Activities and Mechanisms of Eugenol and Esential Oil Betel Leaf (Piper Betle, Linn) Against Some Bacterial Pathogens

Eco. Env. & Cons. 25 (3) : 2019; pp. (1461-1465) Copyright@ EM International ISSN 0971–765X

Activities and mechanisms of Eugenol and esential oil betel leaf (Piper betle, Linn) against some bacterial pathogens

*Muhammad Yanis Musdja1, Ulfa Sadiah1 and Eka Putri1

Department of Pharmacy, State Islamic University, Jakarta, Indonesia

(Received 20 January, 2019; accepted 30 March, 2019)

ABSTRACT Eugenol is one of the ingredients of essential oils of betel leaf and clove pistil, essential oils of betel leaf and eugenol are used by health workers and the community against microbes. This aim of this research was to determine the activity and mechanism of inhibition of the essential oil of betel leaf that was compared to eugenol against Proteus mirabilis, Proteus vulgaris, Salmonella thypimurium, Shigella flexneri, and Streptococcus mutans. Determination of Minimum Inhibitory Concentration (MIC) was done by using micro dilution method. MIC values of essential oil for Proteus mirabilis 9% (v/v), Proteus vulgaris 6% (v/v), Salmonella thypimurium 5% (v/v), Shigella flexneri 11% (v/v), and Streptococcus mutans more than 17% (v/v), and for eugenol was obtained MICvalues for Proteus mirabilis 0,4% (v/v), Proteus vulgaris 0,5% (v/v), Salmonella thypimurium 0,2% (v/v), Shigella flexneri 0,5% (v/v) and Streptococcus mutans more 0,4% (v/v). mechanism of inhibition of the essential oil of betel leaf and eugenol could cause membrane damage that indicated with the accurrence of leakage of cellular metabolites. In this cases, could be seen with the increase leaked nucleic acid and protein from cell of bacteria that indicates an increased absorbance at wavelength of 260 nm and 280 nm by using Ultra Violet-Visible Spectrophotometer. This was reinforced by an increase in leakage ions Ca ,+and K+,with observed by using Atomic Absorption Spectrophotometre (AAS) moreover also occurred plasmolysis on bacterial that was observed by using Scanning Electron Microscope (SEM).

Key words : Piper betle, Proteus mirabilis, Proteus vulgaris, Salmonella thypimurium, Shigellaflexneri, Streptococcus mutans, Antibacteri.

Introduction against bacteria, parasites, viruses and fungi. In this case if the experts fail to find new drugs to fight in- Finding new antimicrobial compounds is very ur- fection, then in the next 20-30 years, to treat infec- gent, because as predicted by WHO, in the near fu- tious diseases will be more difficult. Antimicrobial ture, we will enter the post-antibiotic era. Because resistance happens when microorganisms, i.e bac- the antibiotics that exist now, from time to time, teria, fungi, viruses, and parasites change when have continued to increase resistance to antibiotics they are exposed to Anti-microbial drugs such as that exist today. Therefore, on the world health day antibiotics, antifungals, antivirals, antimalarials, in 2011, the WHO has declared combat to antimicro- and anthelmintics. At this ime, new resistance bial resistance (AMR) worldwide (WHO, 2011) mechanisms are emerging and spreading globally, Antimicrobial resistance (AMR) has threatened threatening our ability to treat common infectious the prevention and treatment of infectious diseases diseases, resulting in prolonged illness, disability

*Corresponding author’s email : yanis.musdja @uinjkt.ac.id 1462 Eco. Env. & Cons. 25 (3) : 2019

and death. Antimicrobial resistance occurs naturally methods (2006) by using micro-dilution (Supprakul over time, usually through genetic changes. Antimi- et al., 2006). crobial resistant-microbes are found in people, Analysis of Proteins and Nucleic Acids was de- animals, food, and the environment (in water, soil termined based on Carson et al., methods (2002) and air). Antibiotic resistance is present in every from suspension of test bacteria that had been incu- country, especially against, resistance in tuberculo- bated for 24 hours in Mueller-Hinton Broth Medium sis (TB), malaria. HIV, influenza etc (WHO, 2018, (MHB) by using UV-VIS spectrophotometer Cowan, 1999). (Carson et al., 2002; Glasel, 1995) Therefore it is very necessary to look for new an- Analysis of leakage of metals were measured in tibacterials to prevent and treat infectious diseases, the form ions Ca2+ and K+ based Carson et al., meth- especially those sourced from natural products. In ods (2002) by using Atomic Absorption Spectrum the essential oil of betel leaf, there are many (Carson et al., 2002) phenol compounds which are strongly suspected to Analysis of changes in bacterial cell morphology have efficacy against microbes. Because of the large was done based on Carlson methods (2002) by us- number of chemical compounds, where the possi- ing Scanning Electron Microscopy (SEM) (Carson bility of work is synergistic. It is likely that mi- et al. methods, 2002; Cox et al., 2001) crobes are more difficult to become resistant to betel leaf essential oil. Eugenol has long been used Results and Discussion as a disinfectant, especially by dentists. Eugenol can be made synthetically or can also be obtained by iso- The result of the identification was carried out by lating from betel leaves, or cloves. (Coralie Pavesi et the Biology Research Center, Indonesian Institute of al., 2018; Abd El-Baky et al., 2016; Hamed et al., Sciences, Bogor, Indonesia, showed that the plants 2012). used was true betel leaves (Piper betle Linn). Usually natural products have smaller side ef- Essential oil of betel leaf was isolated in a fresh fects than synthetic chemical compounds or single condition by using steam distillation methods chemical compounds (Cowan, 1999). However, to which was clear yellowish in color with a distinctive compare the advantages and disadvantages be- odor such as betel nut with a yield value of 0.14% (v tween eugenol and betel leaf essential oil as antibac- / w). terial, other studies are needed, such as safety and Essential oil was analyzed by using GC-MS and side effects, types of bacteria that are more sensitive obtained results in the form of chromatograms. and the prices of these two compounds, etc. Chromatogram results showed the chemical components of essential oil of betel leaf has 57 components Materials and Methods with major components (> 1%) as many as 18 components and minor components (<1%) as many as Betel leaf (Piper bettle Linn) was obtained from the 39 chemical components of essential oil of betel leaf. Bogor Medicinal and Aromatic Plant, Bogor, Indo- nesia. Betel leaf (Piper bettle Linn) was identified at the Center for Biological Research, Indonesian Insti- tute of Sciences, Bogor Indonesia Preparation of test material and isolation of betel leaf essential oil was done with steam distillation methods. Identify the chemical components of essential oils of betel leaf and eugenol was done by using GC-MS (Caburian et al., 2010; Suppakul et al., 2006) Determination of diameter zone inhibition was carried out based on Supprakul et al., methods (2006) by using paper disc diffusion (Supprakul et al., 2006) Determination of Minimum Inhibition Concen- tration was carried out based on Supprakul et al Fig. 1. Chromatogram of of betel oil (Piper bettle Linn) MUSDJA ET AL 1463

As shown in Figure 1. Table 2. The absorbance values of nucleic acid com- Synthetic Eugenol was obtained from the Indone- pounds (260 nm) of essential oils and eugenot sian Institute of Sciences, after analysis by using against Shigella flexneri with UV-Vis. GCMS, showed that the compound was Eugenol Treatment Absorbance Absorbance with very high purity or high quality. As shown in betel oil eugenol Figure 2. Normal Control 0.283 0.283 The test results of the zone of inhibition of vola- 1 MIC 1.018 1.446 tile oil and eugenol, each with a concentration of 2 MIC 1.366 1.842 15% against several gram (+) pathogenic bacteria are shown in Table 1. Table 3. The absorbance values of protein (280 nm) of es- Antibacterial activity test results with concentra- sential oils and eugenot against Shigella flexneri tion 15% of both essential oil of betel leaf & eugenol with UV-Vis. against some bacteria. Determination of MIC value for essential oils of Treatment Absorbance Absorbance betel oil eugenol betel leaf was startted from a concentration of 4% to 11% (v / v), except for Sreptococcus mutans was Normal Control 0,260 0,260 started from a concentration of 4-17% (v / v) and for 1 MIC 1,351 1,552 eugenol was started from a concentration of 0.1% - 2 MIC 1,630 1,709 0 , 8% (v / v). This concentration determination was based on 100% bacterial growth inhibition by essen- Table 4. Measurement of Ca2+ ion levels of Shigella tial oils of betel leaf and eugenol at the lowest con- flexneri by using Atomic Absorption Spectro- centration. As shown in Table 2 photometry (AAS). Protein leak analysis at a wavelength of 260 nm Treatment Iion Ca2+ betel Ion Ca2+ eugenol and nucleic acid at a wavelength of 280 nm was car- (Concentration) oil (ppm) (ppm) ried out using a UV / VIS spectrophotometer. In Normal control 4,21 4,21 this experiment, Shigella flexneri was used as a com- 1 MIC 27 31 parison with no essential oils and eugenol. In this 2 MIC 32 35 case a dose of 1MIC (11%) and 2 MIC (22%) was used for essential oils and a dose of 1MIC (0.5%) and 2MIC(1%) for eugenol. As shown in Table 3. Table 5. Measurement of K+ ion of Shigella flexneri by using Atomic Absorption Spectrophotometri Analysis of Ca2+ and K+ ion leakage were carried (AAS) out by using Atomic Absorption Spectrophotometry (AAS). In this experiment, Shigella flexneri was used Perlakuan Ion K+ betel Ion K+ eugenol as a comparison without giving essential oils and (Konsentrasi) oil (ppm) (ppm) eugenol. In this case a dose of 1MIC (11%) and Normal control 52,3 52,3 2MIC (22%) were used for essential oils and a dose 1 MIC 61 78 of 1MIC (0.5%) and 2MIC (1%) for eugenol, as 2 MIC 86 90 shown in Table 5 Morphological changes in Shigella flexneri cells shown in Figure 2.b and 2.c for essential oils and 2.d was caused by betel oils and eugenol at a dose of 1 and 2.e for eugenol. MIC and 2 MIC was observed by using SEM, as In this case there was a difference between nor-

Table 1. Antibacterial activity test results with concentration 15% of both essential oil of betel leaf & eugenol against some bacteria. No. Bacteria Test sample % (v/v) “ Zone of inhibition 1. Proteus mirabillis 15% Oil of betel leaf 15% Eugenol 3 mm4 mm 2. Proteus vulgaris 15% Oil of betel leaf 15% Eugenol 3 mm7 mm 3. Salmonella thypemerium 15% Oil of betel leaf 15% Eugenol 5 mm6 mm 4. Shigella flexneri 15% Oil of betel leaf 15% Eugenol 5 mm8 mm 5. Streptococcus mutans 15% Oil of betel leaf 15% Eugenol 1 mm10 mm 1464 Eco. Env. & Cons. 25 (3) : 2019

mal control cells and cells given the betel oils and wall become destroyed (Figure 2d). Whereas with eugenol, as shown in Fig. 2a. eugenol treatment at 2 MIC dose on the surface of In cells of Shigella flexneri the dose of betel oil of perforated bacterial cells and morphology of bacte- 1MIC could be seen on bacterial cells with cell walls rial cells becomes irregular because plasmolysis oc- that were not intact (Figure 2.b), whereas in betel oil curs in bacterial cells, as shown in Figure 2.e. with a dose of 2 MIC can be seen bacterial cells have According to Brock (1973): Substances that pro- holes and are destroyed and for eugenol 1 MIC cell duce larger inhibitory zones are not necessarily

(a) Complete cell surface (b)

(e)

Fig. 2. Changes in cell morphology was observed by using SEM. Type: Shigella flexneri normal cells (a), Shigella flexneri cells with 1MIC betel oil treatment (b) and Shigella flexneri cells with 2 KHM betel oil treatment (c). While Shi- gella flexneri cells with 1MIC eugenol treatment (d) and Shigella flexneri cells with 2 MIC eugenol treatment (e). (15,000x magnification) MUSDJA ET AL 1465 more active than substances that produce smaller sian Institute of Sciences (LIPI), Bogor, Indonesia for inhibitory zones (Brock, 1973). The size of the inhibi- their technical assistance in this study tory zone is influenced by sensitivity, media culture, incubation conditions, concentration of References antimocrobial substances on disc paper (Lorian, 1980). Abd El-Baky, R.M. and Hashem, 2016. Eugenol and lina- From the above data it can also be mentioned lool: Comparison of their antibacterial and antifun- that the essential oil of betel leaf was more selective gal activities. Afr. J. Microbiol. Res. 10 (44) : 1860-1872. as an antibacterial against Proteus mirabillis, Proteus Brock, T.D. 1973. Basic Microbiology with Aplications. Lon- vulgaris, Salmonella thypimurium and Shigella flexneri, don: Prentice-Hall International Caburian, A.B. and Osi ,M.O. 2010. Characterization and but it was less sensitive to Streptococcus mutans bac- Evaluation of Antimicrobial Activity of the Essential teria because Streptococcus mutans was a gram posi- Oil from the Leaves of Piper betle L. E-Int. Sci. Res. J. tive where gram-positive bacteria have a thick cell 2094-1749, 2 (1) : 20-12. wall structure (15 - 80 nm), single layered (mono). Carson, C.F., Mee, B.J. and Riley, T.V. 2002. Mechanism of With its cell wall containing lipids, teikoic acid and Action of Tea Tree Oil on Staphylococcus aureus De- peptidoglycan. Peptidoglycan is the main compo- termined by Time Kill, lyses, Leakage, and Salt Tol- nent of the bacterial cell wall. The cell walls of gram- erance Assays and Electron Microscopy. Antimicro- positive bacteria are thicker than gram-negative, so bial Agent and Chemotherapy. 6 : 1914–1920. gram-positive bacteria are more resistant than Coralie Pavesi, Lucy A. Banks and Taghread Hudaib, 2018. Antifungal and Antibacterial Activities of gram-negative. While eugenol is sensitive to all bac- Eugenol and Non-Polar Extract of Syzygium teria tested aromaticum L. J. Pharm. Sci. and Res. 10 (2) : 337-339. Cowan, M.M. 1999. Clinical Microbiology Review. J. Am. Conclusion Soc. Micr (ASM). 12 (4) : 564-582. Cox, S.D., Mann, C.M., Markham, J.L., Gustafson, J.E., 1. Betel oil have antibacterial potential for Proteus Warmington, J.R. and Wyllie, S.G. 2001. (Determin- mirabilis, Proteus vilgaris, Salmonella thypimurium, ing the Antimicrobial action of Tea Tree Oil. Journal Shigella flexneri and Streptococcus mutans. of Molecules. 6 : 87-91. Glasel, J.A. 1995. Validity of Nucleic Acid Purities Moni- 2. Eugenol was stronger than betel oil in inhibiting tored by A2 60/A280 Absorbance Ratios. the growth of Proteus mirabilis, Proteus vulgaris, Biotechniques. 18 : 62-63. Salmonella thypimurium, Shigella flexneri and Gupta, S., Kumar, N. and Gupta, S.M. 2009. Antibacterial Streptococcus mutans. and antifungal activity in extract and oil of Piper betle 3. The mechanism of inhibition of betel oils and (Linn) landrace Bangla Mahoba. Adv. Zool. 31: 16- 20. eugenol against Shigella flexneri occurs through Hamed, S.F., Sadek, Z. and Edris, A. 2012. Antioxidant and destruction of bacterial cell membranes so that Antimicrobial Activities of Clove Bud Essential Oil cell leakage can be observed with the leakage of and Eugenol Nanaparticles in Alcohol-Free cellular metabolites (proteins and nucleic acids), Microemulsion. J. Oleo Sci. 61 (11) : 641-648. leakage of metal ions (K + and Ca2 +) and occur- Lorian, V. 1980. Antibiotic Laboratory Medicine. Baltimore. The Williams & Wilkins Company rence morphological changes in bacterial cells. Suppakul, P., Ead, N.S. and Phoopuritham, P. 2006. Anti- 4. From this study, it was suspected that there was microbial and Antioxidant Activities of Betel Oil. no synergistic effect of the content of com- Kasetsart J. Nat. Sci. 40 (Suppl.) : 91 - 100. pounds in essential oils of betel leaf. WHO, Policy package to combat antimicrobial resistance, 2011. https://www.who.int/bulletin/volumes/ Acknowledgement 89/5/11-088435/en/. WHO, Antimicrobial resistance report, 2018. http:// The author would like to thank Dr. Andria Agusta www.who.int/news-room/fact-sheets/detail/antimicrobial-resistance. and all staff in the Microbiology laboratory. Indone-