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Chemical Compositions and Antimicrobial Activities of Ocimum sanctum L. Essential Oils at Different Harvest Stages

Background: Essential Oils (EOs) possess antibacterial properties and represent a natural source to treat infections and prevent food spoilage. Their chemical composition might be affected by the environmental condition and the developmental growth stages of the plant. Objectives: The current study aimed to determine the variations in chemical compositions and antimicrobial activities of the EOs of Ocimum sanctum L. at different stages of harvesting. Materials and Methods: The oils constituents were analyzed by gas chromatography/mass spectrometry (GC/MS). The effects of three different harvest stages of O. sanctum EOs against most common causes of food-borne were evaluated by broth micro-dilution method as recommended by the Clinical and Laboratory Standards Institute (CLSI). Results: The analysis of the EOs indicated that eugenol was the major compound of the EOs at all developmental stages which reached its maximum level at the second stage. The results showed that the tested EOs exhibited antimicrobial activities against all of the examined pathogens at concentrations of 0.125-32 µL/mL, except Pseudomonas aeruginosa which was only inhibited by high concentrations of the floral budding and full flowering EOs. EO distilled from the second developmental growth stage (floral budding) of O. sanctum exhibited the strongest antibacterial activities against the food borne bacteria. Conclusions: Considering the wide range of antimicrobial activities of the examined EOs, they might have the potential to be used to manage infectious diseases or extend the shelf life of food products.


Background
Foods provide a suitable media for many microorganisms to grow and produce byproducts and metabolites. Consumption of the contaminated food with a toxin or secondary metabolite due to bacterial growth in food, such as Staphylococcus aureus or Aspergillus species, is called food poisoning. On the other hand, certain types of live bacteria such as Entrobacteriacea family might be transmitted through eating contaminated foods and cause food-borne infection. Contaminated foods are among the main concerns of the public health policy makers and food industries. Traditionally, in tropical areas where the food-borne infections are more frequent, the aromatic plants and spices are used in foodstuff for their flavoring and preservative properties (1)(2)(3)(4). It is observed that many of these plants and their aromatic products have potential antimicrobial activities (5)(6)(7)(8).
In the recent decades, there is a global tendency to use organic and chemical-free foodstuffs. The use of aro-matic plants with known medicinal and antimicrobial properties might be a solution to increase the shelf life of products or overcome antibiotic resistance (8).
Parallel to the invention of new generations of antibiotics, clinicians encounter antibiotic resistance which has dramatically increased during the recent years. This problem is increasing worldwide, imposing a huge burden of costs, morbidity, and mortality. Therefore, it is necessary to find new antimicrobial agents to apply in the cases of antibiotic resistance. Methicillin-resistant S. aureus (MRSA), vancomycin-resistant Enterococcus (VRE) species, third-generation cephalosporin-resistant (TGCsR) Escherichia coli, imipenem and quinolone-resistant Pseudomonas aeruginosa, antibacterial-resistant Salmonella and Shigella species are the top resistant pathogens. Plants and their Eos are among the resources of new anti-microbial compounds. The number of studies on antimicrobial activities of plant compounds against many different types of microbes, including food-borne pathogens has increased in the recent decades.
Chemical and biological diversity of the aromatic and medicinal plants differ significantly depending on the factors such as cultivation area, climatic conditions, genetic modification, different plant parts, developmental stages and collection time (19)(20)(21). In the recent years, numerous publications have reported the chemical compositions of the EOs of medicinal and aromatic plants demonstrating that the growth stage and harvesting time have a major impact on the EO content and compositions (21)(22)(23). Therefore, it is necessary to determine the proper time and plant growth phase to harvest by analyzing the EO and its compositions at various growth and developmental stages. To the best of authors` knowledge, literature pertaining to the EO content and composition of Tulsi from Iran is not available. Moreover, there is no report on EO compositions of Tulsi at different collection time such as vegetative, floral budding and flowering stages. It is, therefore, imperative to determine the appropriate harvesting time by analyzing the oil yield and composition of the plant.

Objectives
The current study, for the first time, aimed to assess the EO content and chemical constituents of three different growth and developmental stages of O. sanctum. In addition, the antimicrobial effects of these EOs were evaluated against standard strains and clinical isolates responsible for food borne infections.

Plant Material
This experiment was carried out in Agricultural Research Center of Bushehr province, located in nine miles South-east of Borazjan, Iran, with an altitude of 110 meters, 51°17' longitude and 21° 2' latitude in a 1350 square meters land, which was a fallow land last year. The average annual rainfall in this region is 300 to 350 mm, the maximum temperature is 51°C in August, and minimum temperature is -1°C in February. Some chemical characteristics of the experimental soils are shown in Table 1. Ocimum sanctum aerial parts were collected from the cultivated plants at three stages of growth and development during June and July 2012. The samples were harvested at vegetative, floral budding (emergence of flower buds) and full flowering stages. A voucher specimen (HSU 24989) is deposited in the herbarium of Shiraz University, Shiraz, Iran.

Essential Oils Preparation
At the above-mentioned developmental stages, the essential oil of the aerial parts of the O. sanctum (50 g for each stage) was obtained by hydrodistillation for 2.5hours, using an all-glass Clevenger-type apparatus, according to the protocol outlined by the British Pharmacopoeia (24). The sample oils were dried over anhydrous sodium sulphate and stored in sealed vials at 4°C before gas chromatography (GC) and gas chromatography/mass spectrometry (GC-MS) analysis and antimicrobial assessments.

Essential Oils Analysis by Gas Chromatography/Mass Spectrometry (GC/MS)
The EOs were analyzed by GC-MS (Agilent, USA). The analysis was carried out on a Thermoquest-Finnigan Trace GC/MS instrument equipped with a DB-5 fused silica column (60 m × 0.25 mm i.d., film thickness 0.25 mm). The oven temperature was programmed to increase from 60 to 250ºC at a rate of 4ºC/minute and finally held for 10 minutes; transfer line temperature was 250ºC. Helium was used as the carrier gas at a flow rate of 1.1 mL/minute, with a split ratio equal to 1:50. The quadrupole mass spectrometer was scanned over the 35-465 amu with an ionizing voltage of 70 eV and an ionization current of 150 mA.
GC-FID analysis of the oil was conducted using a Thermoquest-Finnigan instrument equipped with a DB-5 fused silica column (60 m x 0.25 mm i.d., film thickness 0.25 mm). Nitrogen was used as the carrier gas at the constant flow of 1.1 mL/minute; the split ratio was the same as that of GC/MS. The oven temperature was raised from 60 to 250ºC at a rate of 4ºC/minute and held for 10 minutes. The injector and detector (FID) temperatures were kept at 250 and 280ºC, respectively. Semiquantitative data were obtained from FID area percentages without the use of correction factors.

Identification of EO Components
Retention indices (RI) were calculated by using retention times of n-alkanes (C6-C24) that were injected after the oil at the same temperature and conditions. The compounds were identified by comparison of their RI with those reported in the literature (25) and their mass spectrum was compared with the Wiley Library.

Microorganisms
The antifungal activities of the EOs were determined against five standard strains of fungi including

Determination of Minimum Inhibitory Concentration (MIC)
MICs were determined using the broth microdilution method recommended by the CLSI (26,27) with some modifications. Briefly, to determine antifungal activities, serial dilutions of the EOs (0.06-64.0 µL/mL) were prepared in 96-well microtitre plates using RPMI-1640 media (Sigma, St. Louis, USA) buffered with MOPS (Sigma, St. Louis, USA). To determine the antibacterial activities, serial dilutions of the EOs (0.06-64.0 µL/mL) were prepared in Muller-Hinton media (Merck, Darmstadt, Germany). Test fungi or bacteria strains were suspended in the media and the cell densities were adjusted to 0.5 McFarland standards at 530 nm wavelength using the spectrophotometric method (this yields stock suspension of 1-5 × 10 6 cells/mL for fungi and 1-1.5 × 10 8 cells/mL for bacteria). To each well of the micotiter plates, 0.1 mL of the working inoculums was added and the plates were incubated in a humid atmosphere at 30ºC for 24-48 hours for yeasts or at 37ºC for 24 hours for bacteria. To provide control (blank) samples, 200 μL of un-inoculated medium was included. In addition, growth controls (medium with inoculums, without EO) were also included.
The growth in each well was compared with that of the growth control well. MICs were visually determined and defined as the lowest concentration of the EO produced ≥ 50% growth inhibition for fungi and ≥ 95% growth reduction for bacteria compared with the growth control wells. Each experiment was performed in triplicate. In addition, media from wells with fungi showing no visible growth were further cultured on Sabouraud dextrose agar (Merck, Darmstadt, Germany) and from wells with bacteria showing no visible growth on Muller-Hinton agar (Merck, Darmstadt, Germany) to determine the minimum fungicidal concentration (MFC) and minimum bactericidal concentration (MBC), respectively.

Results
The hydro-distillation of the aerial parts of O. sanctum at the vegetative, floral budding, and full flowering stages yielded 0.98%, 0.92% and 1.1% (w/w) EO, respectively. The composition of EOs at different growth stages is shown in Table 2, in the order of their elution from a DB-5 column. A total of 13, 15 and 15 compounds representing 88.44%, 95.09% and 92.21% of the total were detected at vegetative, floral budding and full flowering stages, respectively. GC/MS analyses showed that the main constituents of the EO from the vegetative stage were 1, 8-Cineole and β-bisabolen, while eugenol was found as the main compound of floral budding and full flowering developmental stages.
The antibacterial activities of O. sanctum EOs against the tested bacteria are presented in Table 3. The EOs inhibited the growth of Gram-positive cocci at the concentrations of 0.5-32 µL/mL and removed them at the concentrations ranging from 1 to 32 µL/mL. Furthermore, the EOs exhibited anti-bactericidal activity (MBC) against all of the above-mentioned Gram-positive cocci at the concentrations ranging from 8 to 32 µL/mL, except the vegetative growth stage against E. faecalis. The O. sanctum EOs inhibited the growth of all Gram-negative bacteria at the concentrations of 0.25-4 µL/mL, except P. aeruginosa. The growth of P. aeruginosa was inhibited only in concentrations ≥ 64 µL/mL. The EOs exhibited anti-bactericidal activity (MBC) against all the above-mentioned Gram-negative bacteria at concentrations ranging from 0.5 to ≥ 64 µL/mL. The antimicrobial activities of O. sanctum EOs against the tested fungi are shown in Table 3. The essence of O. sanctum at different growth stages exhibited fungiostatic and fungicidal activities against the Aspergillus species at the concentrations of 0.125-2 µL/mL and 0.125-4 µL/mL, respectively.

Discussion
EOs and their components are generally recognized as safe (GRAS) for human and animal consumption under US Federal Regulations and have antimicrobial activity against a wide variety of foodborne pathogens. The current study showed anti-bacterial and anti-fungal characteristics of a traditional plant called O. sanctum on food-borne Gram-positive, Gram-negative bacteria and Aspergillus species. The compositions of EOs might be affected by the developmental stage of the plant (6,28). Although some authors reported Methyl Chavicol (14,29) as the major compounds of the EO, others reported eugenol as the main constituent of the EO (13,16). In the present study, β-bisabolen was the dominant compound of the oil at the vegetative stage which declined gradually from 20.99% to 13.29% at the floral budding stage. Eugenol which reached its maximum level at the floral budding stage was identified as the main compounds of both floral budding and full flowering stages. The lower concentration of eugenol in this study, compared with that of some previous reports (13,16), may reflect variations due to geographical location. During the various developmental stages of O. sanctum, the concentration of 1, 8-Cineole gradually declined from 20.78% to 20.45%.
The antimicrobial activity of O. sanctum against some microorganisms has been investigated in some previous studies (12). Agarwal et al. in an experimental study demonstrated that O. sanctum extract was effective against Streptococcus mutans (12). Others showed antimicrobial properties of the extract of this plant against S. aureus and enteric bacteria (30). In another study, its efficacy against multi-resistant strains of Neisseria gonorrhea was demonstrated (31). In the present study, the EOs inhibited the growth of all Gram-positive bacteria and removed them at the concentrations up to 32 µL/mL, , except E. faecalis. Similar to the previous studies (30), nearly all the studied Gram-negative bacteria were susceptible to EOs of this plant at the concentrations of 0.25-4 µL/mL, except P. aeruginosa which was only inhibited by high concentrations of the floral budding and full flowering EOs. This bacterium is a well-known nosocomial and burn wound pathogen. It has a low susceptibility to many antibiotics which is caused by the action of multidrug efflux pumps and the low permeability of its outer membrane (32). The higher MICs of the EOs against P. aeruginosa in comparison with the other bacteria might be attributable to the presence of some constructive proteins (such as Protein F) in its outer membrane (32).
The antimicrobial properties of O. sanctum are not confined to bacteria. It possesses anti-fungal and antihelmintic activities. Of Aspergillus species, A. flavus and A. parasiticus are responsible for producing a number of secondary metabolites including aflatoxins. These secondary by-products are well-known for carcinogenic, mutagenic and teratugenic effects on humans and animals (33). In addition, Aspergillus species, in particular A. fumigates and A. flavus, are responsible for severe invasive infections especially in immuocompromised hosts (34). Antifungal activities of the O. sanctum against Candida spp. and dermatophytes were previously indicated (6,14,15). In the current study, the developmental growth stages of the EOs, in particular full flowering stage, exhibited significant fungistatic and fungicidal activities against Aspergillus species. These results support the previously reported data (13), and suggest that the EO of O. sanctum has the potential to be used in the food industries as preservative to prevent the growth of aflatoxicogenic fungi.
Since the EOs exhibited similar antimicrobial effect. One of the main characteristics of EOs is their hydrophobicity, which enables their incorporation into the cell membrane (35). Of the developmental stages, the second stage was more potent against most of the examined bacteria. Its mechanism may yield behind the active ingredients of O. sanctum, since biological activity of the EOs is determined by its various chemical constituents. The most potent antibacterial component of O. sanctum in the second stage was eugenol and the noticeable antibacterial properties of this stage might be related to this compound. Eugenol is a phenylpropanoid compound used in perfumes and flavorings as well as medicines as an antiseptic and anesthetic.
It is proposed that the effective microbial damage of this molecule might be due to disruption of bacterial cell membrane (41,42), which leads to increased cell membrane permeability and protein leakage (39). Other possible mechanisms of antimicrobial action of eugenol are conversion of cytochrome P-450 mediated into cytotoxic quinine methide (40,43,44) and inhibition of energy generation (45). Other ingredients of the EOs with significant antibacterial potency were cineol and β-bisabolen. These two latter ingredients had higher concentrations at the vegetative stage than the other stages. Synergistic activity of the combination of ampicillin with β-bisabolen against S. aureus was confirmed by previous studies (46)(47)(48). Moreover, it was shown that 1, 8-cineol has significant antimicrobial activities alone or in combination with other monoterpens or drugs (49). All together, these facts and results support the broad spectrum antimicrobial activities of O. sanctum EO by the mechanisms different from those of the antibiotics, and its possible application in the cosmetic, medicinal and food products.
Chemical composition and subsequent biological activities of EOs are not only determined by the origin and environmental conditions, but also by developmental stage in which the plant is collected. The EO distilled from the second developmental growth stage of O. sanctum (floral budding) exhibited strong antibacterial activities against several food-borne bacteria as well as Aspergillus species. Therefore, any attempts for clinical or industrial application of this EO should focus on the second developmental stage and its major ingredient (i.e. eugenol). Regarding the considerable antibacterial activities of the tested EOs, they might be the candidates to develop new antibiotics and disinfectants to control infective agents. The desirable flavor and odor of this EO at the examined concentrations is an additional benefit to its antimicrobial activities which makes it a suitable candidate to be used as a food preservative.

Funding/Support
This study is part of the MD thesis of Amir A′lam Kamyab. The study was financially supported by Shiraz University of Medical Sciences (Grant No. 90-01-43-2979). The funding organization is Shiraz University of Medical Sciences, which had no role in the design and conduct of the study; collection, management, and analysis of the data; or preparation, review, and approval of the manuscript.