ANTIBACTERIAL POTENTIAL ETHANOL EXTRACT OF KAYU RACUN LEAF (Rhinacanthus nasutus) AGAINST Staphylococcus aureus AND METHICILLIN RESISTANT Staphylococcus aureus

The excessive use of antibiotics in the treatment of infections or diseases caused by Staphylococcus aureus is the main cause of antibiotic resistance. Methicillin-resistant S. aureus (MRSA) strains are a serious problem because of their wide distribution in the clinical environment and the living community, so it is necessary to search for antibacterial sources from natural products and traditional medicines such as Kayu racun leaves (Rhinacanthus nasutus). Information regarding the content of the active ingredient and the antibacterial potential of the ethanol extract of Kayu racun leaves is still limited. This study aims to test the phytochemical and antibacterial activity of the ethanol extract of Kayu racun leaves against S. aureus and MRSA bacteria by invitro. The research used an experimental method and was divided into two stages, namely; Phytochemical test of the ethanol extract of Kayu racun leaves using the Thin Layer Chromatography (TLC) method and the antibacterial activity test of the ethanol extract of Kayu racun leaves against S. aureus and MRSA using the paper disc diffusion method. Based on the research results, it can be concluded that the ethanol extract of Kayu racun leaves contains alkaloids, phenols, and flavonoids. The best antibacterial activity was found at a concentration of 100 mg/ml with an inhibition zone diameter of 26.20 mm against S. aureus and 17.90 mm against MRSA.


INTRODUCTION
Antibiotic resistance is a global health problem (Shamsuddin et al., 2018). Excessive administration of antibiotics in controlling pathogenic microbes has triggered the emergence of resistance cases (Hussein et al., 2020).
S. aureus is one of the species of pathogenic microbes caused by a skin infection that has good adaptability in epidermal tissue (Hua, et al., 2018;Pawar, et al., 2020). Treatment of S. aureus infection is generally carried out with antibiotic therapy which is still the main support of infection management. It is estimated that around 92% of Indonesians do not use antibiotics properly. So that it causes losses not only in terms of health and economy but also more worrying is the emergence of resistance or resistance to one or more antibiotics (Utami, 2011).
So far the most reported cases of resistance are Methicillin-Resistant Staphylococcus aureus (MRSA). MRSA is a gram-positive bacterium, a strain of S. aureus that has developed through horizontal gene transfer and natural selection. The spread of S. aureus and MRSA is generally through human contact (Kumar and Githa, 2018). MRSA strains are resistant to almost all β-lactam antibiotics, which are the most effective and widely used antimicrobial class for the treatment of infections (Oliveira dan de Lencastre, 2011;Amalia et al., 2017). These groups of antibiotics include; penicillin, tetracycline, methicillin, and vancomycin (Chakraborty et al., 2018). MRSA is also the leading cause of nosocomial infections worldwide thus increasing the length of treatment, medical costs, to the death rate (Hua et al., 2018;LanMai, 2020).
The strategy currently being developed to prevent an increase in antibiotic resistance is to combine the use of antibiotics with natural products and herbal traditional medicines (Pawar et al., 2020). Over the last decade the interest in finding natural products as antimicrobial sources has increased. Plant selection is considered the most promising because it can be selected based on ethnopharmacology and a variety of plants is quite easy to obtain (Diaz, et al., 2019)

METHODS
Kayu racun leaf samples were collected from Nagari Simanau-Solok. The research was conducted at Biota Laboratory and Microbiology Laboratory of Biology Department, Andalas University with an experimental research method. The research stages include; sample preparation, sample ethanol extraction, sample phytochemical analysis, preparation of the S. aureus and MRSA bacterial suspensions, and testing the antibacterial activity of the Kayu racun leaves ethanol extract against the two tested bacteria (Figure 1).

Preparation of Plant Extracts
Three kg of Kayu racun leaves were collected from the field. Furthermore, the leaf sample was dried, ground, and mashed in a blender. The sample is macerated in stages. The first maceration macerated Kayu racun leaf powder with a comparison of the sample and ethanol =1:10 (w/v). A total of 250 g of sample was macerated with 2.5 l of 70% ethanol for 18 hours.
Then filtered to separate the ethanol extract of Kayu racun leaves from the residue, so that the ethanol extract 1 is obtained. The second maceration was carried out on the residue and ethanol by comparison 1:5 (w/v) for 24 hours. In the same way, ethanol extract 2 is obtained. The ethanol extract 1 and 2 are combined then evaporated to obtain a thick ethanol extract (crude extract).

Phytochemical test
The phytochemical test used the Thin Layer Chromatography (TLC) method. The sample was spotted on a 60 F254 silica gel plate using a capillary tube. Furthermore, the TLC plate was sprayed with a specific spray to determine the active compound class. The spots formed on the TLC plate were observed under the light of UV254 and UV365. The specific spray reagent used is a) 1% FeCl3 to detect the presence of tannins and phenolic compounds if green, red or blue colors appear after spraying, b) Lieberman-Burchard to detect steroid compounds when blue appears and terpenoids if red appears, c) Citroborat to detect flavonoid compounds when green and yellow fluorescence appears, d) Dragendorff, to detect alkaloids when yellow fluorescence appears (Harborne, 1987;Indonesia Herbal Pharmacopeia, 2012).

Antibacterial Activity Test
Starting with the preparation of a suspension of S. aureus and MRSA test bacteria in a 0.9% (w/v) NaCl solution and equalizing the turbidity with a standard solution of 0.5 McFarland (10 8 CFU/ml) (Balouiri et al., 2016). Antibacterial activity test was carried out disc paper diffusion method. Each MHA medium (Mueller Hinton Agar) was poured into a sterile 10 ml petri dish, waited for it to cool and solidify. Then 100 µl of S. aureus and MRSA test bacteria were inoculated on each petri dish using the spread plate technique. Place the disc paper that has been dripped with the ethanol extract of Kayu racun leaves at the concentration tested (concentration 20, 40, 60, 80 dan 100 mg/ml), above the surface of the medium. Negative control using DMSO (dimethylsulfoxide). A positive control using the antibiotic Amoxicillin (1mg/ml). Then incubated at 37°C for 24 hours. The formed inhibition zone indicates that the ethanol extract of Kayu racun leaves has antibacterial activity (Purwanto, 2015).

Data Analysis
The data obtained are presented in the form of tables and figures and then described descriptively.

RESULTS AND DISCUSSION Phytochemical Analysis of Ethanol Extract of Kayu racun Leaves
The use of plants as medicinal substances is related to the secondary metabolites contained in plants. The results of phytochemical analysis of the ethanol extract of Kayu racun leaves are shown in Table 1.  (Tricia, et al., 2018;Mayer (2011).

Antibacterial Activity of Kayu racun Leaf Extract
Antibacterial activity test of ethanol extract of Kayu racun leaves was carried out to determine the response of both S.aureus and MRSA test bacteria. The results can be seen in Figure 2 and Figure 3.   This proves that the formation of the inhibition zone is caused by the content of secondary metabolite compounds in the ethanol extract of Kayu racun leaves.
The diameter of the inhibition zone that was formed increased with the increase in the concentration of the Kayu racun leaf extract. This is because the increase in concentration is also followed by an increase in the content of active compounds contained in the extract. In addition, it is also influenced by the ability of active compounds to diffuse. Ariyanti et al., (2012) stated that the diameter size of the inhibition zone is influenced by the diffusion rate of the antibacterial agent. Diffusion of solutes from higher concentrations is faster, compared to lower concentrations. So that the growth of the test bacteria around the extract is stunted. Figure 3. The best concentration of ethanol extract of Kayu racun leaf (R.nasutus) 100 mg/ml against S.aureus (a) and MRSA (b). U = repetition, A= amoxicillin 1 mg/ml Figure 3 shows the antibacterial activity of the ethanol extract of Kayu racun leaves at the best concentration of 100 mg/ml with the highest average inhibition power reaching 26.20 mm against S.aureus which is classified as strong and 17.90 mm against MRSA which is in the moderate category. Cavalieri et al., (2005) classified three categories based on the diameter of the inhibition zone; if the diameter ≤10 mm is categorized as resistant, between 11-19 mm is categorized as moderate, while diameter ≥20 mm is categorized as not resistant (strong).
The inhibition zone formed was greater in S. aureus test bacteria compared to MRSA. This is because MRSA is an infection-causing bacteria originating from an S. aureus strain that is resistant to several antimicrobial groups. MRSA is a strain of S. aureus bacteria that is resistant to all β-lactam and other antibiotics such as all Penicillin and Methicillin derivatives as well as broad-spectrum antimicrobial beta-lactamase (Okwu et al., 2019;Pawar et al., 2020).
In the positive control (K +) with Amoxicillin, a concentration of 1 mg/ml inhibition was formed 9.35 mm against S. aureus and 11.18 mm against MRSA. When compared to the inhibition zone of Kayu racun leaf ethanol extract with K (+) mathematically, it can be seen that the

CONCLUSION
Based on the research that has been done, conclusions can be drawn; Kayu racun leaf extract contains alkaloids, phenols, and flavonoids which contribute to its antibacterial properties. The extract concentration of 100 mg/ml showed the best antibacterial activity with an inhibition zone diameter of 26.20 mm against S. aureus and 17.90 mm against MRSA.
The suggestion for further research is to isolate the active compound of Kayu racun leaves as a new antibacterial source in inhibiting S. aureus and MRSA.