 2004). A study in five different areas of Algeria showed that thymol content is higher in the plants that grow in areas near the sea with low altitudes (Bekhechi et al., 2010). High concentration of thymol in C. copticum oil had been determined by gas chromatography (Gersbach and Reddy, 2002). The effect of cultivation situations on the amount of Iranian C. copticum constituents is shown in Table 1 (Zarshenas et al., 2014; Singh et al., 2004; Naseri et al., 2007). It has been also shown that this plant has other constituents including carbohydrates, fat, protein, fiber, tannins, glycosides, moisture, saponins, flavone, and minerals (Bairwa et al., 2012).
ANTIINFLAMMATORY EFFECTS The antiinflammatory effect of C. copticum has been reported in several studies. Significant inhibitory effects of total alcoholic extract (TAE) and total aqueous extract (TAQ) of C. copticum on both acute (carrageenan-induced paw edema in rat) and sub-acute (cotton-pellet-induced granuloma) rat models of inflammation have been demonstrated, which were comparable with those of aspirin and phenyl butazone. TAQ and TAE can affect synthesis of kinnin, prostaglandin, bradykinin, and lysozyme, which is suggested to contribute in the antiinflammatory effect of the plant (Thangam and Dhananjayan, 2003). Rheumatoid arthritis is an autoimmune disease in which reactive oxygen species (ROS) as well as reactive
(a)
(b)
Figure 2. Molecular shape of (a) thymol and (b) carvacrol. Copyright © 2014 John Wiley & Sons, Ltd.
nitrogen species have likely an important role in its pathogenesis (Fachini-Queiroz et al., 2012). The effect of C. copticum on collagen-induced arthritis in rats was evaluated by measuring elastase (marker for collagen degradation), catalase (CAT), reduced glutathione (GSH), superoxide dismutase (SOD) (Bairwa et al., 2012), and nitric oxide (NO). Administrations of the C. copticum extract resulted in reduction of elastase and subsequent decrease in neutrophil infiltration and activation. The plant inhibited lipid peroxidation and retrieved GSH and SOD levels in tissue. Treatment with C. copticum extract decreased NO level, which increases during rheumatoid arthritis. In terms of histology, articular tissue was recovered, which may be due to the inhibitory effect on infiltrating of inflammatory cells and restoration of antioxidants. It was concluded that the antiinflammatory effect of the C. copticum extract may be due to the existence of flavonoids and phenols (Mahboubi and Kazempour, 2011). In a clinical trial, herbal drops of several plants including C. copticum were used for few eye disorders. These herbal drops were able to treat eye infection and inflammation in studied patients (Biswas et al., 2001). In vitro method for testing antiadhesion of platelets in the blood of human volunteers showed that blood platelet aggregation was inhibited by C. copticum extract. The possible mechanisms of these findings are perhaps due to the effect of the plant on thromboxane production in platelets. The aforementioned study confirmed the traditional use of C. copticum in postpartum period (Srivastava, 1988). Thymus vulgaris essential oil containing thymol as the main component has potent antioxidant and antiinflammatory effects. In human acute monocytic leukemia (THP-1) cell line, T. vulgaris suppresses 5-lipoxygenase (5-LOX) activity more than α-bisabolol (known as having an antiinflammatory effect) and has an inhibitory effect on TNF-α and IL-8 secretion in lipopolysaccharide (LPS)-treated THP-1 cell line. Therefore, T. vulgaris has antiinflammatory properties because of the 5-LOX inhibition and reduction of cytokines such as TNF-α, IL-8, and IL-1β (Tsai et al., 2011). In an in vitro model of macrophage inflammation induced by LPS and interferon gamma (IFN-γ) that led to an increased NO production, T. vulgaris treatment inhibited inducible nitric oxide synthase mRNA expression and has NO scavenging property (Vigo et al., 2004). Thyme extract oils (from T. vulgaris, Thymus zygis, and Thymus hyemalis) also contain bioactive components including thymol and carvacrol. In a study conducted by Ocaña and Reglero (2012), the effect of Phytother. Res. 28: 1739–1748 (2014)
GC/FID GC/FID GC/FID GC/FID GC/FID GC/FID GC/FID GC/FID GC/FID GC/FID GC/FID GC/FID Mashhad Tehran South of Shiraz Northwest of Shiraz Zahedan Kerman Torbat-e-heydaryeh Darab Bandar-abbas Meymand Gorakhpur Fars Province Myrcene (1.32%) Myrcene (0.8%) Carvacrol (0.43%) Carvacrol (0.65%) Limonene (1.32%) Myrcene (0.92%) Thujene (1.51%) α-Pinen (1.31%) β-phellandrene (0.65%) β-Pinen (0.99%) Carvacrol (0.3%) Carvacrol (0.45%) β-Pinen (3.46%) Thymol (1.5%) β-Pinen (1.8%) β-Pinen (1.5%) β-Pinen (2.58%) β-Pinen (3.4%) β-Pinen (3.7%) β-Pinen (3.64%) β-Pinen (1.23%) Carvacrol (1.01%) β-Pinen (1.7%) Thymol dihydroquinone (0.96%)
Copyright © 2014 John Wiley & Sons, Ltd.
GC/FID, gas chromatograph/flame ionization detector.
Thymol (21.29%) β-Pinen (3.04%) γ-Terpinene (19.21%) γ-Terpinene (16.98%) p-Cymene (20.73%) γ-Terpinene (21.31%) γ-Terpinene (23.11%) Thymol (19.58%) γ-Terpinene (12.93%) Thymol (20.62%) γ-Terpinene (23.2%) Hexadecanoic acid (9.69%) γ-Terpinene (32.54%) γ-Terpinene (35.12%) p-Cymene (19.67%) p-Cymene (22.38%) γ-Terpinene (21.71%) Thymol (30.4%) p-Cymene (31.8%) γ-Terpinene (28.04%) p-Cymene (19.15%) p-Cymene (34.64%) p-cymene (30.8%) Linoleic acid (13.56%) p-Cymene (37.3%) p-Cymene (57.31%) Thymol (55.21%) Thymol (53.66%) Thymol (49.16%) p-Cymene (40.2%) Thymol (35.04%) p-Cymene (43.3%) Thymol (63.31%) γ-Terpinene (37.43%) Thymol (39.1%) Thymol (64.59%)
Main constituent
Table 1. Chemical constituents of Carum copticum essential oil in different geologic areas (Zarshenas et al., 2014; Singh et al., 2004; Naseri et al., 2007)
Cultivation location
Method
ANTIINFLAMMATORY EFFECTS OF C. COPTICUM AND ITS CONSTITUENTS
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thyme extract oils on activated THP-1 macrophage by oxidized low-density lipoproteins was assessed. In this study, the extract oils diminished TNF-α, IL-1β, and IL-6 genes expression but increased IL-10 gene expression. Therefore, the extracted oils may be helpful for the treatment of inflammatory diseases. In a cellular model of atherosclerosis (a chronic inflammatory disease), the effect of Origanum vulgare, which contains sabinene hydrate, thymol, and carvacrol, on THP-1 macrophages and levels of pro-inflammatory and antiinflammatory cytokines was evaluated. The plant extract decreased TNF-α, IL-1β, and IL-6; however, IL-10 as an antiinflammatory cytokine was increased, which inhibited translocation of NF-κB into the nucleus (Ocana-Fuentes et al., 2010). Because the pro-inflammatory cytokines such as IL-1, IL-6, IL-8, TNF-α, IL- 12, and IFN-γ increased in intestinal inflammation, in a study, the effect of T. vulgaris L. and Origanum syriacum L. on colitis was examined by measuring the expression of cytokines or cytokine mRNA level. The results showed that a medium dose of these plants can suppress the expression of the aforementioned cytokines and thus showing an antiinflammatory effect (Bukovská et al., 2007). Elastase is an inflammatory marker released by neutrophils involved in inflammatory disorder and elastase inhibitor can protect tissue against inflammation. It was shown that thymol can inhibit calcium channels and subsequently impeding release of human neutrophil elastase (Braga et al., 2006). Thymol contributes to improving the symptoms of arthritis by inhibiting calcium channels and consequently inactivating elastase (Umar et al., 2012). It was shown that thymol can inhibit cyclooxygenase (COX-1) enzyme (an enzyme of prostaglandin E2 synthase) in an in vitro study, indicating its antiinflammatory effects (Marsik et al., 2005). Ku and Lin (2013) assessed the effect of 27 selected terpenoid compounds such as thymol on Th1/Th2 balance in murine primary splenocytes. They measured IL-2, IFN-γ (Th1 cytokines), IL-4, IL-5, and IL-10 (Th2 cytokines) in terpenoid-treated splenocytes. Their results showed that terpenoids have antiinflammatory nature via inhibition of T cells immune response and improved Th1/Th2 ratio. Aqueous and ethanol extracts of Zataria multiflora, which contain thymol and carvacrol, showed antiinflammatory effects against acute and chronic inflammation. Total extract, flavonoid, and the essential oil of Z. multiflora also showed ameliorative effect on carrageen-induced paw edema in rat (Sajed et al., 2013). Veras et al. (2013) showed that essential oil of Lippia sidoides, which contains thymol (84.9%), had a topical antiinflammatory effect on acute ear inflammation induced by arachidonic acid and phenol. They demonstrated that possibly the thymol content of the essential oil has an inhibitory effect on lipooxygenase, COX, and phospholipase A2. Carvacrol (5-isopropyl-2-methylphenol) is a phenolic monoterpene and one of the main components of C. copticum in which its antiinflammatory property in several studies has been reported. The capacity of Thymus herba-barona essential oil containing phenols, carvacrol (54.0%), and thymol (30.2%) for prevention of NO production as inflammatory marker of LPS-stimulated macrophages was assessed. Results Phytother. Res. 28: 1739–1748 (2014)
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showed that the lowest concentrations of T. herbabarona (0.08 μL/mL) did not inhibit NO production in cells exposed to LPS. However, its higher concentration (0.64 μL/mL) decreased nitrite production as compared with the non-LPS-treated group (Zuzarte et al., 2013). The presence of carvacrol in thyme oils can affect expression of COX-2 mRNA and protein and has antiinflammatory effects. Thyme extract can also reduce COX-2 activation up to almost 75%, and its main component, carvacrol, decreased COX-2 level up to 80% (Hotta et al., 2010). Carvacrol is also able to reduce prostaglandin E2 and inflammatory cytokines such as IL-1β by stimulation of IL-10 release (the antiinflammatory cytokine) (Lima et al., 2012). In the presence of stress signals, carvacrol acts as co-inducer of heat shock protein-70 (HSP-70) expression in mammalian cells and thus can reduce the severity of inflammation (Van et al., 2010). Liu et al. (2012) induced in vitro inflammation on porcine alveolar macrophages (PAM) with LPS and assessed the effect of carvacrol on TNF-α, IL-1β, TGF-β, and IL-10 levels in the cell culture supernatants of macrophages. The results showed that carvacrol suppressed TNF-α, TGF-β, and IL-1β. On the basis of this study results, the suppression of TNF-α accounted for the antiinflammatory effect of carvacrol. In paw edema and gastric lesions animal models, carvacrol was able to decrease inflammation and alleviate edema and lesions with effect on secretion or synthesis of various inflammatory mediators (Silva et al., 2012). The effect of carvacrol in hepatotoxicity induction in rat with D-galactosamine and in the measurement of mRNA and proteins expression such as nitric oxide synthase, COX-2, TNF-α, IL-6, and NF-κB was investigated. Expression of all proteins was downregulated by carvacrol, indicating its antiinflammatory effect (Aristatile et al., 2013). The antiinflammatory effect of thymol and carvacrol was also examined by measurement of exudates volume in the pleural cavity and leukocyte migration after carrageenan injection into the pleural cavity of rats. The results showed that thymol and carvacrol decreased exudates volume. In addition, carvacrol reduced leukocyte migration (in vivo and in vitro) with inhibitory effect on COX-1, 2, and 5-lipoxygenase, which reduced prostaglandins, leukotrienes, and inflammatory mediators involved in edema. The results also showed that thymol did not decrease leukocyte migration and had an irritative effect, which may be due to histamine and prostanoid release from the tissues (Fachini-Queiroz et al., 2012). Another study showed that increased white blood cell (WBC) by Escherichia coli infection was decreased with administration of carvacrol, which also led to the reduction of neutrophil and lymphocyte percentage. The effect of carvacrol was also examined on PAM in vitro. In this study, PAM treated with LPS caused increased TNF-α and IL-1β secretion. Carvacrol added to LPS-treated PAM inhibited secretion of TNF-α and IL-1β. However, the effect of carvacrol on PAM in the absence of LPS was opposite as TNF-α and IL-1β increased because these cytokines are beneficial for body protection (Yanhong, 2011). The effect of carvacrol on serum endothelin, total protein, histamine, and NO levels of guinea-pigs sensitized with ovalbumin as well as its effect on tracheal Copyright © 2014 John Wiley & Sons, Ltd.
responsiveness, lung pathological changes, and total and differential WBC count was also examined. The results of these series of studies showed preventive effect of carvacrol on serum endothelin, total protein, histamine, and NO levels in ovalbumin-sensitized animals. Treatment of sensitized animals with carvacrol also lead to reduction in tracheal responsiveness to both methacholine and ovalbumin, lung pathological changes, changes in the total and differential WBC counts specially eosinophil count, which was comparable with the effect of dexamethasone (Boskabady and Jalali, 2013). Therefore, all plants containing carvacrol such as C. copticum could be effective as a preventive drug in inflammatory disorders such as asthma. Romano et al. (2013) studied the effects of phytocannabinoid on murine peritoneal macrophages in vitro. They demonstrated that carvacrol decreased nitrite value in LPS-stimulated macrophages, which indicates the antiinflammatory effect of carvacrol. Table 2 summarized various antiinflammatory effects of C. copticum and its constituents thymol and carvacrol.
IMMUNOLOGICAL EFFECTS There is not any report regarding the immunological effects of C. copticum. However, different herbs are known to modulate the immune system such as plants of the Apiaceae family including C. cyminum and C. caraway, which contain thymol. The effect of C. cyminum on the immune system was demonstrated by Chauhan et al. (2010). They used cyclosporine-A for suppressing immunity in Swiss albino mice and then orally fed the mice with cumulative doses of C. cyminum. Their results showed that administration of this plant caused increased CD4, CD8, and Th1 cytokines expression. This effect is possibly due to enhancement of IFN-γ expression and induction of IL-12 secretion, which has a main role in cellular immunity. It also increased the weight of the spleen and thymus and caused a reduction in adrenal gland size, which reduced serum corticosterone levels (Chauhan et al., 2010). Khajeali et al. (2012) added different doses of Carum carvi L., a member of the Carum genus (Apiaceae family) that contains thymol, to the food of broiler chickens and measured antibody titers against Newcastle disease virus. The results showed significantly higher antibody titers in groups receiving higher doses of caraway seeds powder. The effects of thymol feed supplementation on the immune response were investigated in broiler chickens. This component modified the immune response by increasing hypersensitivity response and IgG but reducing neutrophil to lymphocyte ratio (Hashemipour et al., 2013). In a study, thymol feed supplementation showed beneficial effects on innate immune conditions (Giannenas et al., 2012). A 1% thymol diet in weaning pigs challenged with Salmonella typhimurium reduced serum IgA. S. typhimurium increased IgM but did not affect IgM concentration in thymol-treated group, although thymol in the non-challenged group enhanced serum levels of IgA and IgM (Trevisi et al., 2010). Ariza-Nieto et al. (2011) assessed the effect of sow diets supplementation with oregano essential oil Phytother. Res. 28: 1739–1748 (2014)
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Table 2. Antiinflammatory effect of Carum copticum and its constituents thymol and carvacrol
Carum copticum
Thymol
Carvacrol
Effect
Reference
Effect on synthesis of kinnin, prostaglandin, and bradykinin Inhibitory effect on infiltrating of inflammatory cells Treatment of eye infection and inflammation Antiadhesion of platelets Improvement of arthritis Inhibition of 5-LOX and decrease of cytokines secretion Inhibition of inducible nitric oxide synthase (iNOS) mRNA expression and has NO scavenging property Decrease of TNF-α, IL-1β, and IL-6 genes expression and increase of IL-10 gene expression Inhibition of translocation NF-kB Suppression of pro-inflammatory cytokines expression Inhibitory effect on release of elastase Inhibition of cyclooxygenase (COX-1) Improvement of Th1/Th2 ratio Decrease of nitrite production Reduction of COX-2 activation Co-inducer of HSP-70 expression Stimulation of IL-10 release Suppression of TNF-α Alleviation of edema and lesions Downregulation of iNOS, COX-2, TNF-α, IL-6, and NF-κB expression Reduction of leukocyte migration and exudates volume Modulation of TNF-α and IL-1β secretion Reduction in tracheal responsiveness of sensitized animals
(Thangam and Dhananjayan, 2003) (Mahboubi and Kazempour, 2011) (Biswas et al., 2001) (Srivastava, 1988) (Umar et al., 2012) (Tsai et al., 2011) (Vigo et al., 2004)
(containing thymol and carvacrol) on the immune response of suckling pigs. Because IgG participates in secondary antibody response and IgA has a role in mucosal immunity and T lymphocytes are responsible for acquired immunity, total IgG and IgA concentrations were measured for evaluation of humoral immunity. This diet did not affect serum immunoglobulin levels and T lymphocyte number and killer cells activity in supplementing sow diets. The absence of immune response may be due to the non-specific immunomodulatory agents (e.g., corticosteroids) in colostrums. Thymol is capable of increasing the percentage of CD4+, CD8+, and MHC-II in the peripheral blood of low-weight growing-finishing pigs. It can also decrease the expression of TNF-α in the stomach of post-weaned pigs (Taranu et al., 2012). Farinacci et al. (2008) investigated the effects of T. vulgaris (containing a considerable amount of thymol) on isolated ovine neutrophils. In this study, adhesion and superoxide production were examined in activated neutrophils by phorbol myristate acetate and incubated with extracts. Results showed that hydroethanol extract of T. vulgaris inhibited adhesion and superoxide production. These effects may be due to the presence of flavonoids and phenolic compounds, such as thymol and carvacrol. T. vulgaris had also an antiinflammatory activity on sheep neutrophils. In a study, the effect of adding the essential oil of plants containing thymol to food of weaner pigs on their gut immunoglobulins (IgG, IgA, and IgM) was examined. The results showed increased IgA and IgM levels as well as phagocytosis rates. Therefore, essential oil containing thymol improved immune function in pigs’ gut (Li et al., 2012). Copyright © 2014 John Wiley & Sons, Ltd.
(Ocaña and Reglero, 2012) (Ocana-Fuentes et al., 2010) (Bukovská et al., 2007) (Braga et al., 2006) (Marsik et al., 2005) (Ku and Lin, 2013) (Zuzarte et al., 2013) (Hotta et al., 2010) (Van et al., 2010) (Lima et al., 2012) (Liu et al., 2012) (Silva et al., 2012) (Aristatile et al., 2013) (Fachini-Queiroz et al., 2012) (Yanhong, 2011) (Boskabady and Jalali, 2013)
Amirghofran et al. (2011) evaluated the effect of a few plants including T. vulgaris and Z. multiflora and their major compound thymol on the proliferation of peripheral blood lymphocytes stimulated by phytohaemagglutinin (PHA) in vitro. Results demonstrated that these plants prevent lymphocytes proliferation in vitro, which is likely due to the thymol content. Thus, these plants and thymol may induce the immunosuppressive effects. In a study, in experimental animals (rabbit), Candida albicans antigen was injected 5 days after the last subcutaneous injection of Z. multiflora. Results showed that innate and cellular immunity responses were significantly stimulated compared with the control group (Khosravi et al., 2007). In another study, Z. multiflora essential oil was orally administrated to common carp under thermal stress. This plant increased total WBC and bactericidal activity of serum in treated groups (Soltani et al., 2010). Carvacrol can act as a treatment of immune system disorders. On the basis of the study of Spiering et al. (2012), injection of carvacrol with thermal stress may suppress autoimmune arthritis in a mouse model induced by human proteoglycan. Treatment of dendritic cells isolated from the bone marrow of mouse with carvacrol and thermal stress reduced pro-inflammatory cytokines and antiinflammatory cytokines, which is likely to be due to the production of tolerogenic dendritic cells and their effect on regulatory T cells; these cells, in turn, can adjust the immune system response in pathologic conditions. Carvacrol also showed beneficial effects on innate immune conditions (Giannenas et al., 2012). Because Th1/Th2 balance plays an important role in many diseases such as asthma, the impact of Z. multiflora, in which carvacrol is its main component, Phytother. Res. 28: 1739–1748 (2014)
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on in vitro and in vivo Th1/Th2 balance (IFN-γ/IL4 ratio) was studied. The in vitro study showed that higher concentration of the extract (200 μg/mL) increased IFN-γ gene expression in phytohaemagglutininstimulated human peripheral blood mononuclear cells, whereas all concentrations of the extract decreased IL-4 gene expression and production. The extract also significantly increased the ratio of IFN-γ to IL-4 (gene expression and production), which indicates increased Th1/Th2 balance. In sensitized guinea-pigs, also enhancement in IL-4 and reduction in IFN-γ serum levels were seen. However, in sensitized guinea-pigs treated with the extract of Z. multiflora, the serum level of IL-4 was decreased, whereas the level of IFN-γ and ratio of IFN-γ to IL-4 were increased (Boskabady et al., 2013). The effect of carvacrol on Th2 cytokine, IL-4, and Th1 cytokine, IFN-γ, was also assessed in ovalbuminsensitized guinea-pigs. IL-4 level decreased and IFN-γ increased by high concentration of carvacrol indicating enhancement of Th1/Th2 balance toward Th1, which is a beneficial effect in the improvement of inflammatory disorders associated with decreased Th1/Th2 balance (Jalali et al., 2013). Intracellular proteins including HSP have a protective role against inflammation with an important role in HSP-specific regulatory T cells. Several factors such as immune disorder or aging can decrease or alter the function of these proteins. Burt et al. (2007) examined the effect of carvacrol on protein synthesis in E. coli cells. In their study, it was shown that carvacrol increased the HSP-60 in prokaryotes. Carvacrol also increased FOXP3 expression, which is a marker of regulatory T cells, and thus, it is beneficial for the modulation of the immune system. Carvacrol also affects the immune response by reducing neutrophil to lymphocyte ratio, which was similar to the effect thymol (Hashemipour et al., 2013).
Khosravi et al. (2009) used Z. multiflora essential oil (containing 61.29% carvacrol and 25.18% thymol) in mice infected with C. albicans. According to their results, the colony-forming unit of Candida in the liver, spleen, lungs, brain, and kidneys reduced in the treated group compare with the control group, which likely reflects stimulation of the immune system by this plant. Table 3 summarized the different immunological effects of C. copticum and its constituents thymol and carvacrol.
ANTIOXIDANT PROPERTIES Many plants possess antioxidant property that depends on the species of plant, climate condition, geolocation, and other factors such as the steps of harvesting and processing. The composition and value of phenolic compounds are very important in the antioxidant activity of plants too (Skrovankova et al., 2012). Because ROS and reactive nitrogen species are involved in aging and in the pathogenesis of many diseases such as cancer and heart disease, various plant such as C. copticum are known to have antioxidant effects and can be beneficial in the improvement of such diseases (Fachini-Queiroz et al., 2012). The antioxidant potential of C. copticum fruit extract and its fractions was examined by 1,1-diphenyl-2picrylhydrazyl (DPPH) free radical scavenging assay, and total antioxidant capacity (TAC) was examined by phosphomolybdenum method. Data of this study elucidated that methanol extract had 90.2% activity for scavenging of free radicals and was comparable with 90.3% activity of ascorbic acid (Zahin et al., 2010). In another study, the antioxidant activity of the C. copticum essential oil was assessed by comparison of ferric reducing antioxidant power, activity of DPPH,
Table 3. Immunological effects of Carum copticum and its constituents thymol and carvacrol Effect Cuminum cyminum (Apiaceae family) Carum carvi L. (Apiaceae family) Thymol
Carvacrol
Reference
Increase of CD4, CD8, and Th1 cytokines expression and spleen and thymus weight Increase of antibody titers against Newcastle disease virus
(Chauhan et al., 2010)
Beneficial effects on innate immune conditions Effect on concentration of IgM and IgA No effect on serum immunoglobulin levels and T lymphocyte number and killer cells activity + + Increase percentage of CD4 , CD8 , and MHC-II Inhibition of adhesion and superoxide production on isolated ovine neutrophils Improvement of immune function in pigs’ gut Induce immunosuppressive effect Modifying immune response Suppression of autoimmune arthritis Beneficial effects on innate immune conditions Modulation of Th1/Th2 ratio Beneficial effect in improvement of inflammatory disorders Increase of FOXP3 expression (marker of regulatory T cells) and modulation of immune system Modifying immune response
(Giannenas et al., 2012) (Trevisi et al., 2010) (Ariza-Nieto et al., 2011)
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(Khajeali et al., 2012)
(Taranu et al., 2012) (Farinacci et al., 2008) (Li et al., 2012) (Amirghofran et al., 2011) (Hashemipour et al., 2013) (Spiering et al., 2012) (Giannenas et al., 2012) (Boskabady et al., 2013) (Jalali et al., 2013) (Burt et al., 2007) (Hashemipour et al., 2013) Phytother. Res. 28: 1739–1748 (2014)
ANTIINFLAMMATORY EFFECTS OF C. COPTICUM AND ITS CONSTITUENTS
and H 2O2 radical scavenging with ascorbic acid. Results of this study showed that C. copticum can be applied in foodstuff and can be used for medicinal purposes as non-synthetic antioxidant agent (Chatterjee et al., 2013). The effect of C. copticum on arthritis model in Wistar rats induced by intradermal injection of collagen was examined. In this model, enhanced levels of oxidative stress markers, namely thiobarbituric acid reactive substance and diminished GSH, SOD and CAT were observed. However, treatment with C. copticum restored GSH and decelerated the deterioration of SOD activity and CAT level. The increase of nitrite concentration was also reduced in the group treated with C. copticum. On the basis of the results of this study, C. copticum has antioxidant properties that might be due to flavonoids and phenols content of the plant (Umar et al., 2012). In one study, the extract of C. copticum was used as natural antioxidant in flaxseed and Bahera oils. The amount of peroxide oxygen per kilogram of oil was measured in different periods and different temperatures without and with antioxidant (the extract of C. copticum). Data showed the peroxide value of oil in the presence of the extract of C. copticum significantly decreased in longer time and higher temperature, which indicates a strong antioxidant effect of the extract of C. copticum (Bera et al., 2004). It was also shown that C. copticum is able to eliminate free radicals and enhance the body’s functions, which is perhaps due to its phenolic groups content (Kavoosi et al., 2013). Methanol extract of C. copticum (MCE) with concentration of 50 μg/mL also decreased free radical up to 89.3% compared with ascorbic acid (92.3%), which demonstrates a high antioxidant activity of C. copticum. Effect of pretreatment of MCE against potassium dichromate (K2Cr2O7) in human bronchial epithelial cells (BEAS-2B) and human peripheral blood lymphocytes also showed that C. copticum helps to keep antioxidant enzyme levels such as SOD and GSHPx. In other words, MCE showed a significant reduction effect in ROS levels created by K2Cr2O7. This effect is probably due to phenolic terpenoid compounds in MCE including thymol, which is its most effective constituent (Deb et al., 2011). The phenolic content of the plant has direct connection with its antioxidant activity. Because lipids in food products are oxidized, food factories use synthetic antioxidants for the food preservation. Therefore, Aeschbach et al. (1994) evaluated the antioxidant effect of natural antioxidants such as thymol. They assessed the capacity of these compounds for inhibiting peroxidation of membrane lipids as well as reaction with trichloromethyl peroxyl radical and bleomycin-dependent DNA damage. Results of this study showed that thymol inhibited phospholipid liposome peroxidation, but its effect was less than synthetic antioxidants. In addition, thymol and carvacrol can scavenge proxyl radicals and likely have no pro-oxidant effects, which can lead to DNA damage. The antioxidant feature of thymol and carvacrol at different concentrations and time of autoxidation for triacylglycerols of lard and sunflower oil also demonstrated that thymol had higher antioxidant activity than carvacrol (Yanishlieva et al., 1999). It has been shown that thymol on the basis of concentration and time has different effects on parental and drug-resistant human lung cancer cell lines. Incubation Copyright © 2014 John Wiley & Sons, Ltd.
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of cells with lower concentrations (
REVIEW
Antiinflammatory, Antioxidant, and Immunological Effects of Carum copticum L. and Some of Its Constituents Azam Alavinezhad and Mohammad Hossein Boskabady* Neurogenic Inflammation Research Centre, Department of Physiology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
Carum copticum L. has been used traditionally for its various therapeutic effects. The plant contains various components such as thymol and carvacrol. Different therapeutic effects such as antifungal, antioxidant, antibacterial, antiparasitic, and antilipidemic were described for the plant and its constituents. Therefore, antiinflammatory, antioxidant, and immunological effects of C. copticum and its constituents, thymol and carvacrol, were discussed in the present review. Previous studies have shown potent antiinflammatory, antioxidant, and immunological effects for C. copticum and its constituents, thymol and carvacrol. Therefore, the plant and its constituents have therapeutic values in several inflammatory and immunological disorders as well as in the oxidative stress conditions. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: Carum copticum; thymol; carvacrol; antiinflammatory; antioxidant; immunological effect.
INTRODUCTION Herbal medicines have been used for the prevention and the treatment of chronic disorders such as inflammatory diseases, diabetes, allergy, arthritis, and other similar conditions for a long time. Carum copticum is a grassy plant that belongs to the Apiaceae (Umbelliferae) family with white flowers and grayish seeds (Fig. 1) (Dwivedi et al., 2012). C. copticum is known as Ajowan, with other names in different languages including Trachyspermum copticum, Ammi copticum L., bishop’s weed, Ethiopian caraway, kummel, ajawa, ajwain, and ajamoda (Bairwa et al., 2012). This plant may increase body resistance and has antiinflammatory and antispasmodic properties (Thangam and Dhananjayan, 2003). The relaxant effect of C. copticum and its constituent, carvacrol on tracheal smooth muscle (Boskabady et al., 2003; Boskabady et al., 1998; Boskabady and Jandaghi, 2003), and its possible mechanism including anticholinergic effect, stimulatory effect on the β-adrenergic, and inhibitory effect on histamine receptors were previously demonstrated (Boskabady et al., 2011a, 2011b; Boskabady and Shaikhi, 2000; Boskabady et al., 2011a, 2011b; Boskabady and Moemeni., 2000; Boskabady and Krachian, 2000; Boskabady et al., 2012). Furthermore, bronchodilatory effect of the plant extract on asthmatic patients was also shown (Boskabady et al., 2007). The antitussive effect of C. copticum was also evaluated, which was even greater than codeine at concentrations used (Boskabady et al., 2005).
Carum copticum oil has antifungal, antimicrobial, and antiparasitic effects (Kavoosi et al., 2013), and extracts of this plant showed the highest antioxidant activity among 125 herbs (Huang et al., 2011). Its fruit has stimulant, antispasmodic, and carminative properties, and it is an important therapeutic agent in indigestion and diarrhea (Bentely and Trimen, 1983). Thymol is an aromatic substance with antimicrobial (Singh and Singh, 2000), antispasmodic (cramps), and antiparasitic (Sivropoulou et al., 1996) properties. It is used for the treatment of gastrointestinal ailments, loss of appetite, and respiratory problems (Cortés-Arroyo et al., 2011). With regard to the wide usage of C. copticum in Iranian traditional medicine and because of several studies indicating antiinflammatory, antioxidant, and immunological effects of the plant and its main constituents, thymol and carvacrol, these effects of C. copticum and the two constituents of the plant were reviewed in the present article. These effects of the plant and its constituents are therapeutically important and scientifically valuable to review. The other plants containing thymol and carvacrol, which have antiinflammatory, antioxidant, and immunological effects, were also included in this review, which supports the mentioned effect for thymol and carvacrol.
CHEMICAL COMPOSITION * Correspondence to: Mohammad Hossein Boskabady, Neurogenic Inflammation Research Centre, Department of Physiology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran. E-mail: [email protected]
Copyright © 2014 John Wiley & Sons, Ltd.
The main components of C. copticum oil are thymol (known as 2-isopropyl-5-methylphenol, C10H14O) (Fig. 2a) and glycosyl constituents such as carvacrol or isothymol (know as 5-isopropyl-2-methyl phenol, Received 12 March 2014 Revised 25 June 2014 Accepted 25 June 2014
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Figure 1. Flowers and seeds of Carum copticum.
C10H14O), (Fig. 2b), 2-O-β-D-glucopyranoside, 3,5dihydroxytoluene, and 3-O-β-D-galactopyranoside (Ishikawa et al., 2001; Zahin et al., 2010). The other constituents of C. copticum oil are γ-terpinene, β-pinene, o-cymene, and p-cymene (Zarshenas et al., 2014; Kazemi et al., 2011; Khajeh et al., 2004; Singh et al., 2004). However, acetone extract of C. copticum had different percentages of some constituents from the total extract (68.8%) such as β-pinene (0.2%), p-cymene (1.6%), γ-terpinene (2.6%), thymol (39. 1%), and carvacrol (0.3%), (Singh et al., 2004). A study in five different areas of Algeria showed that thymol content is higher in the plants that grow in areas near the sea with low altitudes (Bekhechi et al., 2010). High concentration of thymol in C. copticum oil had been determined by gas chromatography (Gersbach and Reddy, 2002). The effect of cultivation situations on the amount of Iranian C. copticum constituents is shown in Table 1 (Zarshenas et al., 2014; Singh et al., 2004; Naseri et al., 2007). It has been also shown that this plant has other constituents including carbohydrates, fat, protein, fiber, tannins, glycosides, moisture, saponins, flavone, and minerals (Bairwa et al., 2012).
ANTIINFLAMMATORY EFFECTS The antiinflammatory effect of C. copticum has been reported in several studies. Significant inhibitory effects of total alcoholic extract (TAE) and total aqueous extract (TAQ) of C. copticum on both acute (carrageenan-induced paw edema in rat) and sub-acute (cotton-pellet-induced granuloma) rat models of inflammation have been demonstrated, which were comparable with those of aspirin and phenyl butazone. TAQ and TAE can affect synthesis of kinnin, prostaglandin, bradykinin, and lysozyme, which is suggested to contribute in the antiinflammatory effect of the plant (Thangam and Dhananjayan, 2003). Rheumatoid arthritis is an autoimmune disease in which reactive oxygen species (ROS) as well as reactive
(a)
(b)
Figure 2. Molecular shape of (a) thymol and (b) carvacrol. Copyright © 2014 John Wiley & Sons, Ltd.
nitrogen species have likely an important role in its pathogenesis (Fachini-Queiroz et al., 2012). The effect of C. copticum on collagen-induced arthritis in rats was evaluated by measuring elastase (marker for collagen degradation), catalase (CAT), reduced glutathione (GSH), superoxide dismutase (SOD) (Bairwa et al., 2012), and nitric oxide (NO). Administrations of the C. copticum extract resulted in reduction of elastase and subsequent decrease in neutrophil infiltration and activation. The plant inhibited lipid peroxidation and retrieved GSH and SOD levels in tissue. Treatment with C. copticum extract decreased NO level, which increases during rheumatoid arthritis. In terms of histology, articular tissue was recovered, which may be due to the inhibitory effect on infiltrating of inflammatory cells and restoration of antioxidants. It was concluded that the antiinflammatory effect of the C. copticum extract may be due to the existence of flavonoids and phenols (Mahboubi and Kazempour, 2011). In a clinical trial, herbal drops of several plants including C. copticum were used for few eye disorders. These herbal drops were able to treat eye infection and inflammation in studied patients (Biswas et al., 2001). In vitro method for testing antiadhesion of platelets in the blood of human volunteers showed that blood platelet aggregation was inhibited by C. copticum extract. The possible mechanisms of these findings are perhaps due to the effect of the plant on thromboxane production in platelets. The aforementioned study confirmed the traditional use of C. copticum in postpartum period (Srivastava, 1988). Thymus vulgaris essential oil containing thymol as the main component has potent antioxidant and antiinflammatory effects. In human acute monocytic leukemia (THP-1) cell line, T. vulgaris suppresses 5-lipoxygenase (5-LOX) activity more than α-bisabolol (known as having an antiinflammatory effect) and has an inhibitory effect on TNF-α and IL-8 secretion in lipopolysaccharide (LPS)-treated THP-1 cell line. Therefore, T. vulgaris has antiinflammatory properties because of the 5-LOX inhibition and reduction of cytokines such as TNF-α, IL-8, and IL-1β (Tsai et al., 2011). In an in vitro model of macrophage inflammation induced by LPS and interferon gamma (IFN-γ) that led to an increased NO production, T. vulgaris treatment inhibited inducible nitric oxide synthase mRNA expression and has NO scavenging property (Vigo et al., 2004). Thyme extract oils (from T. vulgaris, Thymus zygis, and Thymus hyemalis) also contain bioactive components including thymol and carvacrol. In a study conducted by Ocaña and Reglero (2012), the effect of Phytother. Res. 28: 1739–1748 (2014)
GC/FID GC/FID GC/FID GC/FID GC/FID GC/FID GC/FID GC/FID GC/FID GC/FID GC/FID GC/FID Mashhad Tehran South of Shiraz Northwest of Shiraz Zahedan Kerman Torbat-e-heydaryeh Darab Bandar-abbas Meymand Gorakhpur Fars Province Myrcene (1.32%) Myrcene (0.8%) Carvacrol (0.43%) Carvacrol (0.65%) Limonene (1.32%) Myrcene (0.92%) Thujene (1.51%) α-Pinen (1.31%) β-phellandrene (0.65%) β-Pinen (0.99%) Carvacrol (0.3%) Carvacrol (0.45%) β-Pinen (3.46%) Thymol (1.5%) β-Pinen (1.8%) β-Pinen (1.5%) β-Pinen (2.58%) β-Pinen (3.4%) β-Pinen (3.7%) β-Pinen (3.64%) β-Pinen (1.23%) Carvacrol (1.01%) β-Pinen (1.7%) Thymol dihydroquinone (0.96%)
Copyright © 2014 John Wiley & Sons, Ltd.
GC/FID, gas chromatograph/flame ionization detector.
Thymol (21.29%) β-Pinen (3.04%) γ-Terpinene (19.21%) γ-Terpinene (16.98%) p-Cymene (20.73%) γ-Terpinene (21.31%) γ-Terpinene (23.11%) Thymol (19.58%) γ-Terpinene (12.93%) Thymol (20.62%) γ-Terpinene (23.2%) Hexadecanoic acid (9.69%) γ-Terpinene (32.54%) γ-Terpinene (35.12%) p-Cymene (19.67%) p-Cymene (22.38%) γ-Terpinene (21.71%) Thymol (30.4%) p-Cymene (31.8%) γ-Terpinene (28.04%) p-Cymene (19.15%) p-Cymene (34.64%) p-cymene (30.8%) Linoleic acid (13.56%) p-Cymene (37.3%) p-Cymene (57.31%) Thymol (55.21%) Thymol (53.66%) Thymol (49.16%) p-Cymene (40.2%) Thymol (35.04%) p-Cymene (43.3%) Thymol (63.31%) γ-Terpinene (37.43%) Thymol (39.1%) Thymol (64.59%)
Main constituent
Table 1. Chemical constituents of Carum copticum essential oil in different geologic areas (Zarshenas et al., 2014; Singh et al., 2004; Naseri et al., 2007)
Cultivation location
Method
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thyme extract oils on activated THP-1 macrophage by oxidized low-density lipoproteins was assessed. In this study, the extract oils diminished TNF-α, IL-1β, and IL-6 genes expression but increased IL-10 gene expression. Therefore, the extracted oils may be helpful for the treatment of inflammatory diseases. In a cellular model of atherosclerosis (a chronic inflammatory disease), the effect of Origanum vulgare, which contains sabinene hydrate, thymol, and carvacrol, on THP-1 macrophages and levels of pro-inflammatory and antiinflammatory cytokines was evaluated. The plant extract decreased TNF-α, IL-1β, and IL-6; however, IL-10 as an antiinflammatory cytokine was increased, which inhibited translocation of NF-κB into the nucleus (Ocana-Fuentes et al., 2010). Because the pro-inflammatory cytokines such as IL-1, IL-6, IL-8, TNF-α, IL- 12, and IFN-γ increased in intestinal inflammation, in a study, the effect of T. vulgaris L. and Origanum syriacum L. on colitis was examined by measuring the expression of cytokines or cytokine mRNA level. The results showed that a medium dose of these plants can suppress the expression of the aforementioned cytokines and thus showing an antiinflammatory effect (Bukovská et al., 2007). Elastase is an inflammatory marker released by neutrophils involved in inflammatory disorder and elastase inhibitor can protect tissue against inflammation. It was shown that thymol can inhibit calcium channels and subsequently impeding release of human neutrophil elastase (Braga et al., 2006). Thymol contributes to improving the symptoms of arthritis by inhibiting calcium channels and consequently inactivating elastase (Umar et al., 2012). It was shown that thymol can inhibit cyclooxygenase (COX-1) enzyme (an enzyme of prostaglandin E2 synthase) in an in vitro study, indicating its antiinflammatory effects (Marsik et al., 2005). Ku and Lin (2013) assessed the effect of 27 selected terpenoid compounds such as thymol on Th1/Th2 balance in murine primary splenocytes. They measured IL-2, IFN-γ (Th1 cytokines), IL-4, IL-5, and IL-10 (Th2 cytokines) in terpenoid-treated splenocytes. Their results showed that terpenoids have antiinflammatory nature via inhibition of T cells immune response and improved Th1/Th2 ratio. Aqueous and ethanol extracts of Zataria multiflora, which contain thymol and carvacrol, showed antiinflammatory effects against acute and chronic inflammation. Total extract, flavonoid, and the essential oil of Z. multiflora also showed ameliorative effect on carrageen-induced paw edema in rat (Sajed et al., 2013). Veras et al. (2013) showed that essential oil of Lippia sidoides, which contains thymol (84.9%), had a topical antiinflammatory effect on acute ear inflammation induced by arachidonic acid and phenol. They demonstrated that possibly the thymol content of the essential oil has an inhibitory effect on lipooxygenase, COX, and phospholipase A2. Carvacrol (5-isopropyl-2-methylphenol) is a phenolic monoterpene and one of the main components of C. copticum in which its antiinflammatory property in several studies has been reported. The capacity of Thymus herba-barona essential oil containing phenols, carvacrol (54.0%), and thymol (30.2%) for prevention of NO production as inflammatory marker of LPS-stimulated macrophages was assessed. Results Phytother. Res. 28: 1739–1748 (2014)
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showed that the lowest concentrations of T. herbabarona (0.08 μL/mL) did not inhibit NO production in cells exposed to LPS. However, its higher concentration (0.64 μL/mL) decreased nitrite production as compared with the non-LPS-treated group (Zuzarte et al., 2013). The presence of carvacrol in thyme oils can affect expression of COX-2 mRNA and protein and has antiinflammatory effects. Thyme extract can also reduce COX-2 activation up to almost 75%, and its main component, carvacrol, decreased COX-2 level up to 80% (Hotta et al., 2010). Carvacrol is also able to reduce prostaglandin E2 and inflammatory cytokines such as IL-1β by stimulation of IL-10 release (the antiinflammatory cytokine) (Lima et al., 2012). In the presence of stress signals, carvacrol acts as co-inducer of heat shock protein-70 (HSP-70) expression in mammalian cells and thus can reduce the severity of inflammation (Van et al., 2010). Liu et al. (2012) induced in vitro inflammation on porcine alveolar macrophages (PAM) with LPS and assessed the effect of carvacrol on TNF-α, IL-1β, TGF-β, and IL-10 levels in the cell culture supernatants of macrophages. The results showed that carvacrol suppressed TNF-α, TGF-β, and IL-1β. On the basis of this study results, the suppression of TNF-α accounted for the antiinflammatory effect of carvacrol. In paw edema and gastric lesions animal models, carvacrol was able to decrease inflammation and alleviate edema and lesions with effect on secretion or synthesis of various inflammatory mediators (Silva et al., 2012). The effect of carvacrol in hepatotoxicity induction in rat with D-galactosamine and in the measurement of mRNA and proteins expression such as nitric oxide synthase, COX-2, TNF-α, IL-6, and NF-κB was investigated. Expression of all proteins was downregulated by carvacrol, indicating its antiinflammatory effect (Aristatile et al., 2013). The antiinflammatory effect of thymol and carvacrol was also examined by measurement of exudates volume in the pleural cavity and leukocyte migration after carrageenan injection into the pleural cavity of rats. The results showed that thymol and carvacrol decreased exudates volume. In addition, carvacrol reduced leukocyte migration (in vivo and in vitro) with inhibitory effect on COX-1, 2, and 5-lipoxygenase, which reduced prostaglandins, leukotrienes, and inflammatory mediators involved in edema. The results also showed that thymol did not decrease leukocyte migration and had an irritative effect, which may be due to histamine and prostanoid release from the tissues (Fachini-Queiroz et al., 2012). Another study showed that increased white blood cell (WBC) by Escherichia coli infection was decreased with administration of carvacrol, which also led to the reduction of neutrophil and lymphocyte percentage. The effect of carvacrol was also examined on PAM in vitro. In this study, PAM treated with LPS caused increased TNF-α and IL-1β secretion. Carvacrol added to LPS-treated PAM inhibited secretion of TNF-α and IL-1β. However, the effect of carvacrol on PAM in the absence of LPS was opposite as TNF-α and IL-1β increased because these cytokines are beneficial for body protection (Yanhong, 2011). The effect of carvacrol on serum endothelin, total protein, histamine, and NO levels of guinea-pigs sensitized with ovalbumin as well as its effect on tracheal Copyright © 2014 John Wiley & Sons, Ltd.
responsiveness, lung pathological changes, and total and differential WBC count was also examined. The results of these series of studies showed preventive effect of carvacrol on serum endothelin, total protein, histamine, and NO levels in ovalbumin-sensitized animals. Treatment of sensitized animals with carvacrol also lead to reduction in tracheal responsiveness to both methacholine and ovalbumin, lung pathological changes, changes in the total and differential WBC counts specially eosinophil count, which was comparable with the effect of dexamethasone (Boskabady and Jalali, 2013). Therefore, all plants containing carvacrol such as C. copticum could be effective as a preventive drug in inflammatory disorders such as asthma. Romano et al. (2013) studied the effects of phytocannabinoid on murine peritoneal macrophages in vitro. They demonstrated that carvacrol decreased nitrite value in LPS-stimulated macrophages, which indicates the antiinflammatory effect of carvacrol. Table 2 summarized various antiinflammatory effects of C. copticum and its constituents thymol and carvacrol.
IMMUNOLOGICAL EFFECTS There is not any report regarding the immunological effects of C. copticum. However, different herbs are known to modulate the immune system such as plants of the Apiaceae family including C. cyminum and C. caraway, which contain thymol. The effect of C. cyminum on the immune system was demonstrated by Chauhan et al. (2010). They used cyclosporine-A for suppressing immunity in Swiss albino mice and then orally fed the mice with cumulative doses of C. cyminum. Their results showed that administration of this plant caused increased CD4, CD8, and Th1 cytokines expression. This effect is possibly due to enhancement of IFN-γ expression and induction of IL-12 secretion, which has a main role in cellular immunity. It also increased the weight of the spleen and thymus and caused a reduction in adrenal gland size, which reduced serum corticosterone levels (Chauhan et al., 2010). Khajeali et al. (2012) added different doses of Carum carvi L., a member of the Carum genus (Apiaceae family) that contains thymol, to the food of broiler chickens and measured antibody titers against Newcastle disease virus. The results showed significantly higher antibody titers in groups receiving higher doses of caraway seeds powder. The effects of thymol feed supplementation on the immune response were investigated in broiler chickens. This component modified the immune response by increasing hypersensitivity response and IgG but reducing neutrophil to lymphocyte ratio (Hashemipour et al., 2013). In a study, thymol feed supplementation showed beneficial effects on innate immune conditions (Giannenas et al., 2012). A 1% thymol diet in weaning pigs challenged with Salmonella typhimurium reduced serum IgA. S. typhimurium increased IgM but did not affect IgM concentration in thymol-treated group, although thymol in the non-challenged group enhanced serum levels of IgA and IgM (Trevisi et al., 2010). Ariza-Nieto et al. (2011) assessed the effect of sow diets supplementation with oregano essential oil Phytother. Res. 28: 1739–1748 (2014)
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Table 2. Antiinflammatory effect of Carum copticum and its constituents thymol and carvacrol
Carum copticum
Thymol
Carvacrol
Effect
Reference
Effect on synthesis of kinnin, prostaglandin, and bradykinin Inhibitory effect on infiltrating of inflammatory cells Treatment of eye infection and inflammation Antiadhesion of platelets Improvement of arthritis Inhibition of 5-LOX and decrease of cytokines secretion Inhibition of inducible nitric oxide synthase (iNOS) mRNA expression and has NO scavenging property Decrease of TNF-α, IL-1β, and IL-6 genes expression and increase of IL-10 gene expression Inhibition of translocation NF-kB Suppression of pro-inflammatory cytokines expression Inhibitory effect on release of elastase Inhibition of cyclooxygenase (COX-1) Improvement of Th1/Th2 ratio Decrease of nitrite production Reduction of COX-2 activation Co-inducer of HSP-70 expression Stimulation of IL-10 release Suppression of TNF-α Alleviation of edema and lesions Downregulation of iNOS, COX-2, TNF-α, IL-6, and NF-κB expression Reduction of leukocyte migration and exudates volume Modulation of TNF-α and IL-1β secretion Reduction in tracheal responsiveness of sensitized animals
(Thangam and Dhananjayan, 2003) (Mahboubi and Kazempour, 2011) (Biswas et al., 2001) (Srivastava, 1988) (Umar et al., 2012) (Tsai et al., 2011) (Vigo et al., 2004)
(containing thymol and carvacrol) on the immune response of suckling pigs. Because IgG participates in secondary antibody response and IgA has a role in mucosal immunity and T lymphocytes are responsible for acquired immunity, total IgG and IgA concentrations were measured for evaluation of humoral immunity. This diet did not affect serum immunoglobulin levels and T lymphocyte number and killer cells activity in supplementing sow diets. The absence of immune response may be due to the non-specific immunomodulatory agents (e.g., corticosteroids) in colostrums. Thymol is capable of increasing the percentage of CD4+, CD8+, and MHC-II in the peripheral blood of low-weight growing-finishing pigs. It can also decrease the expression of TNF-α in the stomach of post-weaned pigs (Taranu et al., 2012). Farinacci et al. (2008) investigated the effects of T. vulgaris (containing a considerable amount of thymol) on isolated ovine neutrophils. In this study, adhesion and superoxide production were examined in activated neutrophils by phorbol myristate acetate and incubated with extracts. Results showed that hydroethanol extract of T. vulgaris inhibited adhesion and superoxide production. These effects may be due to the presence of flavonoids and phenolic compounds, such as thymol and carvacrol. T. vulgaris had also an antiinflammatory activity on sheep neutrophils. In a study, the effect of adding the essential oil of plants containing thymol to food of weaner pigs on their gut immunoglobulins (IgG, IgA, and IgM) was examined. The results showed increased IgA and IgM levels as well as phagocytosis rates. Therefore, essential oil containing thymol improved immune function in pigs’ gut (Li et al., 2012). Copyright © 2014 John Wiley & Sons, Ltd.
(Ocaña and Reglero, 2012) (Ocana-Fuentes et al., 2010) (Bukovská et al., 2007) (Braga et al., 2006) (Marsik et al., 2005) (Ku and Lin, 2013) (Zuzarte et al., 2013) (Hotta et al., 2010) (Van et al., 2010) (Lima et al., 2012) (Liu et al., 2012) (Silva et al., 2012) (Aristatile et al., 2013) (Fachini-Queiroz et al., 2012) (Yanhong, 2011) (Boskabady and Jalali, 2013)
Amirghofran et al. (2011) evaluated the effect of a few plants including T. vulgaris and Z. multiflora and their major compound thymol on the proliferation of peripheral blood lymphocytes stimulated by phytohaemagglutinin (PHA) in vitro. Results demonstrated that these plants prevent lymphocytes proliferation in vitro, which is likely due to the thymol content. Thus, these plants and thymol may induce the immunosuppressive effects. In a study, in experimental animals (rabbit), Candida albicans antigen was injected 5 days after the last subcutaneous injection of Z. multiflora. Results showed that innate and cellular immunity responses were significantly stimulated compared with the control group (Khosravi et al., 2007). In another study, Z. multiflora essential oil was orally administrated to common carp under thermal stress. This plant increased total WBC and bactericidal activity of serum in treated groups (Soltani et al., 2010). Carvacrol can act as a treatment of immune system disorders. On the basis of the study of Spiering et al. (2012), injection of carvacrol with thermal stress may suppress autoimmune arthritis in a mouse model induced by human proteoglycan. Treatment of dendritic cells isolated from the bone marrow of mouse with carvacrol and thermal stress reduced pro-inflammatory cytokines and antiinflammatory cytokines, which is likely to be due to the production of tolerogenic dendritic cells and their effect on regulatory T cells; these cells, in turn, can adjust the immune system response in pathologic conditions. Carvacrol also showed beneficial effects on innate immune conditions (Giannenas et al., 2012). Because Th1/Th2 balance plays an important role in many diseases such as asthma, the impact of Z. multiflora, in which carvacrol is its main component, Phytother. Res. 28: 1739–1748 (2014)
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on in vitro and in vivo Th1/Th2 balance (IFN-γ/IL4 ratio) was studied. The in vitro study showed that higher concentration of the extract (200 μg/mL) increased IFN-γ gene expression in phytohaemagglutininstimulated human peripheral blood mononuclear cells, whereas all concentrations of the extract decreased IL-4 gene expression and production. The extract also significantly increased the ratio of IFN-γ to IL-4 (gene expression and production), which indicates increased Th1/Th2 balance. In sensitized guinea-pigs, also enhancement in IL-4 and reduction in IFN-γ serum levels were seen. However, in sensitized guinea-pigs treated with the extract of Z. multiflora, the serum level of IL-4 was decreased, whereas the level of IFN-γ and ratio of IFN-γ to IL-4 were increased (Boskabady et al., 2013). The effect of carvacrol on Th2 cytokine, IL-4, and Th1 cytokine, IFN-γ, was also assessed in ovalbuminsensitized guinea-pigs. IL-4 level decreased and IFN-γ increased by high concentration of carvacrol indicating enhancement of Th1/Th2 balance toward Th1, which is a beneficial effect in the improvement of inflammatory disorders associated with decreased Th1/Th2 balance (Jalali et al., 2013). Intracellular proteins including HSP have a protective role against inflammation with an important role in HSP-specific regulatory T cells. Several factors such as immune disorder or aging can decrease or alter the function of these proteins. Burt et al. (2007) examined the effect of carvacrol on protein synthesis in E. coli cells. In their study, it was shown that carvacrol increased the HSP-60 in prokaryotes. Carvacrol also increased FOXP3 expression, which is a marker of regulatory T cells, and thus, it is beneficial for the modulation of the immune system. Carvacrol also affects the immune response by reducing neutrophil to lymphocyte ratio, which was similar to the effect thymol (Hashemipour et al., 2013).
Khosravi et al. (2009) used Z. multiflora essential oil (containing 61.29% carvacrol and 25.18% thymol) in mice infected with C. albicans. According to their results, the colony-forming unit of Candida in the liver, spleen, lungs, brain, and kidneys reduced in the treated group compare with the control group, which likely reflects stimulation of the immune system by this plant. Table 3 summarized the different immunological effects of C. copticum and its constituents thymol and carvacrol.
ANTIOXIDANT PROPERTIES Many plants possess antioxidant property that depends on the species of plant, climate condition, geolocation, and other factors such as the steps of harvesting and processing. The composition and value of phenolic compounds are very important in the antioxidant activity of plants too (Skrovankova et al., 2012). Because ROS and reactive nitrogen species are involved in aging and in the pathogenesis of many diseases such as cancer and heart disease, various plant such as C. copticum are known to have antioxidant effects and can be beneficial in the improvement of such diseases (Fachini-Queiroz et al., 2012). The antioxidant potential of C. copticum fruit extract and its fractions was examined by 1,1-diphenyl-2picrylhydrazyl (DPPH) free radical scavenging assay, and total antioxidant capacity (TAC) was examined by phosphomolybdenum method. Data of this study elucidated that methanol extract had 90.2% activity for scavenging of free radicals and was comparable with 90.3% activity of ascorbic acid (Zahin et al., 2010). In another study, the antioxidant activity of the C. copticum essential oil was assessed by comparison of ferric reducing antioxidant power, activity of DPPH,
Table 3. Immunological effects of Carum copticum and its constituents thymol and carvacrol Effect Cuminum cyminum (Apiaceae family) Carum carvi L. (Apiaceae family) Thymol
Carvacrol
Reference
Increase of CD4, CD8, and Th1 cytokines expression and spleen and thymus weight Increase of antibody titers against Newcastle disease virus
(Chauhan et al., 2010)
Beneficial effects on innate immune conditions Effect on concentration of IgM and IgA No effect on serum immunoglobulin levels and T lymphocyte number and killer cells activity + + Increase percentage of CD4 , CD8 , and MHC-II Inhibition of adhesion and superoxide production on isolated ovine neutrophils Improvement of immune function in pigs’ gut Induce immunosuppressive effect Modifying immune response Suppression of autoimmune arthritis Beneficial effects on innate immune conditions Modulation of Th1/Th2 ratio Beneficial effect in improvement of inflammatory disorders Increase of FOXP3 expression (marker of regulatory T cells) and modulation of immune system Modifying immune response
(Giannenas et al., 2012) (Trevisi et al., 2010) (Ariza-Nieto et al., 2011)
Copyright © 2014 John Wiley & Sons, Ltd.
(Khajeali et al., 2012)
(Taranu et al., 2012) (Farinacci et al., 2008) (Li et al., 2012) (Amirghofran et al., 2011) (Hashemipour et al., 2013) (Spiering et al., 2012) (Giannenas et al., 2012) (Boskabady et al., 2013) (Jalali et al., 2013) (Burt et al., 2007) (Hashemipour et al., 2013) Phytother. Res. 28: 1739–1748 (2014)
ANTIINFLAMMATORY EFFECTS OF C. COPTICUM AND ITS CONSTITUENTS
and H 2O2 radical scavenging with ascorbic acid. Results of this study showed that C. copticum can be applied in foodstuff and can be used for medicinal purposes as non-synthetic antioxidant agent (Chatterjee et al., 2013). The effect of C. copticum on arthritis model in Wistar rats induced by intradermal injection of collagen was examined. In this model, enhanced levels of oxidative stress markers, namely thiobarbituric acid reactive substance and diminished GSH, SOD and CAT were observed. However, treatment with C. copticum restored GSH and decelerated the deterioration of SOD activity and CAT level. The increase of nitrite concentration was also reduced in the group treated with C. copticum. On the basis of the results of this study, C. copticum has antioxidant properties that might be due to flavonoids and phenols content of the plant (Umar et al., 2012). In one study, the extract of C. copticum was used as natural antioxidant in flaxseed and Bahera oils. The amount of peroxide oxygen per kilogram of oil was measured in different periods and different temperatures without and with antioxidant (the extract of C. copticum). Data showed the peroxide value of oil in the presence of the extract of C. copticum significantly decreased in longer time and higher temperature, which indicates a strong antioxidant effect of the extract of C. copticum (Bera et al., 2004). It was also shown that C. copticum is able to eliminate free radicals and enhance the body’s functions, which is perhaps due to its phenolic groups content (Kavoosi et al., 2013). Methanol extract of C. copticum (MCE) with concentration of 50 μg/mL also decreased free radical up to 89.3% compared with ascorbic acid (92.3%), which demonstrates a high antioxidant activity of C. copticum. Effect of pretreatment of MCE against potassium dichromate (K2Cr2O7) in human bronchial epithelial cells (BEAS-2B) and human peripheral blood lymphocytes also showed that C. copticum helps to keep antioxidant enzyme levels such as SOD and GSHPx. In other words, MCE showed a significant reduction effect in ROS levels created by K2Cr2O7. This effect is probably due to phenolic terpenoid compounds in MCE including thymol, which is its most effective constituent (Deb et al., 2011). The phenolic content of the plant has direct connection with its antioxidant activity. Because lipids in food products are oxidized, food factories use synthetic antioxidants for the food preservation. Therefore, Aeschbach et al. (1994) evaluated the antioxidant effect of natural antioxidants such as thymol. They assessed the capacity of these compounds for inhibiting peroxidation of membrane lipids as well as reaction with trichloromethyl peroxyl radical and bleomycin-dependent DNA damage. Results of this study showed that thymol inhibited phospholipid liposome peroxidation, but its effect was less than synthetic antioxidants. In addition, thymol and carvacrol can scavenge proxyl radicals and likely have no pro-oxidant effects, which can lead to DNA damage. The antioxidant feature of thymol and carvacrol at different concentrations and time of autoxidation for triacylglycerols of lard and sunflower oil also demonstrated that thymol had higher antioxidant activity than carvacrol (Yanishlieva et al., 1999). It has been shown that thymol on the basis of concentration and time has different effects on parental and drug-resistant human lung cancer cell lines. Incubation Copyright © 2014 John Wiley & Sons, Ltd.
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of cells with lower concentrations (
