The Biological Activities of Lavandula stoechas L . against Food Pathogens

Received: 27 April 2017 – Revised: 14 August 2017 Accepted: 03 June 2017 Abstract: Foodborne pathogens are microorganisms as well as a number of parasites, which are capable of infecting humans via contaminated food or water. In recent years, diseases caused by foodborne pathogens have become an important public health problem in the world, producing a significant rate of morbidity and mortality. In traditional medicine, numerous plants and their extracts have used for thousands of years to treat health disorders. Although many studies were made on natural herbs, those involving the antimicrobial, antioxidant and antimutagenic activity of the herb species are rather rare. This study researches the biological activities of ethanol and methanol extracts of Lavandula stoechas L., which are prevalent in Turkey. In this study, 8 food pathogens were used for antimicrobial activity studies. Antimicrobial activity studies were done by disk diffusion assay and MIC (minimum inhibitory concentration). DPPH method was used for non-enzymatic antioxidant activity. The Lavandula extracts were screened for their antimutagenic activity against sodium azide by Ames test in absence of rat microsomal liver enzyme (-S9). The ethanol and methanol extracts of Lavandula stoechas showed antibacterial activity (7 mm) against most of bacteria. The antifungal activity of L. stoechas was not determined against C. albicans RSKK02029. The lowest MIC value was determined as 3250 μg/mL. The highest radical inhibition was determined as 79 % by Lavandula stoechas flower methanol extract. The flower extract of L. stoechas (12500 μg/plate) was found to have its highest antimutagenic activity for Salmonella Typhimurium TA98. This inhibition value is 42 %. L. stoechas leaves extracts (6250 and 3125 μg/plate) showed a moderate positive inhibitory effect for Salmonella Typhimurium TA98, and TA100. L. stoechas flower extracts (12500 and 6250 μg/plate) showed a moderate positive inhibitory effect (respectively 31 and 30 %) for Salmonella Typhimurium TA100. The extracts of L. stoechas have antimicrobial, antioxidant and antimutagenic activities.


INTRODUCTION
More than 200 known diseases are transmitted through food by a variety of agents that include bacteria, fungi, viruses, and parasites.The Center for Disease Control and Prevention [1,2] estimates that 76 million people get sick, more than 300,000 are hospitalized, and 5,000 Americans die each year from foodborne illness.Food items most likely associated with antibiotic resistant pathogens included dairy products, ground beef, and poultry.Pathogens exhibiting multi-drug resistance to five or more antibiotics were identified in more than half of the outbreaks [3].Scientists have taken up the issue to solve this problem.
Medicinal plants are natural resources, yielding valuable herbal products which are often used in the treatment of various ailments [4].The important advantages claimed for therapeutic uses of medicinal plants in various ailments are their safety besides being economical, effective, fewer side effects and their easy availability [5][6][7].Because of these advantages the medicinal plants have been widely used by the traditional medical practitioners in their day to day practice.Today, it is estimated that about 80% of the world population relies on botanical preparations as medicine to meet their health needs [8].
Genus Lavandula belongs to Lamiaceae family and it includes about 39 species however, the most important species are lavender (Lavandula angustifolia Mill.), lavandin (Lavandula intermedia Emeric.) and spike lavender (Lavandula spica L.) [9].Lavender is native to the Mediterranean and grows in natural sites of lower parts of mountains.Lavender, small shrubby plant, grows 20-60 cm high with irregular, much branched stems.The leaves are opposite, sessile, lanceolate, linear or lance-shaped and hairy.Flowers are produced in the long spikes on long stems.The spikes consist of rings of 6-10 flowers which are bilabiate, small, 0.8 cm long with blue, tubular and ribbed calyx and violet-blue corolla.The majority of the oil, extracted from the flowers, is contained in the glands on the calyx [10].According to Nartowska [11] lavender flowers contain essential oil and its components: linalyl acetate (40%), linalool (30%), limonene, β-ocymene, 1,8-cineole, camphor, α-terpineol, borneol, but also phenolic acids, ursolic acid, cumarins flavonoids and sterols.Lavender oil (Lavandulae aetheroleum) is known for its antibacterial, antifungal, carminative, antiflatulence, antiholic, sedative and antidepressive activities [12].Lavender oil is used in phytotherapy to relief cough, neuralgia, insomnia but also for bath and compress [13][14][15].According to World Health Organization, medicinal plants would be the best source to obtain a variety of drugs.Therefore, such plants should be investigated to better understand their properties, safety and efficacy [16].
The aim of the investigation presented in this paper is to evaluate the antibacterial, antioxidant and antimutagenic activities of extracts of Lavandula stoechas on several food pathogens, as there is a significant lack of information on such activities in literature.

Plant Material
Lavandula stoechas flowers and leaves were collected in May 2014 form 0-700 m height above sea level in Izmir.The identity was confirmed by Dr. Olcay Ceylan, Department of Biology, Mugla Sitki Kocman University.The voucher specimens were deposited at the Herbarium of Department of Biology, Mugla Sitki Kocman University.The identification of these specimens was carried out using the Flora of Turkey [17].

Plant Extraction
The flowers and leaves were washed thoroughly 2-3 times with running water and once with sterile distilled water.Fresh plant materials were air dried, and then the dried materials were powdered in a blender.All samples were stored at ambient temperature until initial sample preparation, after which they were stored at 4 °C until required for analysis.The air dried and powdered flowers and leaves of the plant samples (40 g) were extracted with ethanol and methanol (250 mg/mL) using the Soxhlet apparatus.All experiments were continued for 4 hours.All of extracts were evaporated and then the extracts were dissolved in their solvent and then kept in small sterile opac bottles under refrigerated conditions until used.All of extracts concentrations were set to 100-200 mg/mL.

Microorganisms and Cultivation
The extracts were individually tested against food pathogenic strains such as Bacillus subtilis RSKK245, Staphylococcus aureus RSKK2392, Salmonella Typhimurium RSKK19, Enterococcus faecalis ATCC8093, Escherichia coli ATCC11229, Listeria monocytogenes ATCC7644, Yersinia enterocolitica NCTC11174 and Candida albicans RSKK02029.The bacteria were grown for 24 hours at 37 °C in Mueller-Hinton Broth (Merck).C. albicans was grown for 24-48 hour at 30 °C in Sabouraud Dextrose Broth (Merck).These strains of bacteria and C. albicans were obtained from ATCC (American Type Culture Collection, USA), RSKK (Refik Saydam National Type Culture Collection, Turkey) or NCTC (National Collection of Type Cultures).

In vitro Antimicrobial Activity
Kirby-Bauer method applied for antimicrobial activity [18].The extracts of plant were tested by disc diffusion assay.The concentration and quantity of extracts were used as 45 μL of 100-200 mg/mL.In this study, ethanol and methanol were used as organic solvents.The bacteria were maintained on Mueller-Hinton agar plates (MHA, Merck) at 37 °C and yeast was maintained on Sabouraud Dextrose agar plates (SDA, Merck) [18].Bacteria and C. albicans RSKK02029 cultures were adjusted to 0.5 McFarland.The experiments were performed in triplicate.Bacteria were incubated at 37 °C in 24 hours.C. albicans RSKK02029 was incubated at 30 °C for 24 hours.After incubation, the inhibition zones formed and then the values of zone were measured.Ethanol and methanol used as negative control.Chloramphenicol (30μg), and nystatin (100μg) antibiotics used as positive control.

Determination of Minimum Inhibitory Concentration (MIC)
The MIC was evaluated on plant extracts as antimicrobial activity.The MIC was taken as the lowest concentration that inhibits growth after incubation.The broth dilution assay was performed as described in the CLSI standards [19,20].This test was performed at final concentrations of each extract (6500; 3250; 1625; 812.5; and 406.25 μg/mL).

Determination of non-Enzymatic Antioxidant Activity
The non-enzymatic antioxidant activity was determined using DPPH as a free radical.The stable 2,2-dipheny l-1-picrylhydrazyl-hydrate radical (DPPH) was used for determination of free radical scavenging activities of the flower and leaf extracts.Extract (0.1 mL) was added to 3.9 mL of a 0.1 mM methanol DPPH solution.After incubation for 30 minutes, absorbance of extract was measured at 515 nm using spectrophotometer.Methanol was used as a blank, while methanol with DPPH solution was used as a control [21].Trolox was used for reference antioxidant.The DPPH scavenging capacity expressed in percentage (%) was calculated using the formula.

Determination of Antimutagenic Activity
Antimutagenic activity was evaluated by the Salmonella-microsome assay, using the Salmonella Typhimurium tester strains TA98 and TA100, kindly provided by Dr. B.N. Ames (Berkeley, CA, USA), without (−S9) metabolisation by the pre-incubation method [22].The Salmonella histidine point mutation assay of Maron and Ames [22] was used to test the antimutagenic activity of extracts without S9 mix.In this study, two different tester strains were employed to measure the antimutagenicities of Lavandula stoechas extracts.These strains included Salmonella Typhimurium TA98 and TA100.The calculation percentage of inhibition was done according to the formula given by Ong et al., [23].Sodium azide was used as positive control.Methanol is negative control.Concurrently, a positive control (where mutagen but no extract was added) and a negative control (where no mutagen was added) were also set.The test sample was dissolved in methanol.But mutagen was dissolved in distilled water.In our study, non-toxic concentrations of the test sample used for investigating were 12500, 6250 and 3125 µg/plate.These concentrations were categorized as non-toxic because they showed a welldeveloped lawn, almost similar size of colonies and no statistical difference in the number of spontaneous revertants in test and control plates.

RESULTS and DISCUSSIONS
The antimicrobial activities of ethanol and methanol extracts of Lavandula stoechas were evaluated in vitro against 8 microorganisms test species, which are known to cause some diseases in foods.Results of antimicrobial activities of used plant extracts against the test bacteria are shown in Table 1.Besides, the inhibition zone diameters of the reference antibiotics to the test microorganisms are shown in Table 2.
The results of antibacterial activities were recorded as zone of inhibition in mm for all the materials used as follows.Results show that the ethanol extracts of L. stoechas inhibited the growth of six bacteria and the inhibition zones were 7 mm.Whereas methanol extracts of this plant inhibited the growth of seven bacteria and similarly the inhibition zones were 7 mm.In addition the ethanolic and methanolic extracts of this plant did not determine any anticandidal effects against used yeast.The extracts showed the same effect on B. subtilis, S. aureus, L. monocytogenes and Y. enterocolitica (Table 1).Chloramphenicol and nystatin antibiotics used as positive control.Chloramphenicol very strongly inhibited the bacterial growths (Table 2).The non-enzymatic antioxidant activity of plant extract was evaluated by the DPHH radical scavenging capacity.Table 4 shows the percent of DPPH radical scavenging capacity with trolox as reference.The methanol extract showed 79% inhibition at 200 mg/mL concentration.Trolox equivalent value was 2.2 mM/g (Table 4).The antimutagenic activities of the extracts were evaluated by the against sodium azide by Ames test in absence of rat microsomal liver enzyme (-S9).Table 5, 6, and 7 shows the percent of inhibition.The flower extract of L. stoechas (12500 µg/plate) was found to have its highest antimutagenic activity for Salmonella Typhimurium TA98.This inhibition value is 42%.L. stoechas flower extracts (12500 and 6250 µg/plate) showed a moderate positive inhibitory effect (respectively 31 and 30%) for Salmonella Typhimurium TA100 (Table 5).

DISCUSSION
Medicinal plants have proved to be abundant sources of biologically active compounds, many of which have been used as compounds to develop new pharmaceuticals [24].L. stoechas flowers and leaves were selected based on their relevant ethnomedical use.In the present study, extracts of the plant obtained in solvents were tested against eight microorganisms.The antimicrobial activities were compared with the standard antibiotics.Results show that the ethanol extracts of L. stoechas inhibited the growth of six bacteria and the inhibition zones were 7 mm.Whereas methanol extracts of this plant inhibited the growth of seven bacteria and similarly the inhibition zones were 7 mm (Table 1).Gören et al. [25] reported that antimicrobial activities of extracts of L. stoechas ssp.stoechas were found highly effect on bacteria.
Result of this study showed that tested plant flower extracts were found to same effective against S. aureus RSKK2392 and Bacillus subtilis RSKK245 (7 mm) (Table 1).Oskay et al. [26] showed that the most susceptible organism was methicillin resistant S. aureus (MRSA) which was sensitive to methanol and ethanol extracts.Khosravi and Malecan [27] reported that alcoholic extract of L. stoechas has significantly inhibitory effect on the growth of Staphylococcus aureus.These results are like with our study.
In this study, the extracts were not inhibited the growths of S. Typhimurium and E. coli (Table1).Cherrat et al. [28] reported that Lavandula stoechas is effective against Gram (+) bacteria and Gram (-) bacteria is weakly effective.Various workers have already shown that Gram positive bacteria are more susceptible towards plants extracts as compared to Gram negative bacteria [29,30].These differences may be attributed to the fact that the cell wall in Gram positive bacteria is of a single layer, whereas the Gram negative cell wall is multilayered structure [31].
In addition, the ethanolic and methanolic extracts of this plant did not determine any anticandidal effects against used yeast (Table 1).Zuzarte et al. [32] reported that the oil of Lavandula stoechas had low antifungal activity in their study.According to Adam et al. [33], Lavandula angustifolia showed moderate to low antifungal activities against Malassezia furfur, Trichophyton rubrum, and Trichosporon beigelii.Uzun et al. [34] reported that L. stoechas extract was not inhibited growth of Candida albicans.These results are like with our study.In this study, three bacteria showed the lowest sensitivity to extracts of Lavandula stoechas (3250 µg/mL).Ünsal et al. [35] reported that MIC value of Lavandula subsp.stoechas cariensis was 19.52 µg/mL.Results of our study are higher than their results.In our results, MIC values are 6500 µg/mL for B. subtilis and 3250 µg/mL for S. aureus (Table 3).Nunes et al. [36] determined that MIC values are 9830 µg/mL for B. cereus and 9830 µg/mL for S. aureus.Results of our study are better than their results.
In this study, the extracts of Lavandula stoechas have different free radical inhibition.The flower methanol extract showed 79% free radical inhibition at 200 mg/mL concentration (Table 4).Researchers found several compounds to be present in L. stoechas essential oils are known to possess antioxidant activities.These include eugenol, carvacrol, thymol, terpinolene, α-terpinene, γ-terpinene [37], and terpinen-4-ol [38].Matos et al. [39] reported that chemical composition of L. stoechas were fenchone (42%), camphor (35%) and oxygen-containing monoterpenes (87%).These chemical composition differences might be caused by geographic origins, climatic and seasonal conditions, the time of collection, the stage of development, the method of extraction and even might be correlated to the existence of new chemotypes [40].Some authors reported the antioxidant activity of L. angustifolia and L. luisieri extracts [41,42].Cherrat et al. [28] reported that Lavandula stoechas has high antioxidant properties in their study, which supports our work.
The flower extract of L. stoechas (12500 µg/plate) was found to have its highest antimutagenic activity for Salmonella Typhimurium TA98.This inhibition value is 42 % (Table 5).In determining the antimutagenic potential of a sample, a value smaller than 25% inhibition of the mutagen activity indicates a weak or non-antimutagenic effect, a moderate effect when the value is between 25 and 40% and strong antimutagenicity when the value is greater than 40% [43].

CONCLUSION
It is inferred that extracts of L. stoechas is moderately effective against food pathogens and can be utilized as sources of natural antimicrobial agents.Our results support the use of this plant in traditional medicine and suggest that some of the plant extracts possess compounds with good antibacterial properties that can be used as antibacterial agents in the search for new drugs.
The results obtained in this report clearly demonstrate that greater part of tested extracts exhibited strong antioxidant activities, particularly, to scavenge free radicals generated from DPPH reagent, especially Lavandula flower extract.The methanolic extract of L. stoechas flowers, should be beneficial as an antioxidant protection system for the human body against oxidative damage.This plant with high antioxidant ability can be used on development research and may be a source of natural antioxidants for potential exploit in food, cosmetic and pharmaceutical industries.However, further researches are needed to explore the bioactive compounds in this selected medicinal plant.Undoubtedly, the antimicrobial and antioxidant effects of L. stoechas should be investigated further.Fractionation and characterization of the active compounds should be do further works to investigate.

Table 1 .
Antimicrobial activities of Lavandula stoechas extracts

Table 2 .
Antibiotic profils of food pathogens

Table 3 .
shows MICs of Lavandula stoechas extracts obtained by the broth dilution method.Three bacteria showed the lowest sensitivity to extracts of Lavandula stoechas (3250 µg/mL).