Chemical Compositions, Antioxidant and Antimicrobial Activities of the Essential Oil and Extracts of Lamiaceae Family (Ocimum basilicum and Thymbra spicata) from Turkey

Lamiaceae has numerous proven biological properties. Our aim was to investigate the antioxidant and antimicrobial activities and chemical composition of essential oil and extracts of Ocimum basilicum and Thymbra spicata . For this purpose, we used four Gram-positive ( Staphylococcus aureus, Streptomyces murinus, Micrococcus luteus, Bacillus subtilis ), four Gram-negative ( Klebsiella pneumoniae , Pseudomonas aeruginosa , Yersina enterocolitica , Proteus vulgaris ), a yeast ( Candida albicans ) and a mold ( Aspergilus niger ). Essential oil and the extracts of Thymbra spicata have the most effective and significant activity against the bacteria, yeast and mold. Antioxidant activity of the Thymbra spicata was higher than Ocimum basilicum . Phenolic compounds have good properties like antioxidants acting as reducing agent, hydrogen donors and singlet oxygen quenchers. Fourteen phenolic compounds were analyzed and some phenolics were detected in Ocimum basilicum and Thymbra spicata. These results suggest that Lamiaceae is rich in phenolic compounds and has antimicrobial and antioxidant properties as well. Therefore, it can be a useful herbal source in food and drug industries.


INTRODUCTION
Medicinal plants, since times immemorial, have been used in virtually all cultures as a source of medicine.The use of traditional medicine and medicinal plants in most developing countries has been widely observed as a normative basis for the maintenance of good health.The relationship between plants and people has been discussed for years, and it will be discussed in the future.Infectious diseases account for approximately one-half of all deaths in the tropical regions of the world and at least developed countries.In industrialized and advanced nations, despite the progress made in the understanding of microbiology and their control, incidents of epidemics due to drug-resistant microorganisms and the emergence of up to the present unknown disease-causing microbes, pose enormous public health concerns.Today it is conjecturable that plant materials are available or have ensured the models for 50% Western drugs [1].Many commercially certified drugs used in modern medicine were primarily used in a raw untreated form in conventional or folk healing practices or for another objective that proposed potentially useful biological activity [2].The primary benefits of using plant-derived medicines are relatively safer than synthetic alternatives, offering profound therapeutic benefits and more affordable treatment [2].The extracts and essential oils of many plants/herbs have been shown to exert biological activity in vitro and in vivo, which justified research on traditional medicine focused on the characterisation of antimicrobial activity of these plants [3].
Common basil (Ocimum basilicum L.; O. basilicum), a member of the Lamiaceae family is an annual herb which is known locally as "fesleğen or reyhan", is native to Asia and is widely grown as an ornamental or field crops from seed in the Mediterranean countries including Turkey [4,5].Basil is used in pharmacy for diuretic and stimulating properties, in perfumes and cosmetics for its smell; in fact, it is a part of many fragrance compositions [6].Its oil has been found to be beneficial for the alleviation of mental fatigue, colds, spasms, rhinitis, and as a first aid treatment for wasp stings and snake bites.The essential oil has antioxidant, physicochemical and insect-repelling anticonvulsant, hypnotic, and antioxidant activities [7][8][9].Various researchers have reported chemical composition of the essential oil of O. basilicum [8,9].They are regarded as oil-rich on the basis of their essential oil contents.The genus Thymbra, which belongs to Lamiaceae family, includes many native plants of the Mediterranean region in Turkey and is locally known as Karabaş kekik, Kara kekik, Sater or Zater [9][10][11].It is traditionally used for flavouring different kinds of food products, as herbal tea and in folk medicine as an antiseptic agent [11,12].The leafy part of plants such as sage, thyme, oregano and savoury belonging to the Lamiaceae family have been added to meat, fish and food products for years.In addition to improving flavour, certain spices and essential oils prolong the storage life of foods by increasing an antimicrobial activity [13].The flora of Turkey has two species of Thymbra and it attracted a great deal of research interest due to their potential as a source of natural antioxidants and biologically active compounds [14].
In the present study, different groups of microorganisms were used to evaluate the antimicrobial activity of Lamiaceae and chemical composition of the essential oil and extracted from Lamiaceae and its biological properties were investigated.The antimicrobial activity was tested by using agar disc diffusion and broth microdilution methods.Determination of antioxidant activity was tested by using Total Phenolic Contents (TPC), Total Flavonoid Contents (TFC) and Condensed Tannin (CT) and ferric reducing antioxidant power (RFAP).Fourteen different phenolic compounds were determined using reverse phase-high performance liquid chromatography (RP-HPLC).The chemical composition was tested by using GC-MS.

Plant material
Samples of Ocimum basilicum and Tyhmbra spicata were purchased commercially in Amasya, Turkey and dried at room temperature.

Preparation of extracts for antimicrobial activity
Extraction method described elsewhere was used for the antimicrobial screening with slight modification [15].The plants (90 g) separated into equal three parts.The first parts (30g) were refluxed with 250 mL ethanol, the second parts (30 g) were refluxed with 250 mL dichloromethane and the third parts (30 g) were refluxed with 250 mL n-hexane for 2 h.The infusions were filtered and the solvents were removed under rotary vacuum.Then the residue dissolved in DMSO.Oil and extracts samples were stored at 4°C in sealed glass vials.

Preparation of essential oils
The essential oils of the plants were produced by the Clevenger hydrodistillation method [16].Plant materials (50 g) cut into small pieces and placed in a distillation apparatus with double distilled water and hydrodistilled for 3 h.They were stored at 4 o C until tested.(MHA).Yeast and mold were cultured overnight at 35°C in Sabouraud dextrose agar.Moreover, bacteria species were grown in Mueller Hinton Agar (Merck) and Mueller Hinton Broth (Merck).C. albicans and A. niger were grown in Sabouraud Dextrose Broth (Difco) and Sabouraud Dextrose Agar (Oxoid).The concentration of bacterial suspensions was adjusted to ~10 8 cells/mL and fungal suspension to ~10 7 cells/mL.

Antimicrobial assays
The disc diffusion method was employed for the determination of antimicrobial activities and microdilution method of the essential oil and extracts.

Screening for antimicrobial activities
Antimicrobial activity was measured with diffusion disk plates on agar.In order to test antimicrobial and antioxidant activity, the fractions of O. basilicum and T. spicata samples were dissolved in 70% ethanol.Suspensions (50 μL) with approximately 10 8 bacteria and fungus per millilitre were placed in Petri dishes, over agar and dispersed.Inhibition zones were determined after incubation at 25 °C for 24 h [8,17].For the bacteria ampicillin and cephazolin obtained from a local pharmacy were used as a positive control and alcohol as a negative control.

Determination of minimum inhibitory concentration (mic) with microdilution assay
The agar dilution method, described by Vanden et al in 1991 was used for the antimicrobial screening with slight modifications.Instead of 96 well microtitre plates, 24 well tissue culture (Corning) plates were used.The crude extracts were dissolved in 70% ethanol and physiological Tris buffer (Amresco 0826-500G) mixture (1:4) and mixed with an equal amount of 3% agar solution Sabouraud Dextrose Agar (Oxoid) for fungus at 25 o C and Mueller Hinton Agar (Merck) for bacteria at 37°C.Each of the crude extract samples was tested at concentrations of 50, 25, 12.5, and 6.25 mg/mL.From the test solutions, 400 µL was transferred into each well of the tissue culture plate.After solubilization, each well was inoculated with 10 µL of freshly prepared bacterial suspension of 10 8 bacteria, 10 7 fungi/mL and incubated at 37°C for 24 h.The bacterial and fungal growth was assessed by a stereo microscope after the incubation period [18].

Preparation of the extracts for chemical composition and antioxidant capacity
For extraction, approximately 3 g of dry plant samples were placed into a falcon tube with 50 mL of absolute methanol.The mixture was stirred continuously on a shaker (Heidolph Promax 2020, Schwabach, Germany) at room temperature during 24 hours.Particles were removed using filter paper.The final volume of the solution was adjusted by the addition of methanol.

Determination of antioxidant capacity
The polyphenolic contents of the methanolic samples were evaluated in four different ways; Total Phenolic Contents (TPC), Total Flavonoid Contents (TFC), Condensed Tannin (CT) and ferric reducing antioxidant power (FRAP).Total phenolic compounds were determined by an end-point assay of Folin-Ciocalteu method [19].First, 680 µL distilled water, 400 µL 0.5 N Folin-Ciocalteu reagent and 20 µL methanolic sample extract were mixed in a test tube and well vortexed.Next, 400µL Na2CO3 (10%) was added and well vortexed after incubated for 2 h in the dark at room temperature.After the incubation the absorbance was measured at 760 nm, the results were calculated using a standard graph as mg of Gallic Acid Equivalent (GAE) per 100 grams of sample.
The total flavonoid contents of the samples were measured using a spectrophotometric method as reported elsewhere [20].Briefly, 500 µL methanolic samples, 4.3 mL methanol 100 µL of 10% Al(NO3)3 and 100 of 1 M NH4.CH3COO were added to a test tube and incubated at room temperature for 40-min periods, the absorbance was measured against a blank at 415 nm.A standard calibration curve was plotted using quercetin.The total flavonoid concentration was expressed as mg of Quercetin Equivalent (QE) per 100 grams of sample.
Condensed tannins were determined according to the method by Julkunen-Tıtto [21].For each, 25 μL of various concentrations of plant extract was mixed with 750 μL of 4% vanillin in methanol, to which 375 μL of concentrated HCl was added.The well-mixed solution was incubated in the dark at room temperature for 20 minutes.The absorbance against a blank was then measured at 500 nm.(+)-Catechin was used to make the standard curve (0.05-1 mg/mL).The results were expressed as mg Catechin Equivalent (CE) per 100 grams of sample.
Ferric tripyridyltriazine (Fe-III-TPTZ) complex was used to determine total antioxidant capacity [22].Briefly, working FRAP reagent was prepared as required by mixing 25 mL of 300 mM acetate buffer, pH 3.6, with 2.5 mL of 10 mM TPTZ solution in 40 mM HCl and 2.5 mL of 20 mM FeCl3.6H2O solution.Next, 3 mL freshly prepared FRAP reagent and 100 µL of extract were mixed and incubated for 4 min at 37°C, and the absorbance was measured at 595 nm against a reagent blank containing distilled water.Trolox was used as positive control to construct a reference curve (62.5-1000 µM).FRAP values were expressed as mM Trolox equivalent of 100 grams sample.

Analysis of phenolic compounds by RP-HPLC
Fourteen standards of phenolic compounds were analyzed using HPLC (Elite LaChrom Hitachi, Japan).The sample was injected into the HPLC system, set up with a reverse phase C18 column (150 mm x4.6 mm, 5μm; Fortis).Acetonitrile, water and acetic acid were used for the mobile phase by applying a programmed gradient.The mobile phase consisted of (A) 2% acetic acid in water and (B) acetonitrile: water (70: 30).The samples injection volume was 20 μL, column temperature was 30°C and flow rate was 0.75 mL/ min.The programmed solvent used began with a linear gradient held at 95% for three minutes, decreasing to 80% A at 10 min, 60% A at 20 min, 20% A at 30 min and finally 95% A at 50 min as described elsewhere [23].

Antimicrobial activities
Results of disc diffusion methods were summarized in Table 1-2.Essential oil and extracts of the plants displayed varying degrees of activity against bacteria strains.Essential oil of O. basilicum (F00) was also effective on A. niger, S. murinus, P. vulgaris.Antimicrobial activity of F00 against the K. pneumonia, S. aureus, M. luteus, C. albicans, B. subtilis and P. aureginosa were less effective.Ethanolic extracts from O. basilicum (F01) was also effective on A. niger, S. murinus.Antimicrobial activity of F01 against the Y. enterocolitica, K. pneumoniae, C. albicans, S. aureus, M. luteus, B. subtilis, P. vulgaris and P. aureginosa were found to be less effective than the other microorganisms.Diclorometanoic extracts from O. basilicum (F02) was also effective on Y. enterocolitica, A. niger, S. murinus, S. aureus and C. albicans.Antimicrobial activity of F02 against P. aeruginosa, K. pneumonia, M. luteus, B. subtilis, P. vulgaris and P. aureginosa were found to be less effective than the other microorganisms.N-Hexanoic extracts from O. basilicum (F03) was also effective on Y. enterocolitica, S. murinus, K. pneumonia, S. aureus, C. albicans and P. vulgaris and had inhibition zone with diameters in excess of 38 mm.Antimicrobial activity of the F03 extract against the A. niger, M. luteus, B. subtilis and P. aureginosa were found to be less effective than the other microorganisms.These results were supported by Adıgüzel, 2005.Control treatment dimethyl sulfoxide (DMSO) did not show any inhibitory effect on the bacteria (Table 1-2).
There were a lot of reports on the antimicrobial activity of the essential oil of Lamiaceae.Although our study showed that essential oil and extract of Lamiaceae exhibited antimicrobial activity against all test microorganisms, these works of literature are concerned and weak antimicrobial activity was observed against microorganisms including S. aureus and P. aeruginosa [24,25].

Antioxidant Activity
The present study was carried out in order to evaluate the in vitro antioxidant potential and phenolic compounds of T. spicata and O. basilicum plants.In total four complementary test systems were used in the assessment including total phenolic, flavonoid and condensed tannin contents.Total phenolic contents values by themselves may not be sufficiently in order to draw any definitive conclusions regarding the antioxidant potential of biological samples [26].Therefore, some additional assays, such as FRAP concentration determinations were used throughout this study.
The total amount of phenolic, flavonoid and tannin content present in methanolic O. basilicum and T. spicata extract is shown Table 3.It was found that 100 g of O. basilicum and T. spicata, 731.22 to 969.53 GAE of phenols, 0.12 to 0.14 mg QE of flavonoid and 335.72 to 646.49 mg CE of tannin were detected respectively.In addition, it was found that 100 g of O. basilicum and T. spicata, extract 11.884 to 15.106 of FRAP µmol Trolox.These results indicated that antioxidant activity of the T. spicata was higher than that of the O. basilicum.Fourteen phenolic compounds were analysed and some phenolics were detected in O.basilicum and T. spicata (Table 3).t-cinnamic acid and vanillic acid were identified as the major phenolic compounds in both plants.In addition to syringic acid was identified in T. spicata.Phenolic compounds are substances that have redox properties and those properties make them good antioxidants acting as reducing agent, hydrogen donors and singlet oxygen quenchers [27].Fourteen phenolic compounds were analyzed and some phenolics were detected in O. basilicum and T. spicata (Table 4).t-cinnamic acid and vanillic acid were identified as the major phenolic compounds in both plants.In addition to syringic acid was identified in T. spicata.It was reported that O. basilicum with major compounds rutin and two hydroxycinnamic acid derivates, namely ferulic acid and p-coumaric acid were identified and quantified that the result similar to our result [28].

CONCLUSION
In this study antimicrobial effect of various extracts and essential oil of two plants (Lamiaceae family) were carried out.This investigation denotes that essential oil of T. spicata and O. bacilium have significant activity against microorganisms.The essential oil also shows antioxidant activity.In addition, we saw phenolic profile these plants.These results verify the potential use of plant essential oils and extracts in the pharmaceutical industries.Phenolic profile of these plants could be a benefit for treatment microbial diseases.

Table 1 .
Results of antimicrobial screening of O. basilicum and T. spicata extracts determined by the agar diffusion method (inhibition zone in mm)

Table 2 .
Results of antimicrobial screening of O. basilicum and T. spicata of essential oils and (DMSO) extracts determined by the well diffusion method (minimum inhibitory concentration in mg/ml)

Table 3 .
Antioxidant parametres of O. basilicum and T. spicata

Table 4 .
Phenolic profiles of the O. basilicum and T. spicata