Antibacterial Activity of Kecombrang Flower (Etlingera elatior (Jack) R.M. Sm) Extract against Staphylococcus epidermidis and Propionibacterium acnes

ABSTRACT This study aimed to determine the antibacterial activity from the ethanol extract of Kecombrang flower (Etlingera elatior (Jack) R.M. Smith) against Staphylococcus epidermidis and Propionibacterium acnes. The extract was made by the maceration method with 70% ethanol as a solvent. Antibacterial activity test was carried out by the disk diffusion method with a concentration of 10%, 20%, 40%, and 80%. Meanwhile, the Minimum Inhibitory Concentration (MIC) was done at concentrations of 10%, 8%, 6%, 4%, and 2%. The results showed that the Kecombrang flower (Etlingera elatior (Jack) R.M.Smith) extract had antibacterial activity against S. epidermidis and P. acnes. The MIC for S. epidermidis is at a concentration of 4%, while in P.acnes cannot determine yet.

long-term use of antibiotics can cause resistance, organ damage, and immune -hypersensitivity (Adawiyah et al. 2010;Tan et al. 2018;Dikicier 2019). These problems have led many researchers to discover and develop new sources for antimicrobial agents from natural products, e.g. medicinal plants (Abdallah 2011). Sadeek & Abdallah (2019) stated that some phytochemical compounds extracted from medicinal plants showed effective antibacterial potential against multi-drug-resistant pathogens and these compounds could be exploited as antibacterial drugs.
Indonesia is known as one of the countries that have many medicinal plants. One of them is Kecombrang (Etlingera elatior (Jack) R.M. Smith).
Kecombrang is a spice plant that belongs to the Zingiberaceae Family and has been used in making medicine as well as flavour enhancers. This plant contains secondary metabolites such as phenols, flavonoids, glycosides, saponins, tannins, steroids, and terpenoids (Silalahi 2017;Juwita et al. 2018;Effendi et al. 2019). Those compounds are known as potential sources for antibacterial agents (Abdallah 2011;Sadeek & Abdallah 2019). Based on Farida & Maruzy (2016) report, Kecombrang flower has more antibacterial compounds compare to its rhizome, leaves, or fruit. Kecombrang fruit contains flavonoids only. Kecombrang leaves contain saponin and flavonoids. The rhizome of Kecombrang contains saponin, tannin, sterol, and terpenoid. Meanwhile, Kecombrang flower contains flavonoid, saponin, tannin, and terpenoid.
Some studies reported that Kecombrang flower extract has an antibacterial activity to some bacteria. Mackeen et al. (1997) reported that ethanol extract of Kecombrang flower can inhibit the growth of Pseudomonas aeruginosa, Escherichia coli, Bacillus megaterium, and Cryptococcus neoformans. Wijekoon et al. (2013) reported that Kecombrang flower extract with various solvents (water, 50% ethanol, and 96% ethanol) can inhibit the growth of Bacillus cereus, Bacillus subtilis, Staphylococcus aureus, Listeria monocytogenes, and Klebsiella pneumoniae. Another research by Ghasemzadeh et al. (2015), showed that the ethanol and water extract of Kecombrang flower can inhibit S. aureus, B. subtilis, E. coli, Salmonella sp., Micrococcus sp., and Proteus mirabilis. Naufalin & Rukmini (2018) reported that the ethanol extract of Kecombrang flower has better antibacterial activity thBased on that background, this study was done to determine the antibacterial activity from the ethanol extract of Kecombrang flower against S. epidermidis and P. acnes. Since Kecombrang flower is high in containing polyphenol compounds, so it is best to use ethanolic solvents (Farida & Maruzy 2016). Ethanol can attract more polyphenol compounds than others (Tiwari et al. 2011). The outcome of the study is expected to show that ethanol extract of Kecombrang flower can be used as an alternative for acne treatment. This study was done by several steps, namely, plant source and preparation, extraction, phytochemical screening, disk diffusion test, and dilution method to determine Minimum Inhibitory Concentration (MIC).
The Kecombrang flower used in this research was obtained from Kecombrang plantation in Lubuk Begalung, Padang City, West Sumatra. About eight kg of the flowers was washed with clean water and cut into small pieces, then placed in a container and spread evenly for the drying process. The flowers were dried in the oven with a temperature of 40-50 ºC for 3 x 24 hours. The purpose of drying is to get the simplicia of flower that is not easily damaged and not overgrown with fungus in long-term storage (Sa'adah & Nurhasnawati 2015). The oven was chosen because it can keep at a controlled temperature and gave faster drying (Singh & Laishram 2010). The drying flower then mashed up into a homogenous powder to expand the contact between the solvent and the simplicial. This texture can speed up the extraction process because it was enlarging the contact between the powder and the solvent (Depkes RI 1989).
The extraction of phytochemicals from Kecombrang was done using maceration method. Maceration was chosen because it is a very simple method and could be used for the extraction of thermolabile compounds (Zhang et al. 2018). Kecombrang flower powder as much as 500 g were macerated using 70% ethanol as a solvent with a concentration of 1:10. Ethanol was used because it can attract more polyphenol compounds than others (Tiwari et al. 2011). Kecombrang flower is high in containing polyphenol compounds, so it is best to use ethanolic solvents (Farida & Maruzy 2016). 70% ethanol is known more polar than pure ethanol because adding water to pure ethanol up to 30% could increase the polarity of ethanol. According to Velavan (2015), the higher concentrations of bioactive flavonoid compounds were detected in 70% ethanol due to their higher polarity than pure ethanol. Moreover, the higher the solvent polarity, the yield obtained will also increase (Noviyanty et al. 2019). The maceration was done 1 x 24 hours with occasional stirring. Remaceration was done for 2 x 24 hours using the same solvent. The filtrate from maceration then evaporated using rotary evaporator into a thick extract (Depkes RI 1995). an the ethyl acetate's against B. cereus and E. coli.
Based on that background, this study was done to determine the antibacterial activity from the ethanol extract of Kecombrang flower against S. epidermidis and P. acnes. Since Kecombrang flower is high in containing polyphenol compounds, so it is best to use ethanolic solvents (Farida & Maruzy 2016). Ethanol can attract more polyphenol compounds than others (Tiwari et al. 2011). The outcome of the study is expected to show that ethanol extract of Kecombrang flower can be used as an alternative for acne treatment. This study was done by several steps, namely, plant source and preparation, extraction, phytochemical screening, disk diffusion test, and dilution method to determine Minimum Inhibitory Concentration (MIC).
The Kecombrang flower used in this research was obtained from Kecombrang plantation in Lubuk Begalung, Padang City, West Sumatra. About eight kg of the flowers was washed with clean water and cut into small pieces, then placed in a container and spread evenly for the drying process. The flowers were dried in the oven with a temperature of 40-50 ºC for 3 x 24 hours. The purpose of drying is to get the simplicia of flower that is not easily damaged and not overgrown with fungus in long-term storage (Sa'adah & Nurhasnawati 2015). The oven was chosen because it can keep at a controlled temperature and gave faster drying (Singh & Laishram 2010). The drying flower then mashed up into a homogenous powder to expand the contact between the solvent and the simplicial. This texture can speed up the extraction process because it was enlarging the contact between the powder and the solvent (Depkes RI 1989).
The extraction of phytochemicals from Kecombrang was done using maceration method. Maceration was chosen because it is a very simple method and could be used for the extraction of thermolabile compounds (Zhang et al. 2018). Kecombrang flower powder as much as 500 g were macerated using 70% ethanol as a solvent with a concentration of 1:10. Ethanol was used because it can attract more polyphenol compounds than others (Tiwari et al. 2011). Kecombrang flower is high in containing polyphenol compounds, so it is best to use ethanolic solvents (Farida & Maruzy 2016). 70% ethanol is known more polar than pure ethanol because adding water to pure ethanol up to 30% could increase the polarity of ethanol. According to Velavan (2015), the higher concentrations of bioactive flavonoid compounds were detected in 70% ethanol due to their higher polarity than pure ethanol. Moreover, the higher the solvent polarity, the yield obtained will also increase (Noviyanty et al. 2019). The maceration was done 1 x 24 hours with occasional stirring. Remaceration was done for 2 x 24 hours using the same solvent. The filtrate from maceration then evaporated using rotary evaporator into a thick extract (Depkes RI 1995).
The yield extract in this research was about 17.6%. The yield of an extract shows the number of active compounds extracted from the sample. The higher the yield extract, the active compounds contained in the extract also bigger (Harborne 1984;Hasnaeni et al. 2019). The calculation of the yield extract showed in Table 1. The extracted phytochemical was then screened for phytochemical contents based on Materia Medika Indonesia (Depkes RI 1989) and Pandey & Tripathi (2014). Screening includes testing for alkaloids, flavonoids, tannins, saponins, steroids, and triterpenoids. The results of the phytochemical screening showed that Kecombrang flower extract contains flavonoid, saponin, and tannin. While for alkaloid and steroid/triterpenoid, it shows negative results. These results following with Silalahi (2017) and Juwita et al. (2018), which stated that Kecombrang contains many secondary metabolites from terpenoids and phenolic groups, whereas alkaloid groups have not been reported. The phenolic compounds found in Kecombrang are flavonoid, saponin, tannin, and polyphenol. The result of the phytochemical screening showed in Table 2. The presence of flavonoids, saponin, and tannin in this research indicates that Kecombrang flower extract has the potential as an antimicrobial. As stated by Juwita et al. (2018) that the high potential antimicrobial activity possessed by Kecombrang is due to the presence of flavonoid, phenolic, and terpenoid contents.
The antibacterial activity test was carried out by the disk diffusion method on Mueller Hinton Agar (MHA). The MHA was poured into a petri dish and allowed to stand until the media hardens. After the media has hardened, the suspension of bacteria was pipetted about 1 ml onto a petri dish and spread evenly. The Bacterial suspension (9 x 10 7 CFU/ml) was suspended in NaCl 0.9%. After the bacterial suspension dried, sterile disk paper was inserted into a petri dish and 20 l of the extract was dropped. The concentration of the extract was using 10%, 20%, 40%, and 80% based on  MacKeen et al. (1997) and Kusumawati et al. (2015) with modification. They tested the extract of Kecombrang flower and Kecombrang leaf (respectively) in concentrations of 20%, 40%, and 80% which showed positive results to the tested bacteria. The tested disks were then incubated for 24 hours at 37ºC. The diameter of the inhibition zone was measured using a calliper. The clear zone that appears around the disk was measured as the inhibition zone (IZ) (Hudzicki 2016). The inhibition zones were analyzed statistically by Two-Way Anova and Least Significance Different (LSD) using MS Excel 2010 to see the significant effect of the differences concentrations. The positive control used in this research is two antibiotics, named ciprofloxacin for S. epidermidis and clindamycin for P. acnes. Antibiotics are known as a substance that can inhibit the growth of bacteria (Pratiwi 2008). Ciprofloxacin is known as a broad-spectrum antibiotic from quinolone group. Quinolones are known can inhibit or disrupt the nucleic acid synthesis of the bacterial cell (Hogg 2005;Vidyavathi & Srividya 2018). Meanwhile, clindamycin is known as an antibiotic that can inhibit the protein synthesis of the bacteria. Clindamycin usually prescribes for acne treatment (Smieja 1998;Walsh et al. 2016). The use of different antibiotics in this research was due to in preliminary study ciprofloxacin gave a very wide inhibition zone in the dish containing P. acnes, so it can create ambiguous results. The negative control used in this test is DMSO 10% (v/v). DMSO is a surfactant that can dissolve polar and nonpolar materials. It also showed no antibacterial activity (de Brito et al. 2017). The measurements of the inhibition zone showed in Figure 1 and Table 3.   The data in Table 3 showed that the ethanol extract of Kecombrang flower has the activity to inhibit the growth of S. epidermidis and P. acnes. The results showed that the inhibition zone became larger in line with the greater concentration of the extract. The Inhibition zone at a concentration of 80% showed significant inhibition against those two bacteria compared to other concentrations. The difference in diameter of inhibition zones at each concentration possibly was due to differences in the magnitude of active substances contained in the concentration. The active compounds in higher concentrations are more than the opposite (Lingga et al. 2016). Besides that, the size of the inhibition zone was also influenced by the level of sensitivity of the organism, the culture medium, the incubation conditions, and the diffusion rate of the antibacterial compound (Fitriah et al. 2017). Bacteria have different sensitivity against antibacterial agents. Usually, Gram-positive bacteria are more sensitive to antibacterial agents compared to Gramnegative bacteria. The Gram-negative bacteria have an outer membrane in their cell wall that is not easily penetrated or damaged by certain antibacterial compounds (Breijyeh et al. 2020). The culture medium can influence the inhibition zone because different nutrient contents in media are affecting the bacterial growth. The Composition of culture media exercises a key effect on the susceptibilities of microorganisms, such as free from inhibitor content or the magnesium levels (Daoudi et al. 2020). Besides the nutrient contents in media, agar thickness and inoculum volume can influence the diffusion rate of the antibacterial agent. The higher the thickness of agar layer, the smaller the zone diameter. Similarly, high inoculum concentration shows hazy growth on the media, whereas a low concentration of inoculum shows light and immeasurable zone (Dafale et al. 2016). The particle volume also can influence the diffusion rate. Small particles will diffuse faster than large ones. Other factor that influences the inhibition zone is the incubation conditions, such as pH and temperature. Higher temperatures will increase the diffusion process. The pH of a solvent must be adjusted to neutrality (6.0 -8.0) or dissolved in buffer solutions because the bacterial growth might be inhibited in too acid or too alkaline media (Valgas et al. 2007). Antibacterial agents have four mechanisms to inhibit the growth of bacteria. First, by inhibiting the growth of cell walls. Secondly, changing the cell membrane permeability; then inhibits the protein synthesis; and the nucleic acids (Hogg 2005). According to Fitriah et al. (2017), each group of compounds can have different effects in inhibiting bacterial growth. The difference in the activity that occurs is caused by secondary metabolites contained having synergistic energy that is different depending on the nature and morphology of bacteria.
The Kecombrang flower extract in this research contains flavonoid, tannin, and saponin, which has different mechanisms to inhibit the growth of bacteria. According to Juwita et al. (2018), flavonoid compounds in Kecombrang (Etlingera elatior (Jack) R.M.Smith) have antibacterial activity by targeting of membrane cell wall due to its capability to composite with extracellular and soluble proteins. This mechanism is similar to saponin, which also attacks the bacterial cell membrane. Saponin can dissolve lipids in bacterial cell membranes (lipoprotein), which causes the bacterial cell to become lysis and death (Syafriana et al. 2019). Meanwhile, tannins act by disturbed the DNA gyrase, which is an enzyme that plays a role in DNA replication (Khameneh et al. 2019). However, we cannot determine yet which compounds have a significant effect in inhibiting bacterial growth because this research has only qualitative phytochemical data, not a quantitative one. So, we cannot find out whether these compounds are equal in quantity or one is higher than another.
The data in Table 3 also showed us that a minimum concentration (10%) can inhibit the two bacteria. Because of that, the antibacterial test continued to the Minimum Inhibitory Concentration (MIC) value test. MIC value is the lowest concentration value that can inhibit bacterial growth (Dafale et al. 2016). The test was carried out using the solid dilution method which is observing the growth of the bacteria at the lowest concentration of IZ results onto agar media. The concentration used were 10%, 8%, 6%, 4%, and 2%. The liquid medium was mixed with 1 ml of bacterial suspension and 1 ml of predetermined extract concentration. The mixture of cell suspension, media, and extract was then homogenized and incubated for 24 hours at 37 0 C. The results of the incubation were then observed for the presence or absence of bacterial colony growth on the media. If there is bacterial growth, the extract cannot inhibit bacterial growth. However, if the media remains clear, it shows that the extract could inhibit bacterial growth (Pratiwi 2008;Hudzicki 2016). The data of MIC was showed in Table 4.  Table 4 showed that MIC in S. epidermidis is at a concentration of 4% because, in a concentration of 2%, the bacteria showed growth. Meanwhile, the MIC value of P. acnes cannot be determined yet. It's due to at the lowest concentration of the test (2%), the P. acnes still showed no growth. The data in Tables 3 and 4 showed that P. acnes was more sensitive than S. epidermidis against the extract. This data aligned with Nishijima et al. (2000), which reported that S. epidermidis indeed more resistant than P. acnes when testing against several antibiotics. Based on that data, the tested concentration to P. acnes should be below 2% until it shows a MIC value.
In conclusion, the ethanol extract of Kecombrang flower (Etlingera elatior (Jack) R.M.Smith) can inhibit the growth of S. epidermidis and P. acnes. This data showed a potency of Kecombrang flower extract as an antibacterial agent against S. epidermidis and P. acnes that causes acne vulgaris. This research is a preliminary study, so further research is needed to ensure a more valid antibacterial activity, such as determining levels of flavonoid, tannin, and saponin. Besides that, it is also necessary to study the mechanism of inhibition or cell damage caused by the secondary metabolites to prove that the mechanisms mentioned above are suitable.

AUTHORS CONTRIBUTION
VS and RNP designed the study and carried out the laboratory work. VS, YSD, and RNP analysed the data and write the manuscript.