THE ANTIMICROBIAL ACTIVITY OF HYPERICUM PERFORATUM L. FLOWER EXTRACT AGAINST FOOD PATHOGENS AND ITS NON-ENZYMATIC ANTIOXIDANT ACTIVITY

. Foodborne pathogens pose a significant hazard to food safety. Most cases of foodborne illnesses are caused by bacterial pathogens that have infiltrated the food chain at some point, from farm to kitchen. According to the World Health Organization (WHO), approximately one-third of individuals in developed countries are affected by foodborne pathogens each year. Although there are studies on Hypericum perforatum L. in the literature, research in Turkey remains limited. Therefore, the aim is to contribute to the literature by studying H. perforatum samples from the Yaraş region of Muğla province in Turkey. This study specifically aims to investigate the antimicrobial activities against foodborne pathogens and the antioxidant activity of H. perforatum in Muğla. The in vitro antimicrobial activities of flower components from plants grown in Mugla were evaluated using the disc diffusion method and broth dilution test. Additionally, the extracts underwent ABTS (2,2′-azinobis-(3-ethyl benzothiazoline-6-sulfonic acid)) free-radical testing to evaluate their antioxidant activity. The extract exhibited a maximum inhibition zone of 16 mm against Staphylococcus aureus and Listeria monocytogenes . Notably, S. aureus and L. monocytogenes demonstrated the lowest sensitivity to H. perforatum methanol extract (1625 µg/mL). The methanol extract displayed moderate antioxidant activity, with a 53% ABTS radical scavenging capacity. Consequently, the extracts of H. perforatum exhibited both antimicrobial and antioxidant potential.


INTRODUCTION
Free radicals are atoms or molecules carrying unpaired electrons, highly reactive and capable of rapidly engaging in exchange reactions that destabilize other molecules and generate many more free radicals (MANDAL et al., 2009).In biological systems, free radicals are often derived from oxygen, nitrogen, and sulfur molecules.These free radicals are components of molecular groups referred to as reactive oxygen species (ROS), reactive nitrogen species (RNS), and reactive sulfur species (RSS) (VAJRAGUPTA et al., 2004).If not neutralized, free radicals can cause damage to all cellular macromolecules, including proteins, carbohydrates,

O N L I N E F I R S T
There are numerous studies in the literature on the biological activities of plants.The plants from Hypericum genus are distributed worldwide and in Turkey.H. perforatum is a spontaneously growing plant in almost all of Turkey (ASLAN, 2012).Although there are studies on Hypericum species in the literature, research in Turkey remains limited.Therefore, the aim is to contribute to the literature by studying Hypericum samples from the Yaraş region of Muğla province in Turkey.This study specifically aims to investigate the antimicrobial activities against foodborne pathogens and the antioxidant activity of H. perforatum in Muğla.

Plant material and extraction
In May 2014, H. perforatum flowers were collected from the Yaraş region of Muğla at an altitude of approximately 700 meters above sea level.The coordinates of the region are 37.178211 latitude and 28.464499 longitude.The identification of this plant was carried out by Dr. Olcay Ceylan, and the plant specimen has been preserved in the Herbarium of the Department of Biology, Mugla Sitki Kocman University, with Herbarium No: MUH5692.The plant's identification was performed using the Flora of Turkey (DAVIS, 1988).
The flowers of the plant underwent a thorough washing process, including two rinses in running water and one rinse in sterile water.Following this, the materials were air-dried.Subsequently, they were pulverized using a blender and prepared for the study.The resulting samples were stored at room temperature until the initial preparation, after which they were transferred to a temperature-controlled environment at 4 °C for subsequent analysis.
To extract the plant samples, 30 grams of air-dried and powdered flowers were subjected to methanol (Merck) extraction using the Soxhlet apparatus (Isotex).300 mL methanol was added to the Soxhlet apparatus, and the last concentration was adjusted to 100 mg/mL.Then all experiments were conducted over a 4-hour period.After obtaining the extracts, they were evaporated (Heidolph) and then transferred into sterile amber bottles with their respective solvents (10 mL) to prevent the extract from drying out.These bottles were stored in a refrigerator until used in the study.

Organisms and cultivation
This study focused on the investigation of foodborne pathogenic organisms, specifically 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 strains used in this study were obtained from well-established institutions, including ATCC (American Type Culture Collection, USA), NCTC (National Type Culture Collection), and RSKK (Refik Saydam National Type Culture Collection, Turkey).
To conduct the tests, bacteria were cultured in Mueller-Hinton Broth (MHB)medium (Merck) and incubated at 37 °C for 24 hours, whereas C. albicans was cultured in Sabouraud Dextrose Broth (Merck) and incubated at 30 °C for 48 hours.

Measurement of antimicrobial activity
The antimicrobial activity of flower extract was assessed using the Kirby-Bauer method (BAUER et al., 1966).Methanol was employed as the organic solvent in this study.The extract was applied in a concentration and quantity of 35 µL of 100 mg/mL.
The bacterial and C. albicans cultures were adjusted to the 0.5 McFarland standard to achieve consistent turbidity.All experiments were conducted in triplicates, and the results are O N L I N E F I R S T presented as the mean values.Bacterial cultures were incubated in a 37 °C for 24 hours, while C. albicans cultures were incubated for 24 hours in a 30 °C.After the incubation period, the zones of inhibition around the discs were recorded.Methanol served as the negative control in the study, whereas the positive controls were antibiotics including tetracycline (Bioanalyse; 30 μg), nystatin (Bioanalyse; 100 μg), and penicillin (Bioanalyse; 10 μg).

Measurement of minimum inhibitory concentration (MIC)
An method for evaluating antimicrobial activity involves the Minimum Inhibitory Concentration (MIC) test.MIC is defined as the lowest concentration of the extract that effectively inhibits the growth of bacteria and fungi following an incubation period.The broth dilution test was conducted in accordance with the procedures outlined in the Clinical and the Laboratory Standards Institute (CLSI) standards (CLSI, 2003;CLSI, 2006).
A growth control tube without extract and a sterile control tube without bacterial inoculation were prepared for the study.All cultures were activated in Nutrient Broth (NB) (9 mL) at 37 °C for 18 hours.The turbidity of the inoculums was adjusted to the McFarland 0.5 standard.For this test, the final concentrations of the extract used were 6500, 3250, 1625, 812.5, and 406.25 μg/mL.These concentrations were employed to determine the MIC values for the respective plant extract against the tested microorganisms.
Active cultures (100 µL) were inoculated into tubes containing MHB (4.5 mL), and then 0.5 mL of the extract was added.Subsequently, all tubes were incubated at 37 °C for 24 hours.At the end of the incubation period, concentrations that inhibited the growth of microorganisms in the tubes were observed.The concentration at which there was a 90% or greater reduction compared to controls was recorded as the MIC value.

Measurement of non-enzymatic antioxidant capacity
The experiments utilized an improved 2,2′-azinobis-(3-ethyl benzothiazoline-6-sulfonic acid (ABTS) (Merck) radical decolorization assay (RE et al., 1999).Stock solutions included 7 mM ABTS•+ and 2.45 mM potassium persulfate (Merck).The working solution was prepared by equally mixing these stocks and allowing them to react for 12 hours at room temperature in the dark.This solution was then diluted by adding 10 μL of methanol to 1 mL of the ABTS•+ solution.Absorbance was measured at 734 nm using a spectrophotometer (Optizen), 15 minutes after mixing 10 μL of methanol extract (99.9%) with the ABTS • + solution.Trolox (Sigma-Aldrich) served as the reference standard, and the results are expressed as mM Trolox equivalents (TE)/g dry weight.

Essential oil isolation and analysis
In this study, 40 grams of the dried plant was taken and distilled in 1000 mL of distilled water by boiling in water with the Clevenger Distillation System for 4 hours.The resulting essential oil was dissolved with hexane.The extract was also dried with MgSO4.A yield of 0.03% (w/w) essential oil was obtained from the dried plant material on a dry basis.The essential oil has been supplied to the GC-MS system.The GC-MS analysis of the essential oil was performed using an Agilent Inert MS Detector 5975 & 6890 GCMS, equipped with a DB35 -MS column (

Statistical analyses
In this study, the means of the activities were calculated with Excel 2016.

RESULTS
In this study, the methanol extract of H. perforatum was subjected to in vitro testing against eight foodborne pathogenic microorganisms.The results of the antimicrobial activities of the plant extract are presented in Table 1.Additionally, Table 2 presents the diameters of inhibition zones produced by the reference antibiotics against these microorganisms.
At the conclusion of the antibacterial activity studies, the diameter of the formed inhibition zones was measured in millimeters and recorded.The results revealed that the methanol extract of H. perforatum effectively suppressed the growth of four bacterial strains.The largest inhibition zone diameters were observed for Staphylococcus aureus and Listeria monocytogenes, measuring 16 ± 1.25 mm and 16 ± 0.47 mm, respectively.Furthermore, the methanol extract of this plant exhibited no discernible anticandidal effects against the employed yeast strain.Remarkably, the methanol extract of the flowers demonstrated significant efficacy against both S. aureus and L. monocytogenes, yielding the maximum zone of inhibition (16 mm).However, the methanol extract did not produce any inhibition zones against three bacterial strains (as shown in Table 1).Tetracycline (30μg), nystatin (100μg), and penicillin (10μg) antibiotics were employed as positive controls.Tetracycline exhibited a robust inhibitory effect on the growth of Yersinia enterocolitica (Table 2).
Table 3 displays the Minimum Inhibitory Concentrations (MICs) of H. perforatum flower extract, as determined utilizing the broth dilution method.Among the tested microorganisms, two bacteria exhibited the lowest sensitivity to the methanol extract of H. perforatum, with MIC values of 1625 µg/mL, except for Salmonella Typhimurium, which had a MIC value of 6500 µg/mL.The MIC value of Bacillus subtilis was 3250 µg/mL.Table 4 displays the non-enzymatic antioxidant activity of the plant extract assessed using the ABTS radical scavenging method.Trolox served as the positive control, and all values were expressed in terms of the Trolox equivalent.The flower extract at a concentration of 100 mg/mL exhibited 53% inhibition.At the end of the study, the Trolox Equivalent (TE) was determined to be 0.31 mM/g DW.The results of the chemical analysis of H. perforatum essential oil by using GC-MS methods are listed in Figure 1.Fifty-four components were identified, making 71.34% of total oil ingredients.The main components of H. perforatum oil were: 1-tetra decene (18.52%), 1dodecanal (8.23%), β-selinene (7.66%), α-selinene (5.325%), cyclododecane (4.465%), 2pentadecanonane (3.46%), trans-betafarnesene (2.57%) and 2-tetra decene (2.155%).The results of the RT values of H. perforatum essential oil are listed in Table 5.The components include 1-tetradecene and 2-tetradecene belonging to the alkene group, and 1-dodecanal classified under the long-chain fatty acid aldehyde group.β-selinene, α-selinene, and trans-beta farnesene are part of the sesquiterpene group.Cyclododecane falls within the cycloalkane group.2-pentadecanonane is associated with the methyl tridecyl ketone group.

DISCUSSION
Medicinal plants have traditionally been employed globally in the treatment of various human ailments (CHITME et al., 2004).These plants have been acknowledged as abundant reservoirs of biologically active compounds, many of which have served as foundational

O N L I N E F I R S T
elements for the advancement of novel pharmaceuticals (PALOMBO, 2011).In this study, H. perforatum flowers were selected based on their ethnomedical use.In the current study, the methanol extract of the flowers was tested against eight microorganisms to determine their antimicrobial activities.The results revealed that the methanol extract inhibited the growth of four bacteria (Table 1).The methanol extraction of the Holarrhena antidysenterica drug showed high activity on the pathogens above the 16 mm inhibition zone (AHMAD and AQIL, 2007).Researchers found a lot of compounds in H. perforatum.Numerous flavonoid compounds, including hyperoside, quercitrin, isoquercitrin, rutin, quercetin, campferol, luteolin, and myricetin are found in the aboveground portions of the plant, including the leaves, stalk, flowers, and buds (GREESON et al., 2001).In this study, the methanol extract of the flowers did not inhibit the growth of two Gramnegative bacteria, namely Escherichia coli and Yersinia enterocolitica.Previous studies have consistently found that Gram-positive bacteria are more susceptible to plant extracts compared to Gram-negative bacteria (PAREKH and CHANDA, 2006;GARCIA et al., 2008;NAZZARO et al., 2013;DENG et al., 2020), likely due to structural differences in their cell walls.Gram-positive bacteria possess a single-layered cell wall, while Gram-negative bacteria have a more complex and multilayered cell wall structure (SILHAVY et al., 2010).

O N L I N E F I R S T
The results of this study demonstrated that the tested plant extract exhibited high effectiveness against S. aureus and Listeria monocytogenes.In a study by Oskay et al., it was revealed that H. perforatum L. methanol and ethanol extracts showed high sensitivity against methicillin-resistant S. aureus (MRSA), making it the most susceptible organism (OSKAY et al., 2009).Similarly, the extract of H. perforatum showed an inhibitory effect of 16 mm against Staphylococcus aureus (KELEŞ et al., 2001).These findings align with the results obtained in our study.
In this study, the methanol extract exhibited a 53% inhibition of free radicals at a concentration of 100 mg/mL.These results are similar to the literature (HUCK et al., 2006;TATSIS et al., 2007).Studies have demonstrated that H. perforatum contains compounds exhibiting various biological activities.Among these are flavonoids and phenolic acids, which contribute to its antioxidant activity.Very few studies on the antioxidant activity of H. perforatum have been found in the literature.One of these is the study by Okmen and Balpınar.In their study, Okmen and Balpinar reported that the DPPH scavenging activity of H. perforatum flowers exhibited 32% inhibition (OKMEN and BALPINAR, 2017).Another study was conducted by GÜZEL et al. (2019), where the ABTS radical scavenging activity of H. perforatum was reported to be approximately 20%.The studies support the results of this study.
The components obtained from the composition study and having a high percentage belong to the alkene, long-chain fatty acid aldehyde, sesquiterpene, and methyl tridecyl ketone groups.These groups constitute 52.38% of the oil obtained in this study.Therefore, the presence of antimicrobial and antioxidant activity can be attributed to the high proportion of these groups in the plant's essential oils and their substantial effectiveness (SADDIQE et

O N L I N E F I R S T
The phytochemical composition of H. perforatum has been reported in this study.The main compounds from the methanol extract of H. perforatum were identified to be 1-tetra decene and 1-dodecanal as determined by GCMS (Figure 1).In many studies with other species belonging to Hypericum, different results have been reported (SHAROPOV et al., 2010;SHAFAGHAT, 2011;JAIMAND et al., 2012;PIRBALOUTI et al., 2014;KÜÇÜK et al., 2015;YÜCE, 2016;SCHEPETKIN et al., 2020;GÜLER and OZDEMİR, 2023).However, this study supports the studies in the literature.
In studies on the essential oils of H. perforatum in the literature, different components have been obtained not only from various countries but also from different regions of the same country.Furthermore, the percentages of these components have been found to vary.Some components obtained in this study overlap with those in the literature but have different values (HOSNİ et al., 2008;ÇIRAK et al., 2010;DEVECİ, 2014;CARRUBBA et al., 2021;GÜLER, 2022).Several factors influence the quantity and composition of essential oils in plants.These factors vary depending on which part of the plant the essential oil is derived from, the species of the plant, the geographical conditions of the region where the plant is located, climate, the growth stages of the plant, and variations in extraction methods (BAYAZ, 2014).

CONCLUSION
The methanol extract of H. perforatum demonstrated high efficacy against S.s aureus and L. monocytogenes, highlighting its potential as a natural antimicrobial agent.It exhibited maximum inhibition against foodborne pathogens.These findings support the traditional medicinal use of this plant and suggest that certain extracts possess promising antibacterial compounds, which could be explored as potential agents in the search for new drugs.H. perforatum flower extract showed moderate antioxidant activity in vitro, potentially offering beneficial antioxidant protection against oxidative damage in the human body.Further research is needed to explore the bioactive compounds in this plant and investigate its antimicrobial and antioxidant effects.Determining the active compounds is crucial for a deeper understanding of H. perforatum.
30 m x 0.25 mm x 0.25 μM).Temperature program: Oven temperature 60 °C 10 min 4 °C/min 220 10 min 1 °C/min 240 0 Gas flow: Helium 1 mL/min m/z : Scanned between 40-550.Ion source temperature: 230 o C O N L I N E F I R S T Wiley and NIST libraries on the computer were used to identify the components (SCHWOB et al., 2002; BALEA et al., 2020).

Figure 1 .
Figure 1.Chemical compounds of methanol extract of H. perforatum

Table 2 .
Standard antibiotics susceptibility of tested microorganisms

Table 3 .
Minimum inhibitory concentration values of H. perforatum flower extract

Table 4 .
ABTS radical scavenging capacity of H. perforatum flower extract DW: Dry weight