Antioxidant and antimicrobial activities of Thymus vulgaris essential oil contained and synthesis thymus (Vulgaris) silver nanoparticles

Abstract Several species of thymus have therapeutic properties, so they are used in traditional medicine. In this work was carried out to synthesize Thymus vulgalis silver nanoparticles (TSNPS) and evaluate antioxidant and antimicrobial activities of TSNPS and T. vulgalis essential oil extract (TEOE). The essential oils analyzed by GC-MS and were characterized. Major compounds of phenol, 2 methyl 5 (1 methylethyle) (CAS), thymol and 1,2 Benzene dicarboxylic acid, 3 nitro (CAS) (48.75%, 32.42% and 8.12%, respectively) were detected. Results demonstrated that the TSNPS gave a highest DPPH radical scavenging activity, it was obtained 97.2 at 1000 ug/ml. TSNPS, Thymus + Hexane (T+H), Thymus + Ethanol (T+E) gave the greatest antimicrobial activity than amoxicillin (AM) and ciprofloxacin (CIP). In conclusion: The essential oil of thymus (Vulgaris) and thymus (Vulgaris) silver nanoparticles can be a good source of natural preservatives as an antioxidant and antimicrobial agents for increasing the shelf life of foodstuffs.


Introduction
There are 350 species world wide of the genus Thymus (Figure 1) which belongs to Lamiaceae (Labiatae) family.It is known for its large morphological and chemical variety.In addition, it has a various distinguished composition used widely in herbal medic ne products.The essential oils of different species of the Thymus genus have been lately inspected for their traditional uses and heavily investigated as potential source of anti-inflammatory, antibacterial, antioxidant, antifungal, sedative, anti aflatoxinogenic and antiviral , (Miura and Nakatani, 1989); (Katsiotis, et al., 2009).

Materials and Methods
Thyme (Vulgaris) leaves used in this work were collected from local markets, Dammam, Saudi Arabia.The leaves were washed by water and drying in the air for 5 days, then were kept in the hot air oven at 60 c for 24hrs.After that, they were ground to a fine powder.

Extraction of the essential oil extract
The oil was extracted by soaking in n-hexane at room temperature for 72 hours.The extract was filtered after 24 hours/3 days and evaporated to dryness.

Gas Chromatography-Mass Spectrometry (GC-MS) analysis
The instrument of GC/MSD Polaris Q (Thermo Finnigan, USA) was used to perform the analysis of GC-MS spectrometry.Briefly, capillary column (HP5MS) (thickness of film, 0.32mm; 30m × 0.25 mm i.d ) with program of temperature (40ºC initial oven temperature for 5min; gradient of 4ºC min-1 ramped to 200ºC and held for 5 min; gradient of 20ºC min-1 ramped to 280ºC and held for 10min were used.The temperature of injector-line was 260ºC, 10:1split ratio was applied.The flow rate of carrier gas (Helium) was 1.0 mL min-1.The temperature of interface was 280ºC.The electron ionization system (70eV) was used as GC-MS detection.30-350 amu mass range was used to record Totalion current chromatograms.The components were identified by comparing authentic standards retention times with their relative retention times, and mass spectra with NIST, WILEY GC-MS system library data and literature data (Mathe et al., 2004).

Silver nanoparticles synthesis
Silver nitrate (1 mM) was added to the extracts of plant separately to get 200 ml of final solution, then centrifuged for 25 min at 18,000 rpm.50 to 95ºC was used to heat the supernatants.Within 10-15 minutes during heating process the change in the color of the solution was observed.This changes in the color indicate that silver nanoparticles (SNPs) formation (Savithramma et al., 2011).

Transmission Electron Microscopy (TEM)
Transmission electron microscope was used to study the surface morphology and size of the nanoparticles by.(FEI, TEM, Czech Republic working accelerating voltage was 80Kv).

The activity of DPPH radical scavenging
The activity of Radical scavenging was determined by the method of Moreno et al. (2000), they used 2, be used for large-scale nanoparticles synthesis.For long time, silver has been known for its inhibitory effect toward several bacterial strains and micro-organisms usually exist in medical and industrial process (Krutyakov et al., 2008).Moreover, it has been studied with several pathogens such as cancer cells, arthropods vectors infections, cosmetics, medicine and food.Green chemistry suggests using plant for silver nanoparticles synthesis to avoid producing toxic byproduct.Plant mediated nanoparticles synthesis is a preferred method over other synthesis methods as it is friendly to the environment, cost effective and safer for human therapeutic use (Kumar and Yadav, 2009).
Antioxidants are very important as they prevent oxidative stress which could lead to many deteriorating diseases.Plants are considered the main source of antioxidants and the preservative effect of their spices and herbs indicates that there are anti-oxidative and antimicrobial components in their tissues (Javanmardi et al., 2003).The health-benefits of antioxidants from plants is believed to stem primarily from their protective effects by addressing reactive oxygen species that are believed to play an important role in various pathogens, premature aging, chronic diseases and oxidative degradation of cosmetics, foods, and pharmaceuticals (Kaur and Kapoor, 2001).Traditionally the thymus capitatus is considered an antioxidant.It has a key role in preventing several pathological diseases, such as cardiovascular, cancer, and neurodegenerative diseases, and is believed to be linked with oxidative stress (Losso et al., 2007).The synthetic antioxidants used in food industry are highly unstable and volatile which made their safety and effectiveness being often questioned (Sokmen et al., 2004).It is suspected that synthetic antioxidants such as butylated hydroxy anisole (BHA), tertiary butylhydroquinone (TBHQ), butylated hydroxytoluene (BHT), and propyl gallate (PG) cause or enhance the harmful health effect, therefore, interest has been intensified in finding naturally occurring antioxidants which have the potential to protect humans from damage caused by oxidative stress (Scalbert et al., 2005).
Due to the increased demand on natural product by consumers and their willingness to pay for relatively expensive natural foods (Sebranek and Bacus, 2007), therefore, the present study was carried out with the 20-diphenylpicrylhydrazyl (DPPH) as a free radical with some modifications.The stable radical DPPH was used as a reagent in this spectroscopic assay.Briefly, the methanol was used to dissolve each sample (200mL) of various concentrations and added to the DPPH solution (50mL) (0.15mM in methanol) in microtitre plate (96-well).517nm was used to measure the absorbance decrease of solution after incubated in the dark for 30-min at room temperature.The blank was methanol and control was DPPH, without essential oil.The synthetic references were ascorbic acid, BHA and BHT.Then, the DPPH radical reduction was calculated.For the final results, RC 50 values (the concentrations required for a 50% reduction of DPPH (0.15mM)) were calculated from the absorbance diminished by 50%.The experiment was performed in triplicate.

Antibacterial activity
Sample was dried and grind into powder, 35g of finely ground were dissolved in 375 ml of three different solvents (Hexane, ethanol, water) in order to get the best antibacterial effect.The sample of thyme and the three solvents are placed three days in room temperature.Then used rotary evaporator at 60°C for 24 hrs for solvents evaporation (hexane and Ethanol) and oils extraction, the extracts were filtered by using disposable membrane bacterial filter to get a sterile extract.Antibacterial activity was determined using agar well diffusion method.Petri -dishes with 20 ml of nutrient agar were equipped, and incubated with 1x10 6 cell / ml, 100 µl of a 24 h broth culture of test bacteria.Wells of 6 mm diameter each were made and filled with 100 µml of essential oils.The plates were incubated for 24 h at 37° C.After incubation, the diameter of inhibition zone was measured in mm (National Committee for Clinical Laboratory standard).

Thymus (Vulgaris) silver nanoparticles
The reduction of silver ion into silver nanoparticles during exposure to the plant extract could be followed by color change from yellow (Figure 2A) to brown (Figure 2B), Researchers reported that the bark extracts were place yellow in color before addition of AgNO 3 and these were changed to brownish color suggested the rapid formation of silver nanoparticles after lumen.The change of color indicates the biosynthesis of silver nanoparticles exhibit brown color in aqueous solution due to the surface Plasmon resonance phenomenon.The appearances of -yellowish -brown color in the reaction vessels suggest the formation of silver nanoparticles (SNPs) (Shankar et al., 2004).

Transmission Electron Microscopy (TEM)
Thymus (Vulgaris) silver nanoparticles TEM images are shown in Figure 3.The average particle size about 8-105nm are investigated.The nanoparticles morphology were  spherical in nature.Our results agree with Abd El-Aziz and Yousef (2018), they reported that silver nanoparticles (AgNP S )are spherical with varying size (in the range from 10-31.7 nm).TEM analysis revealed that the AgNP S were primarily spherical in shape.Their size ranges from in between 5-85 nm (Ashraf et al., 2016).The average particle size of AgNPs was about 30-50 nm and spherical shape (Al-Jameel et al., 2018).

Antimicrobial activity
Table 3 shows the antimicrobial activity of TEOE and TSNP S .The results showed all extracts had antimicrobial properties on bacteria and yeast in Table 3 and were able to prevent the growth of all microorganisms (grampositive, gram -negative and fungi) except Streptococcus pyogenes, it is resistance all extracts, AM and CIP.T+H extract is more effect than all extracts and AM.The results show that the effect of TSNP S is more than AgNo 3 with Acinetobacter baumanii and St. epidermidis, also, T+E is more effect with Klebsiella peneumonia, Acinetobacter, St. epidermidis, St. aureus and Candida albicane than E extract.Additionally, T+W was able to prevent the growth of all microorganisms than W extract except Acinetobact, st.agalacticae, st.pyogenes and candida albicane, they were resistance both extracts (T+W and W).These results may be due to the important source of bioactive compounds with antibacterial capacities against several Gram-positive and Gram-negative bacteria in thymus (Vulgaris).Studies of other researchers have also confirmed our results, three studies of the Thymus oil extracted effect against yeasts and bacterial isolate which found that, the highest percentage of inhibition was 63.4%, 61.7%, 60.5%, 59.7% and 59.7% within 30% concentration for yeasts C. albican, C. rropicalis, C. glabrata, C. kefyr and C. guillermondii, respectively (Habib et al., 2007) .Also the highest percent of inhibition was 39%, 36.1% and 27.5% with 20% concentration for Staphylococcus aureus, Streptococcus pyogenes and Escherichia coli, respectively.It was shown that inhibition activity of aqueous extracts against Pseudomonas aeruginosa higher compared with other bacteria.The alcoholic extract was more effect on Pseudomonas aeruginosa than another bacteria Staphylococcus aureus and Escherichia coli.Also, Thyme essential oil has antibacterial effects on bacteria isolated from mastitis (Streptococcus agalactiae, Staphylococcus aureus and Escherichia coli) (Mahdi et al., 2013).The clove bud oil presented a MIC of 600 ppm for all the strains of bacterial tested and Thyme essential oil of 400 ppm.However, S. enteritid is and E. coli more sensibility compared to B. cereus and S. aureus in the medium containing clove essential oil and thyme (Zahraei-salehi et al., 2005).There were no significant anti-bacterial effects on Staphylococcus aureus with Thyme extract and peppermint extract in concentration of 3.12%.The essential oils and antimicrobial properties of plant extracts could be used as natural alternative to antibiotics (Abiramasundari et al., 2011).,4,6a,8a,11,11,14bheptamethyl1,3,4,4a,5,6,6a,7,8,8a,9,10,11,12,12a,14,14a 13dimethyl1,2,3,4,7,8,9,10,11,12,13,14,15,16tetradecahydro cyclopenta[a]phenanthren17one 65.20 0.53 Thymus vulgaris essential oil contained and synthesis thymus (Vulgaris) silver nanoparticles aim of: 1-Determination of chemical composition of the essential oil of thymus (Vulgaris) extracted by GC/MS.2-Characterization of synthesized prepared silver nanoparticles.3-Determination of antioxidant and antimicrobial activities of thymus (Vulgaris) essential oil and thymus (Vulgaris) silver nanoparticles.

Figure 2 .
Figure 2. The color changed of plant extract (A) before and (B) after synthesis of silver nanoparticles.

Table 1 .
Antioxidant and antimicrobial activities of essential oil and nanoparticles of thymus (Vulgaris) Main components (%) detected by GC/MS.