Biofarmasetikal Tropis (The Tropical Journal of Biopharmaceutica.
2024, 7.
, 25-33 e-ISSN 2685-3167 p-ISSN 2828-6685 Antibacterial Activity Of Staphylococcus aureus From Chinese Betel Leaf Juice (Peperomia pellucida L.
Kezia T.
Wantah1.
Jabes W.
Kanter1*.
Selvana S.
Tulandi2.
Jeane Mongi1.
Reky R.
Palandi2.
Priska Pakingki1 Department of Pharmacy.
Faculty of Mathematics and Natural Sciences.
Christian University of Indonesia in Tomohon Department of Biology.
Faculty of Mathematics and Natural Sciences.
Christian University of Indonesia in Tomohon *Corresponding author : jabeskanter@gmail.
Accepted: 12 September 2024.
Approved : 25 Oktober 2024
ABSTRAK
Penyakit infeksi, khususnya di negara berkembang seperti Indonesia, menjadi masalah kesehatan yang sering dihadapi, salah satunya disebabkan oleh bakteri.
Penelitian ini menguji aktivitas antibakteri sari daun sirih cina terhadap bakteri Staphylococcus aureus, dengan kandungan metabolit sekunder seperti alkaloid, tanin, dan flavonoid yang berperan sebagai antibakteri.
Penelitian ini merupakan eksperimen laboratorium menggunakan rancangan acak lengkap dengan tiga kali pengulangan pada lima konsentrasi sari, serta satu kontrol positif dan negatif.
Metode yang digunakan adalah metode difusi agar, dan hasil pengujian menunjukkan bahwa sari daun sirih cina memiliki potensi antibakteri terhadap Staphylococcus aureus pada berbagai konsentrasi yang diuji.
Konsentrasi yang digunakan adalah 6AAL/disc, 8AAL/disc, 10AAL/disc, 12AAL/disc, dan 14AAL/disc, di mana semua kategori ini menghasilkan zona hambat yang termasuk dalam kategori sedang menurut Susanto .
Analisis statistik menggunakan uji Kruskal-Wallis menghasilkan nilai signifikan 0,035 (< 0,.
, menunjukkan adanya perbedaan signifikan antara masing-masing konsentrasi yang Dari hasil penelitian, dapat disimpulkan bahwa sari daun sirih cina memiliki aktivitas antibakteri yang efektif terhadap Staphylococcus aureus.
Kata kunci: Antibakteri.
Staphylococcus aureus.
Peperomia pellucida L.
ABSTRACT
Infectious diseases, especially in developing countries such as Indonesia, are a common health problem, one of which is caused by bacteria.
This study tested the antibacterial activity of Chinese betel leaf extract against Staphylococcus aureus bacteria, with secondary metabolite content such as alkaloids, tannins, and flavonoids that act as antibacterials.
This study was a laboratory experiment using a completely randomized design with three repetitions at five concentrations of extract, as well as one positive and negative control.
The method used was the agar diffusion method, and the test results showed that Chinese betel leaf extract had antibacterial potential against Staphylococcus aureus at various concentrations tested.
The concentrations used were 6AAL/disc, 8AAL/disc, 10AAL/disc, 12AAL/disc, and 14AAL/disc, where all these categories produced inhibition zones included in the moderate category according to Susanto .
Statistical analysis using the Kruskal-Wallis test produced a significant value of 0.
035 (<0.
, indicating a significant difference between each concentration tested.
From the research results, it can be concluded that Chinese betel leaf extract has effective antibacterial activity against Staphylococcus aureus.
Keywords: Antibacterial.
Staphylococcus aureus.
Peperomia pellucida L.
INTRODUCTION
of the health problems that many people face One of the factors causing this infection is the presence of bacteria1.
Skin and soft tissue Infectious diseases, especially in developing countries such as Indonesia, are one Biofarmasetikal Tropis (The Tropical Journal of Biopharmaceutica.
2024, 7.
, 25-33 infections occur in 10% of cases of bacterial infections being the cause of hospitalization.
Most bacterial infections of the skin and soft tissues are resolved within 7 to 10 days, it is difficult to estimate the exact incidence of skin and soft tissue infections as the clinical manifestations and duration of infection vary 2,3.
The incidence of the disease is increasing due to an aging population and people with serious illnesses.
Other causes of increased immunosuppressive drugs, malignancies, organ transplants, medical interventions, and surgical wound infections2.
Staphylococcus aureus is known as a pathogenic bacterium that infects humans.
addition, this bacterium is an opportunistic pathogen and commensal bacterium4.
These bacteria cause infections that spread to blood vessels and produce toxins resulting in shock This type of bacteria is grampositive and occurs naturally in various parts of the human body, including the skin, mucous membranes of the nose, mouth and colon.
When a person's immune system is weakened, the bacteria can become pathogenic and cause boils, abscesses, and various inflammation-related Empirically in the Minahasa area, especially in Lemoh Village.
Chinese betel leaf (P.
pellucida L.
) is used by the local community as a traditional prevention and treatment of infectious diseases caused by bacteria by taking the leaves of the Chinese betel leaf plant with a hand grip and then mashing it until smooth after that it is attached to the injured skin surface area.
Therefore, it is necessary to prove on a laboratory scale whether Chinese betel leaves have antibacterial activity.
e-ISSN 2685-3167 p-ISSN 2828-6685 GF254 KLT plate, capillary pipette, cotton, sterile gauze, aluminum foil, mortar and pestle, 10cm petri dish, gloves, mask, lab coat, stationery, camera, container, knife, 8mm paper disc, spatula, tweezers, pipette, 1.
5ml eppendorf.
The materials used in this study are:
Chinese betel leaf (P.
pellucida L.
Staphylococcus aureus bacteria (ATCC 25.
obtained from the Laboratory of the Faculty of Mathematics and Natural Sciences.
Christian University of Indonesia Tomohon.
Nutrient Agar (NA).
Nutrient Broth (NB), 95% alcohol, distilled water, ampicillin.
McFarlan 0.
standard solution, ethyl acetate, n-hexane, chloroform, dragendoff.
FeCl3 5%, ammonia.
Research Type and Research Design This type of research is laboratory experimental research, using a Completely Randomized Design (CRD) by doing three repetitions on 5 concentration series, 1 positive control and 1 negative control for one type of Staphylococcus The antibacterial test method used in this study is the diffusion method with the Kirby Bauer technique, namely the agar diffusion method with paper discs.
The concentrations of Chinese betel leaf juice (P.
pellucida L.
) used were:
6AAL/disc, 8AAL/disc, 10AAL/disc, 12AAL/disc and 14AAL/disc.
The positive control used was 10AAL/disc ampicillin antibiotic and the negative control was sterile distilled water.
Research Procedure Tool Sterilization The tools to be used were sterilized using an autoclave for 30 minutes at 121AC with a pressure of 1 atm.
Sterilization is an activity whose purpose is to kill microorganisms.
In the process of making media, sterilization of tools is carried out to avoid contamination7.
RESEARCH METHODS
Place and Time of Research This research was conducted at the Pharmaceutical Biology and Pharmaceutical Chemistry laboratories of the Faculty of Mathematics and Natural Sciences.
Universitas Kristen Indonesia Tomohon.
The research was conducted in January 2023-March 2024.
Chinese Betel Leaf Sampling The samples used in this study were fresh green Chinese betel leaves obtained from Lemoh Village.
Minahasa Regency which were taken by Samples of Chinese betel leaves that have been taken, collected, wet sorted / cleaned from impurities, washed under running water and drained to reduce water content, then mashed using a mortar and pestle, then placed in sterile gauze and then squeezed to get Chinese betel Tools and Materials The tools used in this study are: Laminar air flow, autoclave, desiccator, incubator, ultra violet lamp, scale, beaker glass, measuring cup, bunsen, erlenmayer, test tube, chamber, silica 60 Biofarmasetikal Tropis (The Tropical Journal of Biopharmaceutica.
2024, 7.
, 25-33 leaf juice.
Then the juice obtained is transferred into a 1.
5ml Eppendorf.
e-ISSN 2685-3167 p-ISSN 2828-6685 until homogeneous.
Then the level of turbidity is seen, namely comparing according to the McFarland 0.
5 standard solution visually.
Positive Control Preparation The positive control solution to be used is ampicillin .
@), with a concentration of 10AAL/disc.
This solution was made by grinding ampicillin tablets using a mortar and pestle and then weighing them to obtain ampicillin powder equivalent to 50 mg and dissolving it in 50ml of distilled water.
Solid Media Preparation The solid medium used is Nutrient Agar (NA).
This solid media is made by weighing 2.
grams of Nutrient Agar (NA) media then dissolving it with 90 mL of distilled water in an Erlenmeyer, stirring until the powder dissolves.
Next, it was sterilized using an autoclave at 121AC for 15 minutes.
Figure 1.
Chinese Betel Leaf Plant Identification of Compounds by Thin Layer Chromatography Alkaloids Samples were photographed on a TLC plate with a mobile phase of n-hexane: ethyl acetate .
: .
v/v with dragendoff stain reagent after being sprayed with dragendorff reagent, it produces orange or brownish red spots if it is positive for alkaloid compounds.
Flavonoids Samples were photographed on a TLC plate with a mobile phase of methanol: chloroform .
v/v with an ammonia vapor stain If it is positive for flavonoid compounds, it will be blue when viewed at UV lamp 366 nm and yellow after ammonia Tannins Samples were photographed on a TLC plate with a mobile phase of n-hexane: ethyl acetate .
: .
v/v and 5% FeCl3 stain reagent.
If it is positive for tannin compounds, then a black colored stain is formed.
yaycnycycycaycuycayce ycycycaycyceycoyceycc ycayc ycycycaycnycu Rf = Then the Rf value or retention factor of each spot that appears and is colored is calculated 8.
Antibacterial Test Procedure Pipette 1 mL of bacterial suspension into an erlenmeyer containing 30 mL of NA that is already warm .
emperature 44AC - 50AC).
Then stirred until homogeneous and then poured into a petri dish that has been sterilized, under aseptic conditions, then left to solidify.
The disc paper that has been saturated with the test solution has previously been allowed to dry at room Samples were prepared by pipetting 6AAL, 8AAL, 10AAL, 12AAL, and 14AAL in the disc paper.
The ampicillin antibiotic solution as a positive control was pipetted as much as 10AAL, and as a negative control a paper disk that had been pipetted with 6AAL of sterile distilled water was used.
Then the dried disc paper is affixed to the test media in a Petri dish that has been marked then incubated at 34AC- 37AC for 24 hours in an inverted position.
Furthermore, after 24 hours of incubation, observe whether there is an inhibition zone formed around the disc paper, measure the inhibition zone formed using a caliper and do this test 3 times.
Preparation for Test Bacteria Suspension The test bacteria were taken and then suspended into an Erlenmeyer containing 100 ml of distilled water with 0.
8g of sterilized NB and then stirred, then the bacterial suspension was incubated at 30AC for 24 hours.
Bacterial growth is characterized by turbidity in the media.
After incubating for 24 hours, it was taken and shaken Data Analysis After the data is collected, the results of observations and measurements are tabulated in tables and figures9.
Testing the antibacterial activity of Chinese betel leaf juice against staphylococcus aureus.
Subsequently, it was analyzed statistically.
Using the Kruskal-Wallis non-parametric test and further analysis using yaycnycycycaycuycayce ycycycaycyceycoyceycc ycayc yceycoycyceycuyc Biofarmasetikal Tropis (The Tropical Journal of Biopharmaceutica.
2024, 7.
, 25-33 Mann Whitney analysis with the Statistical Product Service Solution (SPSS) program with a confidence level of 95% or = 0.
The respective results obtained in this test Alkaloids Observations on the TLC plate before being given the reagent and UV irradiated produced yellow green stain spots, after UV 366 nm irradiation revealed orange, fluorescent stain Furthermore, the results given by the dragendoff reagent obtained results before UV irradiation are green and at UV 366 nm the results are blackish orange and at UV 254 nm get the results of green stain spots.
By using the mobile phase which is n-hexane: ethyl acetate .
The appearance of stains on the KLT plate before and after giving dragendoff reagent with a brownish green or brownish orange color is positive for alkaloid compounds12.
The color that appears and is formed due to the coordinate covalent bonds in the K ions of potassium tetraiodobismutate forming an alkaloid potassium complex13.
The n-hexane eluent is a non-polar solvent while ethyl acetate is a polar solvent.
The eluent can separate many stains and there are stains that indicate the presence of alkaloid compounds14.
So, the results of this study indicate that Chinese betel leaf juice is detected to have tannin alkaloid compounds.
The TLC results showed yellow green stain spots before UV irradiation and at UV 366 nm showed orange stain spots, and at UV 254 nm green stain After being sprayed with 5% FeCl3 reagent, the results before UV irradiation showed blackish green stains and at UV 366 nm showed black stains, and at UV 254 nm the results obtained yellowish green stains, so that the results of TLC with this Chinese betel leaf juice sample are suspected of containing tannin By using the mobile phase which is n-hexane: ethyl acetate .
The appearance of stains on the TLC plate before and after giving 5% FeCl3 reagent with concentrated black and purple colors is positive for tannin compounds12.
The n-hexane eluent is a non-polar solvent while ethyl acetate is a polar The eluent used is the best eluent and can show stains that are tannin compounds15.
Flavonoids The TLC results obtained yellowish green stain spot results before UV irradiation and there are blue and black, fluorescent stain spots at UV 366 nm and then obtained a green stain spot at UV 254 nm.
By using the mobile phase which is methanol: chloroform .
The appearance of Inhibition zone diameter calculation 11.
ya yaA ya Formula: D = a .
Description:
D: diameter of inhibition zone A: vertical diameter B: horizontal diameter C: diameter diagonal RESULTS AND DISCUSSION Thin Layer Chromatography (TLC) TLC result:
(A)
(B)
Figure 2.
TLC Results of Alkaloids (A) Before being sprayed with Reagent, (B) After being sprayed with Dragendoff
Reagent
(A)
e-ISSN 2685-3167
p-ISSN 2828-6685
(B)
Fegure 3.
TLC Results of Tannin (A) Before being sprayed with Reagent, (B) After being sprayed with 5% FeCl3 Reagent Fegure 4.
Flavonoid TLC Results After Evaporation of Ammonia Reagent Biofarmasetikal Tropis e-ISSN 2685-3167 p-ISSN 2828-6685 (The Tropical Journal of Biopharmaceutica.
2024, 7.
, 25-33 stains on the TLC plate before and after giving ammonia vapor reagent with yellow, green and blue colors is positive for flavonoid compounds.
Methanol eluent is a polar solvent while chloroform is a non-polar solvent, which is an eluent containing a solvent that can attract flavonoid compounds well12.
The results of the TLC test shown in Figures 4, 5, and 6, obtained the Rf value of each according to the distance traveled by the eluent.
The alkaloid test had three stains with Rf values 11, 0.
15: 0.
Stains with Rf values of 0.
15 are suspected of having alkaloid compounds because they are included in the alkaloid test Rf range of 0.
The flavonoid test had six stains with Rf values of The stain with an Rf value of 0.
35 is suspected to have flavonoid compounds because it is included in the flavonoid test Rf range of 0.
2 - 0.
The tannin test has four stains with Rf values of 0.
The four Rf values are included in the Rf range of the tannin test, which is 0.
Tests of TLC metabolite compounds in this study on samples of Chinese betel leaf juice have alkaloid, flavonoid and tannin compounds because they are included in the Rf range in the resulting stain patches.
The results of the Rf values are different because they are influenced by the eluent used, the eluent greatly affects the Rf value because it is the mobile phase on the TLC and because it is influenced by the level of polarity.
Antibacterial Activity Test The results of the antibacterial activity test showed as follows:
Figure 5.
Inhibition zone results of Chinese betel leaf juice on Staphylococcus aureus bacteria Table 1.
Results of Antibacterial Activity Testing on Staphylococcus aureus Bacteria.
Concentration .
6 AAL/disc 8 AAL/disc 10 AAL/disc 12 AAL/disc 14 AAL/disc 10 AAL/disc 6 AAL/disc Description .
Chinese betel
leaf juice Ampicillin
Aquadest
8,24
10,04
9,86
10,06
10,13
10,06
9,14
10,11
10,14
10,21
10,28
Based on the measurement of the average diameter of the inhibition zone, it is known that the concentration of 6AAL/disc inhibits 8.
81 mm, the concentration of 8AAL/disc inhibits 9.
46 mm, the concentration of 10AAL/disc inhibits 9.
75 mm, the concentration of 12AAL/disc inhibits 10.
mm, and the concentration of 14AAL/disc inhibits 12 mm.
The positive control concentration as a comparator inhibited 10.
14 mm and in the negative control no zone of inhibition was Based on the results of the average diameter of the inhibition zone formed at each concentration, it is categorized as being able to i .
8,24 9,22 9,28 10,01 10,11 10,03 Average .
8,81 9,46 9,75 10,05 10,12 10,14 inhibit the growth of Staphylococcus aureus with moderate intensity, namely the diameter of the inhibition zone of 5-10 mm, according to the Clinical and Laboratory Standard Institute (CLSI) antibacterial category19.
Alkaloids are special naturally occurring metabolites with nitrogen as a characteristic element present in their chemical structure.
The rich biological potential of alkaloids is attributed to the different arrangement of atoms in their chemical structure20.
And the mechanism of Alkaloid as an antibacterial is by interfering with the constituent components of peptidoglycan in Biofarmasetikal Tropis e-ISSN 2685-3167 p-ISSN 2828-6685 (The Tropical Journal of Biopharmaceutica.
2024, 7.
, 25-33 bacterial cells so that the cell wall layer is not formed intact and ultimately results in incomplete cell formation21.
Flavonoids are natural chemicals found in Flavonoid compounds are distributed in plant parts such as seeds, flowers, leaves, roots and stems22.
Flavonoids are phenolic compounds that are polar in nature23.
As an antibacterial, it can be divided into 3 categories: inhibiting nucleic acid synthesis, inhibiting cell membrane function, and inhibiting energy metabolism.
Flavonoids will damage the cytoplasmic membrane and cause leakage of important metabolites, thus inactivating the bacterial enzyme system24.
Tannins, on the other hand, can act as antibacterials by reacting with the cell membrane to inactivate enzymes and disable the function of genetic material.
In addition, tannin compounds also have antibacterial mechanisms that can inhibit reverse transcriptase and DNA topoisomerase enzymes, thus preventing the formation of bacterial cells24.
The structure and composition of bacterial cells also play an important role in antibacterial Staphylococcus aureus bacteria are widespread flora that often cause disease and have the same potential as invasive pathogens based on coagulase synthesis.
Staphylococcus aureus is one of the most resistant types of bacteria because it is based on bacteria that do not produce spores25.
The results of data analysis using the Kruskal Wallis test in Table 5 show a P value or Asymptotic significance value of 0.
035 <0.
which means that it is smaller than the specified significant value of 0.
05 with a confidence level of 95%, it can be concluded that there is a significant difference or meaningful difference from each concentration, the effect of the inhibition zone of Chinese betel leaf juice on the growth of S.
aureus bacteria that can inhibit bacterial growth affects the analysis.
Table 2.
Kruskal Wallis Test Results Test Statisticsa,b Inhibitory Power Staphylococcus aureus Chi-Square Asymp.
Sig.
The results of the Kruskal Wallis test showed significant differences, so further tests were carried out using the Mann Whitney test to see the differences between each treatment group, in this case the concentration of Chinese betel leaf juice, namely, .
AAL/disc, 8AAL/disc, 10AAL/disc, 12AAL/disc and 14AAL/dis.
, positive control .
mpicillin 10AAg/dis.
, and negative Table 3.
Mann Whitney Test Results Concentration .
6AAL
8AAL
10AAL
12AAL
14AAL
Positive Negative 6AAL
0,513
0,275
0,275
0,077
0,127
0,037
8AAL
10AAL
12AAL
14AAL
Positive .
Negative .
0,275 0,275 0,050 0,127 0,037 0,376 0,184 0,127 0,037
0,275
0,275
0,037
0,827
0,037
0,037
Showing at concentration 1-2, namely .
ositive control and negative contro.
concentration 2-3, namely .
egative control and 6AAL/dis.
, concentration 2-4, namely .
egative control and 8AAL/dis.
, concentration 2-5, namely .
egative control and 10AAL/dis.
, concentration 2-6, namely .
egative control and 12AAL/dis.
, and concentration 2-7, namely .
egative control and 14AAL/dis.
obtained a P value of 0.
037 <0.
05, which means that there is a significant difference or there is a meaningful difference, meaning that the clear zone or inhibition formed looks different against staphylococcus aureus bacteria.
It is said that there is a significant difference because the P value < 0.
While at concentrations 1-3, namely .
ositive control and 6AAL/dis.
, concentrations 1-4, namely .
ositive control and 8AAL/dis.
, concentrations 1-5, namely .
ositive control and Biofarmasetikal Tropis (The Tropical Journal of Biopharmaceutica.
2024, 7.
, 25-33 10AAL/dis.
, obtained a P value of 0.
127> 0.
which means that there is no significant difference or there is no meaningful difference, namely the clear zone or inhibition formed looks different against Staphylococcus aureus At concentrations 1-6 namely .
ositive control and 12AAL/dis.
, concentrations 3-5 .
AAL/disc 10AAL/dis.
, concentrations 3-6 namely .
AAL/disc and 12AAL/dis.
, .
AAL/disc and 10AAL/dis.
, concentrations 4-6 namely .
AAL/disc and 12AAL/dis.
, concentration 6-7, namely .
AAL/disc and 14AAL/dis.
, obtained a P value of 0.
275> 0.
05, which means that there is no significant difference or there is no meaningful difference, namely the clear zone or inhibition formed looks different against staphylococcus aureus bacteria.
And then concentrations 1-7, namely .
ositive control and 14AAL/dis.
, obtained a P value of 0.
827> 0.
meaning that there is no significant difference or there is no meaningful difference, namely the clear zone or inhibition formed looks different against staphylococcus aureus bacteria.
Concentrations 3-4, namely .
AAL/disc and 8AAL/dis.
, obtained a P value of 0.
513> 0.
meaning that there is no significant difference or there is no meaningful difference, namely the clear zone or inhibition formed looks different against staphylococcus aureus bacteria.
Concentrations 3-7, namely .
AAL/disc and 14AAL/dis.
, obtained a P value of 0.
077> 0.
concentrations 4-7, namely .
AAL/disc and 14AAL/dis.
, obtained a P value of 0.
050 = 0.
there is no significant difference or there is no meaningful difference, namely the clear zone or inhibition formed looks different against Staphylococcus aureus bacteria.
Concentrations 5-6, namely .
AAL/disc and 12AAL/dis.
, obtained a P value of 0.
376> 0.
05, meaning that there is no significant difference or there is no meaningful difference, namely the clear zone or inhibition formed looks different against Staphylococcus aureus bacteria.
Concentrations 5-7 namely .
AAL/disc and 14AAL/dis.
obtained a P value of 0.
184> 0.
05, meaning that there is no significant difference or there is no meaningful difference, namely the clear zone or inhibition formed looks different against Staphylococcus aureus bacteria.
These bacteria can cause an infection characterized by inflammation that forms an Infections caused such as acne, boils and wound infections on the skin, these bacteria e-ISSN 2685-3167 p-ISSN 2828-6685 can cause more dangerous infections such as urinary tract infections, food poisoning and can cause death25.
Staphylococcus aureus bacteria are bacteria that infect a lot, usually infections caused by these bacteria are found on the skin and nose27.
The many impacts on human health caused by Staphylococcus aureus bacteria make us more vigilant in finding ways to prevent and control the growth of these bacteria.
Based on the results of the study that the antibacterial activity test with Staphylococcus aureus bacteria.
The activity formed starting from the concentration of 6AAL/disc, 8AAL/disc, 10AAL/disc, 12AAL/disc and 14AAL/disc is moderate according to the category of inhibition zone
CONCLUSIONS
Based on the results of the study, it can be concluded that Chinese betel leaf juice shows antibacterial activity against Staphylococcus aureus bacteria.
The results of the concentration treatment tested, namely 6AAL/disc, 8AAL/disc, 10AAL/disc, 12AAL/disc and 14AAL/disc are categorized as moderate inhibition zone diameter, with the results of the Kruskall Wallis analysis test getting a significant value of 0.
< .
05 or a chi-square value of 13.
564 > r.
592 with a confidence interval value of 95% and an error rate of 5%, meaning that there is a real or significant difference from each concentration carried out.
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