Antibacterial Activity of Essential Oil from Rosemytle Leaves ( Rhodomyrtus tomentosa ( Ait . ) Hassk )

Rosemytle leaves (Rhodomyrtus tomentosa (Ait.) Hassk) have been used by society to treat various diseases related to bacterial infections, such as dysentery and typhoid fever caused by Shigella dysenteriae and Salmonella typhi, respectively. This study aimed to evaluate the antibacterial activity of essential oils from rosemytle leaves against both bacterias. Extraction was performed with a macerating device using n-hexane, ethyl acetate, and ethanol extracts, sequentially. This study used the agar diffusion method to test the antibacterial activity applied to the essential oils with concentrations of 1000, 500, 250, 125, 62.5, and 31.25 μg/mL. The antibacterial test results showed that n-hexane and ethyl acetate extracts were active against both bacterias while ethanol extract was not. Then, isolates N1 and E1 were produced respectively from n-hexane extract and ethyl acetate extract. The MIC values of both N1 and E1 for S. dysenteriae, and S. typhi were the same, namely 125 μg/mL. Isolate N1 was an essential oil containing menthol (59.60%), caryophyllene (25.77%), and cubenol (14.63%) while isolate E1 was an essential oil containing (73.93%), pentanone (8.30%), alpha calacorene (7.58%), and calacorene (3.78%). Rosemytle leaves have the potential to be developed as a drug to treat dysentery and typhoid fever.


INTRODUCTION
Nature provides raw materials for both traditional and modern medicines. The presence of various plants that support life has led researchers to discover their benefits to treat certain infectious diseases. WHO reported that 80% of the world population had been using conventional medicines, which are mostly plant-based, to care for their health (Kamazeri, 2012).
Found in Southeast Asia, Rhodomyrtus tomentosa (Ait.) Hassk, so-called rosemytle, comes from the family Myrtaceae. People use its roots, leaves, and fruits for conventional medicine. Extracts from the aerial part of rosemyrtle contain different bioactive phytochemicals and have been found to have antibacterial, antifungal, anti-inflammatory, antimalarial, antioxidant, and osteogenic activities (Hazrulrizawati & Zeyohannes, 2017). Salni and Marisa (2019) found that n-hexane and ethyl acetate extracts from rosemytle were active against S. typhi and S. dysentriae bacteria, with MIC values of of 250 μg/mL. Rosemytle leaves contain rhodomyrtone, a natural antibiotic or antibacterial compound that is derived from phloroglucinol (Dachriyanus et al., 2002), that is used for treating staphylococcal skin infections. It has a strong in vitro activity against various grampositive and gram-negative bacterias. With ethanol extracted from the same origin, it has a strong antibacterial activity against gram-positive bacterias, including B. cereus, B. subtilis, E. faecalis, S. aureus, S. pyogenes, and S. salivarius (Limsuwan et al., 2009). Ethanol extract from rosemytle leaves is active against staphylococcal bacteria isolated from acne. Both substances are active against acnecausing bacteria Propionibacterium acnes. Since rhodomyrtone shows very low toxicity to skin cells, ethanol extract from rosemytle can be a candidate for a treating agent for acne (Saising & Vorayutthikunchai, 2012).

Isolation of Active Compounds
As much as 100 grams of powdered rosemyrtle leaves were extracted using macerating tools with solvents, started with 1L n-hexane for 2x24 hours, ethyl acetate, and ethanol, respectively. Each extract was evaporated in a rotavapor until becoming a paste and then tested for its antibacterial activity. The most active extracts were fractionated using the vacuum liquid chromatograpic method with sloping eluent consisting of n-hexane, ethyl acetate, and ethanol in 12 fractions. The fractionation began with non-polar solvents, 100% n-hexane, followed by 12 combinations of solvents in the form of n-hexane and ethyl acetate, ethyl acetate and ethanol, and, finally, 100% ethanol. The fractions were tested for antibacterial activity by the diffusion method. Furthermore, the active fractions were purified using a gravity column containing silica gel with eluent from n-hexane and ethyl acetate (9: 1).

Bioautography Tests
To obtain the Rf value of antibacterial compounds, the active fractions were examined using the TLCbioautographic method. An active fraction was loaded onto two TLC plates, which were placed in vessels with a ratio of n-hexane and ethyl acetate of 8:2. The first chromatogram was placed with a bacterial culture on a petri dish and left attached to the media for 1 hour. The petri dish was then incubated for 24 hours. Bright spots caused by the active compound was observed to calculate the Rf value. An H2SO4 solution was sprayed on the second chromatogram. Based on the color formed, the class of active compounds can be estimated: yellow for phenols, purple for terpenoids, and brown for tannin groups (Farnsworth,1996).
Antibacterial activity test n-hexane extract, ethyl acetate extract, and ethanol extract from leaves of Rhodomyrtus tomentosa were tested for their ability to inhibit the growth of S. dysenteriae, and S. typhi bacteria. Antibacterial activity tests were carried out on paper discs using the agar diffusion method by placing 50 μL of bacterial suspension into 10 mL of each medium that had previously been diluted in a petri dish. The media were then allowed to become stable. Paper discs with a diameter of 6 mm were placed on the surfaces of reliable media. A total of 20 μL of the tested mixture was dropped on each disc and allowed to spread for 30 minutes. Then, the discs were placed in an incubator with a temperature of 37 ºC. MIC was determined using the diffusion method with various concentrations of active compounds of 1000, 500, 250, 125, 62.5, and 31.25 μg/mL. The solvent used to dissolve isolates N1 and E1 was dimethylsulfoxide.

RESULT AND DISCUSSION Isolation of Active Compounds
The test results of antibacterial activity against bacterias causing dysentery and typhoid fever, namely S. dysenteriae and S. typhi, respectively, using n-hexane, ethyl acetate, and ethanol extracts indicated that only n-hexane and ethyl acetate extracts were active against bacteria, while ethanol extract was not. Both extracts were fractionated by the vacuum liquid chromatography method with eluent level consisting of n-hexane, ethyl acetate, and ethanol extracts. The results of the antibacterial activity test of n-hexane extract showed that the active fractions were fraction 3 (HC), fraction 6 (HG), and fraction 7 (HI). Furthermore, fraction 3 (HC) was added in the gravity column with eluent n-hexane and ethyl acetate with a ratio of (9:1). Active isolate (isolate N1) was obtained in bottles 6 to 8, as in Figure 1 (a). The results of the antibacterial activity test of ethyl acetate extract showed that active fractions were found in fractions 2 (EB) to 6 (EF) and fractions 12 (EL) to 13 (EM). Fractions 2 (EB) to 6 (EF) were applied in a column chromatography containing eluent n-hexane and ethyl acetate with a ratio of 9:1. The active fractions were shown in bottles 5 to 9 (E1 isolate), Figure 1(b). Isolates N1 and E1 were obtained in the forms of brownish yellow paste. Bioautography tests were performed on both isolates to determine the Rf values and the classes of their active fractions.

Bioautography Tests
Bioautography test results showed that isolates N1 and E1 had Rf values of 0.22 and 0.33, respectively. After being sprayed with 10% H2SO4 solution, both isolates showed purple spots, indicating the existence of terpenoids (essential oils), as shown in Table 1 and Figure 2. The figure also shows the presence of obstacles (bright areas) in bacterial cultures that indicates the inability of bacteria to grow due to the presence of antibacterial compounds.
From the colors, it can be concluded that the active compounds contained by both isolates N1 and E1 were terpenoids (essential oils). Both isolates were active against S. dysenteriae and S. typhi that respectively cause dysentery and typhoid fever. The antibacterial activity of n-hexane and ethyl acetate extracts was expected due to the presence of essential oils from rosemytle leaves, that had never been reported before. Thus, both extracts had the potential as raw materials for drugs to deal with dysentery and typhoid fever.
Essential oils also contain several types of terpenoid compounds. According to the results of previous studies, essential oils consisting of terpenoids showed antibacterial activity. The composition of essential oils can vary in different parts of the same plant. Research by Benchaar et al. (2008) showed that essential oils are mixtures consisting mainly of terpenoids, especially monoterpenes (C10) and sesquiterpenes (C15). However, diterpenes (C20) can also be in the forms of acids, alcohols, aldehydes, acyclic esters or lactones, and extraordinary compounds containing N-and S, coumarin, and phenylpropanoid homologs.
Essential oils that are found to have antibacterial activity contain terpenoids. The results of this study are supported by the research of Trombetta et al. (2005), which found three monoterpenes, namely linalyl acetate, menthol, and thymol active against S. aureus and E. coli, which are a gram-positive and gram-negative bacterias, respectively. Research on the antibacterial activity of essential oils thymol, carvacrol, eugenol, and menthol on four strains of bacterias that cause nosocomial infections, namely E. coli, P.aeruginosa, K. pneumonia, and S. aureus, showed that thymol, carvacrol, and eugenol have significant antibacterial activity. Thymol has a high antibacterial activity against S. aureus and E. coli with MIC values of 0.35 mg/mL while menthol has low activity against all bacterias tested with MIC values greater than 6 mg/mL (Atki et al., 2019).

Characterization of Isolates N1 and E1
Characterization of isolate N1 was done using GC-MS. The obtained GC-MS chromatogram is shown in Figure 3. In the figure, there are three dominant peaks identified. The dominant peaks 16.919, 12.621, and 15.236 indicates content percentages of menthol (59.60%), caryophyllene (25.77%), and cubenol (14.63%). The contents of isolate N1 is shown completely in Table 2.
Isolate E1 was characterized by GC-MS. The results obtained can be seen in Figure 4. Table 3 shows the essential oils contained by isolate E1 from the characterization. Isolate E1 contains, mainly, menthol 73.93%, pentanone 8,73%, and alpha calacorene 7.58%,. Like isolate N1, the menthol compound also has the highest content percentage in E1.    Another previous study also showed contents similar to those of isolates N1 and E1. Salvia triloba in South Brazil has been reported to have essential oils, including β-caryophyllene, along with α-thujone, 1,8-cineole, and camphor, exhibiting extraordinary bacteriostatic and bactericidal activity against B. cereus, B. megatherium, B. subtilis, A. hydrophila, A. sobria, and K. oxytoca (Delamare, Moschen, Atti, & Echeverrigaray, 2007). Soetjipto, Dewi & Prayitno (2008) investigated the chemical contents in antibacterial compounds from Japanese lavender (Tithonia diversifolia (Hemsley) A. Gray) and gave similar results to this study. They showed that the essential oils contained 29 components consisting of, mainly, caryophyllene (27.76%), nerolidol (21.81%), caryophyllene oxide (7.06%), copaene (6.41%) and bicylogermacrene (4.90%). To determine the antibacterial activity, a bioautography test was performed. Bioautography test results of two antibacterial spots gave Rf values of 0.49 and 0.61. The spots with an Rf value of 0.49 were a mixture of 21 compounds with henisicon, nonacosana, and tetratetracontane as the main compounds, while spots with an Rf value of 0.61 consist of 22 compounds with nerolidol as the main compound.

Minimum Inhibitory Concentration (MIC) of The Isolates
From the results of the isolation of the antibacterial compounds, the active compounds of isolate N1 were obtained from n-hexane extract, whereas, the active compounds of isolate E1 were obtained from ethyl acetate extract. Both N1 and E1 were essential oils. The results of determining the MIC value of isolate N1 can be seen in Table 4 and Figure 5. The following shows the MIC value of isolate E1 in Table 5 and Figure 6.
As shown in Table 4, isolate N1 at a concentration of 1000 µg/mL produced the largest inhibitory diameters of 19.33 mm for S. dysenteries and 18.33 mm for S. typhi. The test results in table 4 also show that the decrease in isolate N1 concentration resulted in smaller inhibitory diameter. The smallest inhibition against S. dysenteriae and S. typhi was obtained at a concentration of 125 μg/mL, meaning that the MIC value of isolate N1 was 125 μg/mL.  The antibacterial activity of isolate E1 showed that the largest inhibitory diameter of bacterial growth was found at a concentration of 1000 µg/mL with inhibitory diameters of 17.00 mm for S. dysenteriae and 16.00 mm for S. typhi. The test results also showed that the decreasing concentration of isolate E1 decreases inhibitory diameter produced. The smallest resistance against S. dysenteriae and S. typhi was obtained at a concentration of 125 μg/mL, hence it can be stated that the MIC value of E1 isolate is 125 μg/mL. The MIC values of isolates N1 and E1 containing menthol against S. thypi and S. dysenteriae bacteria were 125 μg/mL, smaller than MIC values of essential oils thymol against S. aureus and E. coli, which were 310 μg /mL and 500 μg/mL, respectively. Against the same bacterias, menthol had MIC values of 620 mg/mL and 250 μg/mL, while linalyl acetate had MIC values of 125 mg/mL and 500 μg/mL (Trombetta et al., 2005).
MIC values of isolates N1 and E1 were classified as antibacterial with strong activity. Holetz et al. (2009)  MIC values of isolates N1 and E1 against both bacterias were 125 μg/mL, which are the same as that of fennel (Foeniculum vulgare Mill.) showed by the investigation by Diao, Hua, Zhanga, & Xuab (2014), which also revealed that MIC values of essential oils from fennel showed the antibacterial activity against S. typhimurium, S. dysenteriae and E. coli. These essential oils showed the most active reaction to S. dysenteriae when reaching the lowest MIC value of 125 μg/mL. Besides, the killing time test also showed that the essential oils of fennel were the fastest in killing S. dysenteriae. Based on the results of tests and observations of using electron microscope, the essential oils worked against S. dysenteriae on its membrane integrity and caused its electrolyte leakage and loss of contents (protein, sugars, and materials sized 260 nm). The mechanism of antibacterial action is carried out by three monoterpenes, namely menthol, thymol, and linalyl acetate by causing interference with the plasma membrane lipids of microorganisms that result in changes in cell membrane permeability. This effect depends on the lipid content and the surface charge of the microbial cell membrane. Changes in permeability cause drug ingredients to cross the cell membrane, penetrate the interior of the cell, and interact with intracellular material that is important for antibacterial activity (Trombetta et al., 2005).
The main components of essential oils N1 and E1 are the same as those found in peppermint oil. Peppermint oil with antiseptic properties derived from the family Labiatae has antibacterial activity. The MIC values of peppermint oil for various types of microorganisms are in the range of 0.125-2 μL/mL. Candida albicans is the most sensitive microorganism, and Pseudomonas aeruginosa is the least sensitive. Peppermint oil shows the same activity as vancomycin, gentamicin, and amphotericin B. It can be used as a natural antibiotic and can reduce the dose of an effective antibiotic. Menthol, menthone, and methyl acetate are the main components of peppermint oil, followed by carveone, neomenthol, 1,8-cineole, and limonene (Mahboubi & Kazempour, 2014).