COMPARISON OF THE EFFECTIVENESS OF BANDOTAN LEAVES ( Ageratum conyzoides Linnaeus ) AND SINTRONG LEAVES ( Crassocephalum crepidioides ) EXTRACTS ON THE GROWTH OF THE BACTERI Staphylococcus aureus ATCC 6538

Staphylococcus aureus is a leading cause of infections and infant mortality in Indonesia, especially pneumonia. According to data from the Indonesian Ministry of Health in 2022, pneumonia accounted for 14.4% of cases in infants and 9.4% in toddlers, with 1,475 cases reported in Cirebon city. Allergic reactions from antibiotics have become a serious issue in treatment, prompting the need for natural alternatives such as bandotan leaf ( Ageratum conyzoides Linnaeus ) and sintrong leaf ( Crassocephalum crepidioides ). The compounds alkaloids, flavonoids, saponins, and tannins present in these leaves are believed to effectively inhibit bacterial growth. The study aims to compare the effectiveness of bandotan leaf extract ( Ageratum conyzoides Linnaeus ) and sintrong leaf extract ( Crassocephalum crepidioides ) on the growth of Staphylococcus aureus bacteria. Experimental research with posttest-only control group design from April to July 2023. The study used 8 groups consisting of bandotan leaf extract (Ageratum conyzoides Linnaeus) and sintrong leaf extract ( Crassocephalum crepidioides) at concentrations 50%, 75%, and 100%, as well as chloramphenicol (positive control) and DMSO 10% (negative control). Data were analyzed using One-way ANOVA, Post-hoc Tukey HSD, and Independent T-test. There is a comparison of the effectiveness of bandotan leaf extract ( Ageratum conyzoides Linnaeus ) and sintrong leaf extract ( Crassocephalum crepidioides ) against the growth of Staphylococcus aureus bacteria. Sintrong leaf extract ( Crassocephalum crepidioides ) exhibits an average inhibitory zone effectiveness of ± 10.44083 mm (P-value 0.000). The Crassocephalum crepidioides extract group was the most effective in inhibiting the growth of Staphylococcus aureus bacteria.


INTRODUCTION
Pneumonia is a major infectious disease that causes death in infants.One of the etiologies of pneumonia is Staphylococcus aureus (Gram positive) bacteria that infect tissues by producing enterotoxins (Cheung et al., 2021; 169,791 cases with 1,475 of them coming from Cirebon City (Kemenkes, 2022;Sugiarto, 2022).
In previous research conducted by Hasyim (2020) on the inhibition test of ethanol extract of bandotan leaves (Ageratum conyzoides Linnaeus) as an antibacterial in inhibiting the growth of Staphylococcus aureus that causes boils, the inhibition zone was 26.94 mm with a concentration of 35% (Hasyim, 2020).As in other studies conducted by Maimunah et al (2020) on the antibacterial activity test of sintrong leaf extract (Crassocephalum crepidioides) against Staphylococcus aureus bacteria, the inhibition zone result was 6.5 mm at 10% extract concentration (Maimunah et al., 2020).Based on these descriptions, it can be seen that the two plants have potential as antibacterials, especially in inhibiting the growth of Staphylococcus aureus bacteria.

Type of Research
This research is a true experimental design with a posttest only control group design whose research subject is Staphylococcus aureus bacteria.The independent variables were bandotan leaf extract and sintrong leaf extract with concentrations of 50%, 75%, and 100% while the dependent variable was the zone of inhibition of Staphylococcus aureus antibacterial activity on MHA media.The equipment was dried and put into an autoclave at a heating temperature of 121°C for 15 minutes at a pressure of 2 atm.Tweezers and ose were heated directly using a flame to make them sterile (Hasyim, 2020).

b. Extraction
The dried simplisia was blended into powder.The powdered simplisia was weighed as much as 500 grams and soaked using 70% ethanol solvent in a closed container for 3-5 days at room temperature with occasional stirring.The results of the soak were filtered using filter paper and collected in a container, then, concentrated in a rotary vacuum evaporator at 40°C until it became a semi-viscous extract (Almira et al., 2021).The semi-viscous extract was thickened with a waterbath at 60°C (Nofita, 2020).

c. Concentration series
The thick extract was diluted using 10% DMSO with the following dilution formula (Trisuci et al., 2020)

b. Positive control
The positive control solution was prepared by opening the shell of a 250 mg chloramphenicol capsule and dissolving it in 50 ml of distilled water to obtain a chloramphenicol solution in the amount of 5 mg/ml (Alouw et al., 2022;Bawondes et al., 2021).

Antibacterial effectiveness test
Prescribe disk paper in bandotan (Ageratum conyzoides Linnaeus) leaf extract at 50%, 75%, and 100% concentration.Do the same for sintrong (Crassocephalum crepidioides) leaf extract at 50%, 75%, and 100% concentration.The positive control solution of chloramphenicol and the negative control solution of 10% DMSO solution were also treated with disk paper.Prescribing was done by dripping 0.1 ml of solution on each paper disk.The disc paper that has been prescribed, planted on the inoculation medium and incubated for 24 hours in an incubator at 37°C (Wardaniati & Gusmawarni, 2021).The test bacteria that have been incubated, measured the inhibition zone using a caliper and recorded the measurement results in millimeters (mm) (Hasyim, 2020).

Interpretation of inhibition zone
The zone of inhibition was obtained from the equation in Figure 1 (Winastri et al., 2020).The calculation results were interpreted into the antibacterial sensitivity levels in the following

Data Analysis
Statistical analysis of the results of the study used the Shapiro-wilk normality test, Levene's homogeneity test, one way ANOVA bivariate test, and Tukey HSD.As well as the Independent T test multivariate test.

Result Mean Inhibition of Antibacterial Activity
The results of this study showed a comparison of the effectiveness of bandotan leaf extract and sintrong leaf extract against the growth of Staphylococcus aureus bacteria after incubation in the treatment groups.The mean inhibition of antibacterial activity can be found in Table 2.

Table 2. Mean Inhibition of Antibacterial Activity
Based on table 2, the inhibitory activity of sintrong leaf extract (Crassocephalum crepidioides) is greater than that of bandotan leaf extract (Ageratum conyzoides Linnaeus) by 13.28125 mm for P6 and 6.4125 for P3, by 10.525 mm for P5 and 4.625 mm for P2, as well as 7.78125 mm for P4 and 3.25 mm for P1.The inhibition value formed by the control treatment group has an average of 24.3125 mm in the positive control group of chloramphenicol (K (+)) while for the negative control group of 10% DMSO (K (-)) there is no inhibition zone.The difference in effectiveness between the bandotan and sintrong leaf extract treatment groups against the growth of Staphylococcus aureus bacteria in forming the inhibition zone can be seen as in Figure 2  Based on Figure 2, it can be concluded that the inhibition zone formed is in direct line with the increase in extract concentration.

Bacteria
Effective concentration is the lowest concentration that is sensitive in inhibiting bacterial growth (Dewi et al., 2018).In the results of the study, the average inhibition zone of 50% bandotan leaf extract was 3.25 mm and included in the weak category according to Davis-stout.The average inhibition zone of 75% bandotan leaf extract was 4.625 mm and categorized as antibacterial with weak sensitivity according to Davis-stout while the average inhibition zone of 100% bandotan leaf extract obtained an inhibition zone of 6.4125 mm and included the moderate category according to Davis-stout (Almira et al., 2021;Winastri et al., 2020).
Based on this description, it can be concluded that bandotan leaf extract is effective in inhibiting the growth of Staphylococcus aureus bacteria.The group with 100% bandotan leaf extract has high effectiveness, while the group with the lowest concentration that is still effective is 50% bandotan leaf extract.

Effectiveness of Sintrong Leaf Extract on the Growth of Staphylococcus aureus Bacteria
Effective concentration is the lowest concentration that is sensitive in inhibiting bacterial growth (Dewi et al., 2018).In the results of the study of the effectiveness of sintrong leaf extract, the average inhibition zone of 50% sintrong leaf extract was 7.78125 mm, including the moderate category according to Davis-stout sensitivity calculations.The average inhibition zone produced in 75% sintrong leaf extract was 10.525 mm and was classified as a moderate category.The average inhibition zone formed at 100% sintrong leaf extract was 13.28125 mm and included the strong sensitivity category (Almira et al., 2021;Winastri et al., 2020).
Based on this description, it can be concluded that sintrong leaf extract is effective in inhibiting the growth of Staphylococcus aureus bacteria.The lowest sensitive effective concentration is found in 50% sintrong leaf extract, while the best effectiveness extract group is 100% sintrong leaf extract.

Comparative Analysis of Bandotan Leaf Extract and Sintrong Leaf Extract on the Growth of Staphylococcus aureus Bacteria
The results of the analysis of differences in the inhibitory power of 1543 Asian Journal of Engineering, Social and Health Volume 2, No. 12 December 2023 bandotan leaf extract and sintrong leaf extract against the growth of Staphylococcus aureus bacteria can be seen in Figure 3 below.

Mean of Inhibition Zone Figure 3. Graph of Average Inhibition Power of Bandotan Leaf Extract and Sintrong Leaf Extract against Staphylococcus aureus Bacteria
Figure 3 shows that the average inhibition of the 50%, 75%, and 100% sintrong leaf extract groups is higher than the 50%, 75%, and 100% bandotan leaf extract groups.Comparison of effectiveness for the sintrong leaf extract group obtained an average of ± 4.76250 mm while the effectiveness of the bandotan leaf extract group obtained an average of ± 10.44083 mm.Based on these results, it can be concluded that sintrong leaf extract has better effectiveness than bandotan leaf extract in inhibiting the growth of Staphylococcus aureus bacteria.

Effectiveness of bandotan (Ageratum conyzoides Linnaeus) leaf extract on the growth of Staphylococcus aureus bacteria
Based on the results of the study, bandotan leaf extract (Ageratum conyzoides Linnaeus) has the ability to inhibit the growth of Staphylococcus aureus bacteria.This is evidenced in bandotan leaf extract concentrations of 50%, 75%, and 100% forming a clear zone that has potential as an antibacterial.The results of this study are in accordance with research conducted by Dewi, et al (2018) on the difference in the inhibition zone of Staphylococcus aureus growth at various concentrations of biduri leaf ethanol extract in vitro which states that the effective concentration is the lowest concentration that has sensitivity in inhibiting the growth of Staphylococcus aureus bacteria.50% bandotan leaf extract is the lowest concentration that is sensitive to the growth of Staphylococcus aureus bacteria with an average inhibition of 3.25 mm (Dewi et al., 2018).
In the observation of the antibacterial test of bandotan leaf extract (Ageratum conyzoides Linnaeus), an increase in antibacterial inhibition was obtained at each increase in extract concentration.This is in line with research conducted by Jungjunan, et al (2023) on the test of activity and antibacterial effectiveness of ethanol extract of bandotan leaves (Ageratum conyzoides Linnaeus) against Staphylococcus aureus bacteria which shows that the increase in antibacterial inhibition is proportional to the increase in extract concentration (Jungjunan, 2022).Based on the results of the research that has been done, sintrong leaf extract (Crassocephalum crepidioides) has the ability to inhibit the growth of Staphylococcus aureus bacteria.This is evidenced in the 50%, 75%, and 100% concentrations of sintrong leaf extract, a clear zone is formed which has the potential to inhibit the growth activity of Staphylococcus aureus bacteria.The results of this study are in line with research conducted by Dewi, et al (2018) on differences in the inhibition zone of Staphylococcus aureus growth at various concentrations of biduri leaf ethanol extract in vitro which states that the effective concentration is the lowest concentration that is sensitive in inhibiting the growth of Staphylococcus aureus bacteria (Jungjunan, 2022).50% sintrong leaf extract is a group of sintrong leaf extracts with the lowest concentration in this study and is sensitive in inhibiting the growth of Staphylococcus aureus bacteria by forming an average inhibition of 7.78125 mm.

Staphylococcus aureus bacteria
The results of the inhibition test of sintrong leaf extract in this study are in accordance with the results of previous research conducted by Malik, et al (2022) on the analysis of secondary metabolites and antibacterial sintrong leaves (Crassocephalum crepidioides (Benth.)S. Moore) against Escherichia coli which shows sintrong leaf extract has antibacterial activity at concentrations of 5%, 10%, 20%, and 30% by forming clear zones of 12.20 mm, 12.67 mm, 19.15 mm, and 20.85 mm respectively (Malik, 2022).

Staphylococcus aureus bacteria.
Sintrong leaf extract is a group of extracts that have greater effectiveness than the effectiveness of bandotan leaf extract at the same type of concentration.This can occur because bandotan leaf extract (Ageratum conyzoides Linnaeus) and sintrong leaf extract (Crassocephalum crepidioides) have different amounts of secondary metabolite components.The content of secondary metabolite compounds in bandotan (Ageratum conyzoides Linnaeus) leaf extract quantitatively in the phytochemical screening results showed the composition of alkaloids (0.31 mg/g), saponins (0.13 mg/g), total phenol compounds (8.46 mg TAE g-1), tannins (3.86 mg TAE g-1), and flavonoids (5.80 mg QE g-1) (Adeniji, 2021;Domithesa et al., 2021).As for the composition of secondary metabolite compounds in sintrong (Crassocephalum crepidioides) leaf extract quantitatively consists of tannins (1.05 mg TAE g-1), flavonoids (7.59 mg QE g-1), phenolic compounds (8.68 mg TAE g-1), steroids (6.39 mg/100 g), and oxalic acid (7.8 mg/100 g) (Adeniji, 2021;Domithesa et al., 2021) Flavonoids are polar compounds that can penetrate the peptidoglycan layer of bacteria.These compounds work by forming complexes that bind to proteins and cause the bacterial cell membrane to be damaged by releasing intracellular compounds.Flavonoid compounds also inhibit cell membrane function by damaging membrane permeability and inhibiting the binding process of ATPase and Phospholipase enzymes (Yevani et al., 2023).
Phenolic compounds (saponins) have antibacterial ability by disrupting the stability of the cell membrane through the content of glycosyl groups (polar) and steroid groups (non-polar).The content of glycosyl groups and steroid groups in saponins can cause intracellular fluid to escape and lyse bacterial cells (Sogandi, 2018).
Tannin compounds have the ability to destroy the formation of bacteria through a system of inhibiting bacterial metabolic enzymes.This compound, will precipitate a protein that interferes with reverse transcriptase and DNA topoisomerase enzymes which ultimately results in the inactivation of microbial cell adhesins and disrupts protein transport (Hasanah & Novian, 2020).
The antibacterial mechanism of triterpenoids works by forming polymeric bonds to transmembrane proteins of the outer cell wall which results in damage to the porin and reduces the permeability of the cell wall so that, bacteria become deprived of nutrients and there is a decrease in the number of bacteria.Steroid phytochemical compounds in the extract can react with lipids in bacteria which results in leakage of lysosomes in bacteria (Herrialfian et al., 2020).
The structure and composition of Staphylococcus aureus bacterial cells also play an important role in the mechanism of antibacterial inhibition.Staphylococcus aureus bacteria have thick peptidoglycan walls and contain teichoic acid which functions in the entry and exit of ions into bacterial cells (Hamida et al., 2023;Magvirah et al., 2020).This is in line with the research of Hamida, et al (2018) on the antibacterial activity of 96% ethanol extract of kecapi seeds (Sandoricum koetjape (Burm.F.) Merr.) against Propionibacterium acnes and Escherichia coli stating that differences in peptidoglycan components in bacterial cells can affect the effectiveness of the tannin mechanism of action.Tannins have stronger effectiveness against Grampositive bacteria compared to Gramnegative bacteria.
Statistical analysis in this study showed that the inhibition zone data from bandotan and sintrong leaf extracts were

Results of Inhibitory Zone from Bandotan Leaf Extract and Sintrong Leaf Extract Group
below Comparison of The Effectiveness of Bandotan Leaves (Ageratum conyzoides Linnaeus) and Sintrong Leaves (Crassocephalum crepidioides) Extracts on The Growth of The Bacteri Staphylococcus aureus ATCC 6538 Page 1542 Asian Journal of Engineering, Social and Health Volume 2, No. 12 December 2023Figure 2.
Comparison of The Effectiveness of Bandotan Leaves (Ageratum conyzoides Linnaeus) and Sintrong Leaves (Crassocephalum crepidioides) Extracts on The Growth of The Bacteri Staphylococcus aureus ATCC 6538 Page 1544Asian Journal of Engineering, Social and Health Volume 2, No. 12 December 2023 Comparison of The Effectiveness of Bandotan Leaves (Ageratum conyzoides Linnaeus) and Sintrong Leaves (Crassocephalum crepidioides) Extracts on The Growth of The Bacteri Staphylococcus aureus ATCC 6538 In an effort to compare the effectiveness of bandotan leaf extract and sintrong leaf extract at the same concentration, an Independent T-test was conducted and resulted in a p value = 0.000 (p<0.05).This indicates a statistically significant difference between the ability of the two extracts to inhibit the growth of Staphylococcus aureus bacteria.Nutritional Quality and Phytochemical Assessment of Some Under-Utilized Traditional Green Leafy Vegetables.Almira, J., Yusransyah, Y., Kuncoro, B., Putri, R., & Fhatonah, N. (2021).