THE ANTIBACTERIAL ACTIVITIES OF SYZYGIUM AROMATICUM (L.) MERR. & PERRY AGAINST ORAL BACTERIA AND ITS ANTIOXIDANT AND ANTIMUTAGENIC ACTIVITIES

THE ANTIBACTERIAL ACTIVITIES OF SYZYGIUM AROMATICUM (L.) MERR. & PERRY AGAINST ORAL BACTERIA AND ITS ANTIOXIDANT AND ANTIMUTAGENIC ACTIVITIES

Gulten Okmen ^*, Mahabbat Mammadhkanli and Mustafa Vurkun

Department of Biology, Mugla Sitki Kocman University, Faculty of Science, Mugla 48000, Turkey.

ABSTRACT: Plants are an important source of substances which are claimed to induce biological activities. Although there are a few studies on antimicrobial and antioxidant activities of this plant, antimutagenic activity has not been studied and there is no study in Turkey. Antibacterial activities of Syzygium aromaticum against oral pathogens have not been reported until today. The scope of this work was to investigate the biological activities of S. aromaticum different extracts. The various extracts were screened for antibacterial activity. The bacteria were isolated from oral flora by traditional methods. The plant extracts were tested by Kirby-Bauer method. Other antibacterial activities tests are MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration). In addition to, the antioxidant activities of plant extracts were screened by the stable DPPH (2, 2-diphenyl- 1-picrylhydrazyl hydrate) free-radical. The antimutagenicity of the plant extracts were determined by Ames test using Salmonella typimurium strains. The highest antibacterial activity was determined as 20 mm inhibition zone from methanol extracts. The highest DPPH scavenging activity was found as 82% from aqueous extract. S. aromaticum extracts have antibacterial, antioxidant and antimutagenic potentials. Our results support the use of this plant in traditional medicine and show that some of the plant extracts possess compounds with good biological activities.

Syzygium aromaticum, Antibacterial activity, Antioxidant activity, Antimutagenic activity

INTRODUCTION: Dental caries are one of the public health concerns for several reasons. Teeth affected with dental caries are sources of infection, which can cause an inflammation of dental pulp, periodonteum and gums. If left untreated, this disease gradually leads to teeth loss, which causes chewing difficulties and aesthetic problems ¹. It remains one of the most widespread diseases of the mankind. In developing countries, dental caries is often at epidemic proportions, especially among the poor.

Nowadays microorganisms have become resistance to many antibiotics due to increased use of drugs, which is decreasing efficiency of conventional medicines. So, it has become necessary to find out new antimicrobial agents. Prevention of pathogenic microorganisms in dental caries are usually achieved by using chemical preservatives but they are responsible for many carcinogenic and teratogenic attributes as well as residual toxicity and with growing concern of microbial resistance towards conventional preservatives, consumers tend to be suspicious of chemical additives and thus the exploration of naturally occurring antimicrobial for mouth preservatives receives increasing attention ². Presently, the major problem is that we can not use chemical preservatives safely now a day due to carcino-genic nature of these chemicals ³.

Higher aromatics plants have traditionally been used in folk medicine; antimicrobial properties of these plants are well documented against bacteria, fungi and yeasts ⁴. Most of the medicinal properties of these plants are directly correlated with the essential oils produced by these plants. Essential oils and extracts of these plants are able to control microorganisms related to skin diseases, dental caries and food spoilage ⁵.

Syzygium aromaticum (clove) is one of the most valuable spices that have been used from centuries as food preservative and for many medicinal purposes. Nowadays cloves are cultivated in several parts of the World ⁶. Syzygium species (Fam. Myrtaceae) have been reported to possess biological activities ⁷. Clove’s Botanical name is Caryophyllus aromaticus which is derived from the Latin "clavus", which means nail due to its resemblance with the shape. The clove tree is an evergreen tropical plant, which flowers twice every year. Cloves are the unopened buds and harvested when the outer green leaves have changed from green to a yellow pink ⁸. The cloves are highly antiseptic ⁹, antimutagenic, anti-inflammatory, antioxidant, anti-ulcerogenic, antithrombotic, anti-parasitic ¹⁰, antibacterial ¹¹, antifungal ¹², and antiviral ¹³. Bud oil of clove has natural behavior and the main properties include antioxidant, insecticidal, antifungal and antibacterial properties ³. Flower bud have many medicinal properties like antimicrobial, general stimulating, carminative and anesthetic ^{14, 15}.

The active ingredients of plants against microorganisms are mostly some of the secondary metabolites ⁸. The most important constituent of clove is the eugenol due to which it has strong characteristic aroma ¹⁶. Several compounds from S. aromaticum have been found to possess growth inhibitory activity against oral pathogens, namely 5, 7-dihydroxy-2-methylchromone-8-C-β- D-gluco-pyranoside, biflorin, kaempferol, rhamnocitrin, myricetin, gallic acid, ellagic acid and oleanolic acid ¹⁷. Recently, flavonoide triglycosides have been isolated ¹⁸. The present study was scoped to evaluate the antibacterial, antioxidant and antimutagenic potency of Syzygium aromaticum plant from Turkey, therefore justifying the use of this plant in ethno-medicine for treatment of various ailments.

MATERIALS AND METHODS:

Organisms: In this study, 8 bacteria were used in experiments. Bacteria were isolated aseptically from mouth flora of different people. The identifications of bacteria were studied by traditional methods by Assoc. Prof. Dr. Gulten Okmen ^{19, 20, 21}. The bacterial growth were provided at Mueller- Hinton Broth (MHB; Merck).. Incubation was at 37 ºC for 24 h.

Plant Material: Syzygium aromaticum dried flower table was obtained from akhtars in Mugla on October 2017. The identity of plant was confirmed by Prof. Dr. Guven Gork. The specimens were stored at the Herbarium of Department of Biology, Mugla Sitki Kocman University (Voucher no: OC 1250). The identification of the plant was carried out with the Flora of Turkey ²².

Plant Extraction: The dried flower tables were washed with flowing water and once with sterile distilled water. This process was done 2-3 times. Then this material was powdered in a blender. All samples were stored at ambient temperature until initial sample preparation, after which they were stored at 4 ºC until required for analysis. The samples (30 g) were extracted with ethanol, methanol and aqueous (350 mg/mL) using by Soxhlet. These experiments were continued for 4 h. All of the 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 the extracts concentrations were set to 350 mg/mL.

Cultivation of Microorganisms: The extracts were tested against oral pathogens. The oral pathogens were grown at 37 ºC at Mueller- Hinton Broth (Merck). Duration of incubation was 24 h.

In-vitro Antibacterial Activity: Antibacterial activity studies were done with Kirby-Bauer method ²³. The extracts of plant were tested by disc diffusion assay. The concentration and quantity of extracts were taken as 350 mg/ml and 30 µL. In this study, ethanol, methanol and aqueous were used as organic solvents. The active cultures of bacteria were inoculated on Mueller-Hinton agar plates (MHA, Merck). The concentrations of cultures were adjusted to 0.5 McFarland. The experiments were performed in triplicate. The incubation of bacteria were done at 37 ºC in 24 h. Then, the inhibition zone values were measured. Ethanol, methanol and water are negative controls. In this study, a lot of antibiotics used for positive control.

Determination of Minimum İnhibitory Concentration (MIC): The other antibacterial activity study is MIC. The broth dilution method was done as described in the CLSI standards ^{24, 25}. In this test, final concentrations of each extract were performed as 26000, 13000, 6500, 3250 and 1625 μg/mL.

Determination of Minimum Bactericidal Concentration (MBC): MBC was determined by using the broth dilution technique ²⁶ by assaying the test tubes resulting from MIC determinations. A loopful of the content of each test tube was independently inoculated by streaking on a solidified nutrient agar plate incubated at 37 ºC for 24 h and then observed for bacterial growth. The lowest concentration of the subculture with no growth was considered the minimum bactericidal concentration.

Determination of Non-Enzymatic Antioxidant Activity: The stable 2,2-diphenyl-1-picrylhydrazyl hydrate radical (DPPH) was used for determination of free radical scavenging activities of the extracts. Extract (0.1 mL) was added to 3.9 mL of a 0.1 mM methanol DPPH solution. After incubation for 30 min, absorbance of extract was measured at 515 nm by spectrophotometer. Methanol is blank. The methanol with DPPH solution was used as control ²⁷. Trolox is reference antioxidant. The DPPH scavenging capacity expressed in percentage (%) was calculated using the formula.

Determination of Antimutagenic Activity: Antimutagenic activity tests were evaluated by the Salmonella - microsome method. Salmonella typimurium tester strains TA98 and TA100 were used in this study. The bacteria kindly provided by Dr. B. N. Ames (Berkeley, CA, USA), without (−S9) metabolization by the pre-incubation method ²⁸. In this study, these strains included Salmonella typimurium TA98 and TA100. The Salmonella histidine point mutation assay of Maron and Ames ²⁸ was used to determine the antimutagenic activities of Syzygium aromaticum extracts without S9 mix. The percentage of inhibition was calculated according to the formula given by Ong et al., ²⁹ 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 50000, 25000, 12500, 6250, 3125 and 1562 µg/plate.

RESULTS: The antibacterial activities of extracts of Syzygium aromaticum were tested against 8 microorganisms, which are known to cause diseases in teeths. MBKK1 and MBKK2 were Gram negative bacteria. The other bacteria were Staphylococcus and Gram positive. These bacteria are including Serratia sp. MBKK1 and MBKK2, Staphylococcus sp. MBKK3, S. aureus MBKK4 and MBKK5, Staphylococcus epidermidis MBKK6, MBKK7 and MBKK8. The table of identification not shown. Results of antibacterial activities of used plant extracts against the test pathogens are shown in Table 1. Besides, the inhibition zone diameters of the reference antibiotics to the test organisms are shown in Table 2.

TABLE 1: ANTIBACTERIAL ACTIVITIES OF SYZYGIUM AROMATICUM EXTRACTS AGAINST ORAL PATHOGENS (350 mg/mL)

EE: Ethanol extract ME: Methanol extract AE: Aqueous extract  (-): zone did not occur E: Ethanol M: Methanol A: Aqueous

The results of zones of inhibition were recorded as in mm for all the materials used as follows. Results show that Syzygium aromaticum extracts inhibit the growths of bacteria and the inhibition zones were between 8 to 20 mm. The highest inhibition zone was found against Staphylococcus sp. MBKK3.

The inhibition zone was 20 mm. Additionally, all of the extracts were determined antibacterial effects against used test bacteria Table 1. Reference antibiotics used as positive control. A lot of antibiotics very strongly inhibited the bacterial growths Table 2 and 3.

TABLE 2: REFERENCE ANTIBIOTICS PROFILES OF ORAL PATHOGENS

nt: not tested

TABLE 3: REFERENCE ANTIBIOTICS PROFILES OF OTHER PATHOGENS

(-): No inhibition nt: Not tested

Table 4 shows MIC values of Syzygium aromaticum extracts. The lowest MIC value was 1625 µg/mL for two bacteria.

TABLE 4: MINIMUM INHIBITORY CONCENTRATIONS OF SYZYGIUM AROMATICUM EXTRACTS (µg/mL)

nt: Not tested (-): No inhibition EE: Ethanol extract ME: Methanol extract AE: Aqueous extract

Table 5 shows MBCs of Syzygium aromaticum extracts obtained by the broth dilution method. The lowest MBC value was 3250 µg/mL for two bacteria.

TABLE 5: MINIMUM BACTERICIDAL CONCEN-TRATIONS OF SYZYGIUM AROMATICUM EXTRACTS (µg/mL)

nt: Not tested (-): No inhibition EE: Ethanol extract ME: Methanol extract AE: Aqueous extract

Table 6 shows the percent of DPPH radical scavenging capacity with trolox as reference. The aqueous extract showed 82.7% inhibition at 350 mg/mL concentration. Trolox equivalent value was 2.2 mM/g Table 6.

TABLE 6: ANTİOXİDANT ACTİVİTİES OF SYZYGİUM AROMATİCUM (350 mg/mL)

TE: Trolox equivalent (mM/g DW); DW: Dry weight EE: Ethanol extract ME: Methanol extract AE: Aqueous extract

In this study, these concentrations were categorized as non-toxic because they showed a well-developed lawn, almost similar size of colonies and no statistical difference in the number of spontaneous revertants in test and control plates. The antimutagenic activities of the extracts were evaluated by the against NaN₃ (sodium azide) by Ames test in absence of rat microsomal liver enzyme (-S9). Table 7 and 8 shows the percent of inhibition. The methanol extracts of Syzygium aromaticum (6250 µg/plate) was found to have its lowest antimutagenic activity for Salmonella typhimurium TA98. This inhibition value is 15% Table 7. S. aromaticum extracts (6250 µg/plate) detected a moderate positive effect (27 %) for S. typhimurium TA100 Table 8.

TABLE 7: ANTIMUTAGENIC ACTIVITY OF SYZYGIUM AROMATICUM EXTRACTS (6250 µg/plak)

Sa: Syzygium aromaticum

TABLE 8: ANTIMUTAGENIC ACTIVITY OF SYZYGIUM AROMATICUM EXTRACTS (6250 µg/plak)

Sa: Syzygium aromaticum

DISCUSSION: Herbal medicines have been shown to have genuine utility and about 80% of rural population depends on its primary health care. Bioactive compounds are playing an important role for the treatment of different diseases. As a results of findings, the Syzygium aromaticum flower tables contain bioactive compounds that explain the importance of S. aromaticum as medicinal plant. Results show that the S. aromaticum extracts inhibit bacterial growths. The highest inhibition zone was found against MBKK and the inhibition zone was 20 mm Table 1. Soni and Dahiya ³⁰ reported that antimicrobial activities of Syzygium caryophyllatum essential oil was found between 7 to 22 mm inhibition zone. High levels of eugenol present in S. caryophyllatum essential oil is responsible for strong antimicrobial activity. This phenolic compound can denature proteins and reacts with cell membrane phospholipids changing their permeability ^{31, 32, 33}.

In this work, antibacterial activities of S. aromaticum extracts were found against Gram positive bacteria Table 1. All of these bacteria are Staphylococcus. Abdelkader and Halawani ³⁴ reported that Staphylococcus aureus ATCC 25923 were affected strongly from S. aromaticum extracts. A previous study in Turkey ³⁵ showed that the chemical composition of S. aromaticum oil had about 87% eugenol, 8% eugenyl acetate and 3.6% β-caryophyllene. The modes of action by which microorganisms are inhibited by essential oil and their chemical compounds seem to involve different mechanisms. It has been hypothesized that the inhibition involves phenolic compounds, because these compounds sensitize the microbial cytoplasmic membrane causing increased permeability, unavailability of vital intracellular ingredients ³⁶ and / or impairment of bacterial enzymes systems ³⁷. Previous studies also showed that clove had strong antibacterial activity against Gram positive bacteria ^{38, 39, 40, 41}. Our results are in agreement with those reported by these studies.

In our study, extracts were affected two Gram negative bacteria Table 1. The antimicrobial activity of Syzygium caryophyllatum oil showed strong antibacterial activity against all bacterial isolates tested with maximum activity against Pseudomonas aeruginosa. Klebsiella pneumoniae, Serratia marcescens, Salmonella typhi, Shigella dysentriae and Vibrio cholerae were found resistant ¹³. The antibacterial activity of S. caryophyllatum is attributed to eugenol. High tannin content (10 - 19%) in S. caryophyllatum also provides additional antimicrobial activity ⁴². The antibacterial activity of flavonoids can be explained by the toxicity of this compound towards non- specific interactions in showed susceptibility, such as the establishment of hydrogen bonds with the cell walls proteins or enzymes, the chelation of metal ions, inhibition of bacterial metabolism, sequestration of substances necessary for the growth of bacteria.

Also, the β-ring of flavonoids is important in the intercalation with nucleic acids, thus inhibits DNA and RNA synthesis. It can also inhibit the DNA gyrase of Escherichia coli ^{43, 44}. Previous studies also found that clove had strong antibacterial activity against Gram positive bacteria ^{13, 38, 39, 40, 41, 45, 46}. Our results are in agreement with those reported by these studies.

In this study, two bacteria were showed the lowest sensitivity to extracts of Syzygium aromaticum (1625 µg/mL). Therefore MBC value was 3250 µg/mL Table 4. Abdelkader and Halawani ³⁴ reported that ethanolic extracts of S. aromaticum exhibited maximum activities against S. aureus ATCC 25923 with MIC = 62.5 μg/mL while MBC value was 125 μg/mL. Barakat ³⁸ reported that MIC value for S. aureus was 1500 µg/mL. Dua et al., ⁴¹ reported that MIC values for S. aureus and E. coli were 0.98 and 3.90 mg/mL, respectively. Whereas Karunamoonthy et al., ⁴⁷ were studied by S. benthamianum, and MIC values were found 250 and 500 µg/mL for E. coli and S. aureus, respectively. Results of our study are similar with this results.

In these study, the extracts of S. aromaticum have different free radical inhibition. The aqueous extract of flower tables showed 82.7% inhibition at 350 mg/mL concentration Table 6. Previous studies also showed that clove had strong antioxidant activity, and a high level of phenolics ^{48, 49}. Our results are in agreement with those reported by these studies. These DPPH scavenging activity 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 ¹³.

In our study, the extracts of S. aromaticum (6250 µg/plate) were found to have its low antimutagenic activity for Salmonella typhimurium TA98. This inhibition value is 17% Table 6. Whereas, ethanol extract of plant were shown moderate effect for S. typhimurium TA100 Table 7. In determining the antimutagenic potential of a sample, a value smaller than 20% inhibition of the mutagen activity indicates a weak or non-antimutagenic effect, a moderate effect when the value is between 20 and 40% and strong antimutagenicity when the value is greater than 40% ⁵⁰. Karunamoonthy et al., ⁴⁷ reported anticancer activity for S. benthamianum. This also supports our results.

CONCLUSION: In the present study, extracts of Syzygium aromaticum inhibited the bacterial growths, but their effectiveness varied. Ethanolic, methanolic and water extracts of S. aromaticum showed considerable antibacterial properties against the tested organisms. 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 S. aromaticum aqueous extract. The aqueous extract of S. aromaticum, should be useful as an antioxidant protection system. Furthermore the extracts of S. aromaticum have weak anti-mutagenic activity for Salmonella typhimurium TA98. Whereas, the extracts of S. aromaticum have moderate antimutagenic activity for S. typhimurium TA100.

It may be suggested from the present findings that S. aromaticum extracts can be used as a potential source of natural antimicrobial compound possessing strong antioxidant potential. However, further research is needed for the identification of biologically active compounds present and in vivo studies using animal model. In subsequent researches, fractionation and characterization of the active components should be do further works to investigate.

ACKNOWLEDGEMENT: This study was supported by Mugla Sitki Kocman University Research Funds (Project number: 17/072). The authors wish to thank Prof. Dr. M. Guven Gork for identification of this plant (Mugla Sitki Kocman University, Turkey).

CONFLICT OF INTEREST: The authors declare no conflict of interest.

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    How to cite this article:

    Okmen G, Mammadhkanli M and Vurkun M: The antibacterial activities of Syzygium aromaticum (L.) Merr. & Perry against oral bacteria and its antioxidant and antimutagenic activities. Int J Pharm Sci & Res 2018; 9(11): 4634-41. doi: 10.13040/IJPSR.0975-8232.9(11).4634-41.

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