Total Phenolic Content, Antioxidant Activity and Antifungal Property in Two Parts of Garden Thyme Shoot

Mohammad Mehdi Sabetsarvestani1, Shahram Sharafzadeh2*, Ardalan Alizadeh3, Abbas Ali Rezaeian4

1. Department of Horticulture, Jahrom Branch, Islamic Azad University, Jahrom, Iran 2. Department of Agriculture, Firoozabad Branch, Islamic Azad University, Firoozabad, Iran 3. Department of Horticulture (Biotechnology and Molecular Genetic of Horticultural Crops), Estahban Branch, Islamic Azad University, Estahban, Iran 4. Department of Microbiology, Jahrom Branch, Islamic Azad University, Jahrom, Iran

Corresponding author: Shahram Sharafzadeh

ABSTRACT: The experiment was conducted using the two years old thyme plants. Upper section of shoots were divided into two 10 cm parts (top and bottom). Methanolic extract was prepared for determination of total phenolic content and antioxidant activity. Hydrodistillation was used to isolate the essential oils. The antimicrobial activity of the oil was evaluated using agar disc diffusion and broth microdilution method against Candida albicans and Fusarium solani. Total phenolic contents were 19.65 and 19.06 mg GAE/g dw in top part and bottom part of shoots, respectively. Antioxidant activities, represent as IC50, were 8.12 and 8.23 μg/ml in top part and bottom part of shoots, respectively. Both microorganisms showed inhibition when tested by essential oils of garden thyme.

Keywords: Thymus vulgaris, Candida albicans, Fusarium solani, antioxidant activity, antifungal activity

INTRODUCTION

Thyme (Thymus vulgaris L.) belongs to the Lamiaceae family and is an aromatic and medicinal plant. Thyme essential oils has been reported to be among the top 10 essential oils, showing numerous effects such as antibacterial, antifungal and antioxidant activities (Jackson and Hay, 1994; Letchamo et al., 1995). It has a very important role in phytotherapy (Razic et al., 2003). Recently, thyme has become one of the most important medicinal plants used as a natural additive in poultry and livestock feeding studies (Hernandez et al., 2004; Bolukbasi et al., 2007). Such studies have shown that thyme plant could be considered as an alternative natural growth promoter for poultry instead of antibiotics (McDevitt et al., 2007). Thymol and carvacrol, which are the principal constituents of thyme oil (Atti-Santos et al., 2004; Goodner et al., 2006) have been reported to act as antioxidant (Dorman et al., 1995; Jukic and Milos, 2005; Kulisic et al., 2005), antimicrobial agent (Deans and Ritchie, 1987; Prabuseenivasan et al., 2006), antifungal agent (Klaric et al., 2007; Zyani et al., 2011), treatment for respiratory tract diseases (Inouye et al., 2001), wound healing, a stomachic carminative, diuretic and urinary disinfectant (Boskabady et al., 2006). Essential oils have been widely used for bactericidal, virucidal, fungicidal, antiparasitical, insecticidal, medicinal applications and food industries. They contain a variety of volatile molecules such as terpenes and terpenoids, phenol-derived aromatic components and aliphatic components (Bakkali et al., 2008). Intl J Farm & Alli Sci. Vol., 2 (22): 1017-1022, 2013

Vegetables contain antioxidant compounds called polyphenols that are known to reduce oxidative stress. The antioxidant properties of these compounds are responsible for their anticancer, antiviral and anti-inflammatory properties (Ninfali et al., 2005). Candida albicans is an opportunistic fungus responsible for a variety of human diseases manifestation ranging from superficial skin lesions to disseminate infections. It is the most common cause of fungal disease in humans (Schaberg et al., 1991). Fungi have long been recognized as causal agents of plant diseases such as fruit rot (Fusarium solani). Fusarium is one of the most important fungi causing spoilage of foodstuffs. Fungi are also responsible for off- flavour formation and production of allergenic compounds and mycotoxins which lead to qualitative losses (Ownagh et al., 2010). Chemical fungicides are known to be highly effective to control the postharvest diseases in various vegetables and fruits. However, they are not considered as long-term solutions due to the concerns associated with exposure risks, health and environmental hazards, residue persistence, and development of tolerance (Amzad Hossain et al., 2008 ). Essential oils as antimicrobial agents present two main characters: their natural origin generally means more safety to people and environment; and they can be considered at low risk for development of microbial resistance since they are mixtures of compounds which may present different mechanisms of antimicrobial activity (Karbin et al., 2009). The objective of this study was determination of total phenolic content, antioxidant activity and antifungal property of thyme extract and oil in two parts of the shoots.

MATERIALS AND METHODS

Plant materials and experimental conditions The study was conducted using the two years old thyme plants collected from experimental field of Islamic Azad University, Estahban Branch, Iran (29°632' N, 54°142' E; 1760 m above sea level). Plants were harvested at full bloom stage. Upper section of shoots were divided into two parts (top and bottom) with 10 cm in length and dried at room temperature. The experiment was carried out with three replicates. t-test was used for comparing the total phenolic content and antioxidant activity between upper and lower parts of shoots.

Extract preparation Methanolic extracts of the plants were prepared as follows: ground shade dried plant material (7.5 g) was weighed in a glass and then defatted with petroleum benzene for 3 hours. Each Sample was extracted during 24 hours at room temperature, twice, with 200 ml of 90% aqueous methanol. Each extraction was filtered through Whatman filter paper (Whatman Ltd., England). Supernatants were combined and evaporated to dryness using a rotary evaporator to a volume of about 1 ml. The concentrated extracts were freeze-dried and weighed to determine the yield. These samples were stored in refrigerator for further experiments regarding the total phenolic content and antioxidant activity.

Total phenolic content determination The amount of total phenolics in T. vulgaris extracts was determined with the Folin-Ciocalteu reagent using the method of (Singleton and Rossi, 1965). Briefly, 200 μl of the plant extract dissolved in methanol (1 mg/ml) was mixed with 2.5 ml of Folin-Ciocalteu reagent (diluted 10 times in distilled water) in glass tubes in triplicate. The samples were incubated at room temperature for 5 min and vortex mixed at least two times. Then, 2 ml of Na2CO3 (7.5%) was added and the glass tubes were incubated in the dark for 90 min with continuous shaking. The absorbance of samples were measured at 765 nm using a spectrophotometer (LABOMED. INC. UV/VIS double beam PC.UVD). The distilled water was used as a blank. Different concentrations of gallic acid in methanol were tested in parallel to obtain an standard curve. Total phenolic contents were expressed as milligrammes of gallic acid equivalent per gram of dry weight (mg GAE/g dw).

Free radical scavenging capacity The antioxidant activity was determined by DPPH free radical scavenging assay as described previously with some modifications (Brand-Williams et al., 1995). Briefly, 4 different concentrations (100, 200, 400 and 800 l) of the plant extract dissolved in methanol (10 mg extract in 10 ml methanol) were incubated with a methanolic solution of DPPH 100 M (3900, 3800, 3600 and 3200 l respectively) in a total volume of 4 ml. After 30 min of incubation

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at room temperature, the absorbance was recorded at 517 nm. Methanol was used as blank and all measurements were carried out in triplicate. Trolox, a water-soluble equivalent of vitamin E, and quercetin were used as reference compounds. All solutions were made daily. The percent inhibition of DPPH free radical was calculated by the formula:

Percentage inhibition (%I) = [(A blank – A sample) / A blank] × 100%

Where, A blank is the absorbance of the control reaction (DPPH alone), and A sample is the absorbance of DPPH solution in the presence of the test compound. IC50 values denote the concentration of the sample required to scavenge 50% of DPPH free radicals. IC50 was calculated from the linear regression algorithm of the graph plotted for inhibition percentage against extract concentration. All experiments were carried out in triplicate.

Essential oil isolation Isolation of essential oils was performed using hydrodistillation of dried shoots using a Clevenger-type apparatus over 3 hours. The oils were dried over sodium sulphate. Essential oils were used for determination of antifungal activities.

Determination of antifungal activity by disc diffusion method Standard strain of Candida albicans (ATCC No. 10231), and plant fungi Fusarium solani were obtained from Persian Type Culture Collection (PTCC) in Iranian Research Organization for Science and Technology. Antifungal activity of the essential oil of T. vulgaris was determined using disc diffusion method, according to the National Committee for Clinical Laboratory Standards (NCCLS, 2001) using 100 μl of each suspension of the tested microorganisms, containing 2.0 × 105 CFU/ml (0.5 McFarland) spores for fungi strain. The fungus inocula were prepared by suspending colonies from 48h old potato dextrose agar (PDA) cultures. Fungal suspension were seeded into petri dishes (9cm) containing 20 ml sterile potato dextrose agar (PDA) using a sterile cotton swab. The sterile paper discs (6 mm in diameter) were individually impregnated with 20 μl of the oil and then placed onto the agar plates which had previously been inoculated with the tested microorganisms. The plates were inoculated at 30 °C for 48 h. After incubation, the mean inhibition zone diameter for each concentration was measured in millimeters. All the studies were performed in triplicate. Blank discs containing 20μl DMSO were used as negative controls. Benomil (20 μg/disc), Ketoconazole (20 μg/disc) and Nystatin (30 μg/disc) were used as positive reference standards to determine the sensitivity of the microorganisms. The assessment results were resistant (lower than 7mm in diameter), dose-dependent (7-11mm in diameter) and sensitive (larger than 11mm in diameter).

Determination of the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) A broth microdilution method was used to determine the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) according to the National Committee for Clinical Laboratory Standards (NCCLS, 2001). The essential oil was dissolved in dimethylsulfoxide (DMSO) and diluted in a 2-fold manner to make the concentrations of 5, 2.5, 1.25, 0.625, 0.312 and 0.156 μl/disc. The potato dextrose agar (PDA) containing different amounts (logarithmic, serially and 2-fold diluted) of T. vulgaris oil and the various controls were inoculated with actively dividing microorganisms cells. The cultures were incubated for 48 h at 30°C. The MIC is defined as the lowest concentration of the essential oil at which the microorganism does not demonstrate visible growth. The minimum fungicidal concentration (MFC) is defined as the lowest concentration of the essential oil at which inoculated microorganisms were completely killed. MFC was determined by sub-culturing a 0.01 ml aliquot of the medium drawn from the culture tubes showing no macroscopic growth at the end of 48 h culture on PDA plates.

RESULTS AND DISCUSSION

The results indicated that total phenolic contents were 19.65 and 19.06 mg GAE/g dw in top part and bottom part of shoots, respectively. Antioxidant activities, represent as IC50, were 8.12 and 8.23 μg/ml in top part and bottom part of shoots, respectively (Table 1). Comparison of total phenolic content and antioxidant activity between top and bottom parts of shoots by t-test did not show significant differences. By decreasing IC50, antioxidant activity increase. Total phenolic content was slightly higher in top part of shoots resulted in slightly higher antioxidant activity (lower IC50).

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There is a high correlation between total phenolic content and antioxidant activity. On the other hand, antioxidant activity of plant extracts is not limited to phenolics. Activity may also come from the presence of other antioxidant secondary metabolites, such as volatile oils, carotenoids, and vitamins (Javanmardi et al., 2003). As a general result, both microorganisms showed inhibition when tested by essential oils of garden thyme. Minimum inhibitory concentration against the Candida albicans and Fusarium solani in every part of shoot was 1.56 μg/ml (Table 2). Antimicrobial activity of the standard antibiotics were shown in Table 3. Comparison of Table 2 and Table 3 indicated that essential oils of thyme can inhibit growth of C. albicans and F. solani in lower concentrations when compared to antibiotics. On the other hand, these substances have more safety for people and environment and at low risk for microbial resistance. Broth microdilution method showed that the minimum fungicidal concentration (MFC) was equal to the minimum inhibitory concentration (MIC).

Table 1. Total phenolic content and antioxidant activity of garden thyme extract Total phenolic content Antioxidant activity Part of shoot (mg GAE/g dw) IC50(μg/ml) Top part 19.65 8.12 Bottom part 19.06 8.23 t-test ns ns Each value in the table was obtained by calculating the average of three replicates. ns, not significant

Table 2. Antimicrobial activitiy of the essential oils of garden thyme in different concentrations Inhibition zone diameter (mm) Microorganism Part of shoot Essential oil concentration (μg/ml) 200 100 50 25 12.5 6.25 3.12 1.56 C. albicans Top part 77 68 60 50 43 32 20 17 C. albicans Bottom part 77 64 57 48 40 30 20 15 F. solani Top part 77 77 36 28 22 16 15 13 F. solani Bottom part 77 75 35 27 20 16 14 12 Each value in the table was obtained by calculating the average of three replicates. Diameter of inhibition zone including disc diameter of 6mm.

Table 3. Antimicrobial activity of the standard antibiotics. Inhibition zone diameter (mm) Benomil Ketoconazole Nystatin Microorganism (20μg/disc) (20 μg/disc) (30 μg/disc) Candida albicans na 32 25 Fusarium solani 23 na na Each value in the table was obtained by calculating the average of three replicates. na, not active.

Thymol and carvacrol, major components of thyme oil, can inhibit microbial activity and the other minor components may possess some antimicrobial activity or synergistic effects (Azaz et al., 2004; Souza et al., 2007). Researchers indicated that thyme oil provided irreversible damage to cell wall (degenerative changes), cytoplasm membrane (irregular, dissociated from cell wall, invaginated) and nuclear membrane (folding) of Aspergillus parasiticus (Rasooli and Owlia, 2005). Active antifungal substances from natural sources were reviewed by (Jose Abad et al., 2007). In this review they showed the antifungal effect on Candida albicans growth of the essential oils from several species of the Lamiaceae family. The greatest efficiency was obtained with the essential oil from the T.vulgaris thymol chemotype. The essential oils of Lavandula stoechas L. ssp. stoechas was effective on the inactivation of Fusarium oxysporum. A study indicated that minimum inhibitory concentration (μg/ml) of Thymus essential oils against Candida albicans were 31.25, 62.5, 31.25, 62.5 and 62.5 for Thymus zygioides var. lycaonicus, Thymus longicaulis subsp. 1020

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longicaulis var. subisophyllus, Thymus longicaulis subsp. chaubardii var. chaubardii (chemotype I), Thymus longicaulis subsp. chaubardii var. chaubardii (chemotype II) and Thymus pulvinatus, respectively (Azaz et al., 2004). In our study, minimum inhibitory concentration was 1.56 μg/ml. This shows that genotype and aroma profile of essential oils can alter the results. In an experiment, the essential oil of Melissa officinalis was analysed, and the main constituents were citronellal (25.2%), geraniol (16.4%) and citronellol (11.0%). The essential oils inhibited the growth of Candida albicans (MIC = 300 μg/ml) (Kedzia et al., 1994). This indicates that the essential oil constituents have an important role since in present study, MIC is lower than above result. The essential oils of Ocimum gratissimum was particularly active against Candida albicans. Oil of O. basilicum showed moderate antibacterial and antifungal effects. Thymol was the major constituent of the essential oil of O. gratissimum leaves. (Ndounga et al., 1997). An investigation introduced a formulation contained essential oil of Thymus against Candida albicans (Manou et al., 1998). Fourteen out of the 16 plants were found to be active against C. albicans tested with minimal inhibitory concentration (MIC) values ranging from 150 to 2300 μg/ml and the growth inhibition zone ranging from 16 to 55 mm using disc diffusion method. The essential oils of Zataria multiflora, Thymus kotschyanus, Cuminum cyminum and Plargonium graveolens showed significant activity against C. albicans (Naeini et al., 2009). The MIC in this experiment are higher than present study. Thymus pannonicus All. essential oil from Vojvodina province (north of Serbia at flowering stage was analysed. Geranial (41.42%, w/w) and neral (29.61%, w/w) were the major components. The maximum activity of T. Pannonicus oil was observed against C. albicans (MIC = 50 µg/ml) (Maksimovic et al., 2008). An investigation with Thymus kotschyanus, Agastache foeniculum, and Satureja hortensis showed that thyme oil was the most effective essential oil with the MIC of 62.5 μl/ml against F. solani (Ownagh et al., 2010). These two experiments show that differences between species can alter the results. In conclusion, the results indicate that essential oils have antioxidant and antifungal activities and could find a practical use in the food preservatives and inhibition of fungi growth.

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