Berebat (Spatholobus ferrugineus) is a climbing shrub that grows by wrapping its stems around larger plants and is found in wet tropical biomes (Saputri et al., 2023). In East Belitung, Berebat stems are used empirically by the community to treat various diseases, one of which is sprue mouth. People use boiled Berebat stems to drink and also as a mouthwash (Abdussalam et al., 2021).

Berebat is a plant in the Fabaceae family. The Fabaceae family is a group that produces secondary metabolites from the flavonoid and isoflavonoid groups (Cahyana & Adiyanti, 2021). Preliminary research was conducted by (Marliana, 2007) regarding the antioxidant activity of the active compounds contained in Berebat. Based on the results of phytochemical screening, It is known that Berebat stems contain alkaloids, flavonoids, polyphenols, and terpenoids/steroids. (Saputri et al., 2023) have successfully isolated pterocarpan derivative compounds from the bark of Berebat, namely the compounds variabilin and medicarpin. In another study conducted by (Aliviyanti et al., 2021), 96% ethanol reflux extract was proven to increase the LC50 of Berebat and contains anti-breast cancer substances.

Other research previously carried out on members of the Spatholobus genus included Spatholobus littoralis Hassk extract against Pseudomonas aeruginosa bacteria. Spatholobus littoralis Hassk has antibacterial effectiveness at a concentration of 100% and has a clear zone of 22.4 mm, which can be expressed as a strong inhibitory response (above> 20) against the growth of Staphylococcus aureus with 96% ethanol solvent. It has inhibitory power at concentrations of 10%, 20%, and 30% of 7.63 mm, 8.26 mm, and 8.83 mm. Spatholobus littoralis Hassk contains phenolic compounds, flavonoids, alkaloids, terpenoids, and tannins ((Latu & Wahid, 2023); (Weldy et al., 2022)).

The use of Berebat stems, which has been empirically used but lacks scientific evidence of its efficacy, prompted this study to evaluate its antibacterial activity against Streptococcus sanguinis, a common oral commensal that has been implicated in recurrent aphthous stomatitis and other oral microbial imbalances (Okahashi et al., 2022)(Seoudi et al., 2015). Extraction using maceration and fractionation was chosen to prove the solvent variation on the resulting compound's characteristics and maximize its antibacterial activity.

The maceration of 500 g of Berebat stem powder yielded 82.3 g of ethanol extract, corresponding to an extraction yield of 16.46% relative to the initial plant material. The fractionation was performed using 20 g of the crude ethanol extract as the starting material. Based on this crude extract, the n-hexane fraction yield was 14.84%, and the ethyl acetate fraction yield was 35.1%. Extract yields are fundamental to calculating plant-to-extract ratios (Monagas et al., 2022). The extraction solvent plays a crucial role in influencing the properties of extracts. Various extraction solvents may have various effects on the solubility, the amount of extractable material in the starting plant, extraction yield, and pharmacological activity of the plant (Azabou et al., 2020)(Monagas et al., 2022). Thus, a suitable solvent should be selected for the extraction of compounds targeted.

The phytochemical profiles of all samples (Berebat powder, extract, and fractions) were screened using various reagents to confirm the presence of the significant phytochemicals group, as depicted in Table 1.

A clear zone around the disc paper indicates that the extract can inhibit bacterial growth, as depicted in Table 2. Pan et al. (2009) also classify antibacterial activity based on the inhibition zone diameter, as depicted in Table 3.

So, it was concluded that the ethyl acetate fraction had the best antibacterial activity against Streptococcus sanguinis bacteria. As a semi-polar solvent, ethyl acetate has more complex chemical compounds than polar and nonpolar solvents (Esati et al., 2024). The Kruskal–Wallis test revealed a statistically significant difference in the inhibition zone diameters among the ethanol extract, n-hexane fraction, and ethyl acetate fraction of Spatholobus ferrugineus stem against Streptococcus sanguinis (H = 28.883, df = 2, p < 0.001).

Based on the mean rank values, the ethyl acetate fraction exhibited the highest antibacterial activity (mean rank = 34.17), followed by the ethanol extract (mean rank = 25.93), while the n-hexane fraction showed the lowest antibacterial activity (mean rank = 8.90). These findings indicate that fractionation with ethyl acetate enriched antibacterial constituents and showed the highest antibacterial activity among the tested samples.

The fractionation method and semi-polar solvents yielded better extractions, which may tell us that the Berebat stem is enriched with semi-polar secondary metabolites. Fractionation is a process of separation of plant extracts into various fractions. The fractionation method can maximize the extraction process, extracting compounds based on their polar properties (Abubakar & Haque, 2020). The ethyl acetate solvent is semi-polar, causing the extraction of compounds to be larger, whereas non-polar and polar compounds will also be attracted to the ethyl acetate fraction (Herdiana & Aji, 2020).

Ethanol extract can dissolve flavonoids, tannins, saponins, alkaloids, and triterpenoids in ethyl acetate fraction, flavonoids, tannins, saponins, and alkaloids. Meanwhile, flavonoids, tannins, alkaloids, and triterpenoids were also found in the n-hexane fraction. Flavonoids and alkaloids are semi-polar and are found in all three solvents. They can dissolve in all types of solvent polarity (Setyaningsih et al., 2024). Flavonoids have sugar groups (simple carbohydrates) because they can dissolve in water, which causes them to dissolve quickly in polar or semi-polar solvents. Alkaloids contain nitrogen in their cyclic part and bond with various groups, including amine, amide, phenol, and methoxy groups, which causes alkaloids to be semi-polar (Putri et al., 2023).

Saponin is a surface-active compound that is easily detected through its ability to form foam. The glycosidic bond component contained in saponin causes this compound to tend to be polar. Therefore, saponin is not found in the non-polar fraction (Suleman et al., 2022). The ethyl acetate fraction showed the strongest antibacterial activity, even though it was negative for triterpenoids in the phytochemical screening. This suggests that the inhibitory effect was more likely due to the combined or synergistic action of flavonoids, tannins, and alkaloids detected in the extract and fractions. These compounds are known to contribute to antibacterial activity through multiple mechanisms, including membrane disruption, protein precipitation, enzyme inhibition, and interference with bacterial genetic material. (Setyaningsih et al., 2024). More compounds were present in simplicia powder, which had not undergone an extraction process, and in ethanol extract. Secondary metabolites could be reduced during the extraction process. The extraction process can affect the concentration and therapeutic effect of simplicia due to the stability and nature of secondary metabolites in simplicia (Issusilaningtyas et al., 2023).

Ethyl acetate can attract more flavonoids. Flavonoids are metabolite compounds often contained in the Fabaceae family (Cahyana & Adiyanti, 2021). Berebat is included in this family. This was proven based on the extraction results, which showed that the yield of the ethyl acetate fraction was greater than that of the ethanol extract and n-hexane fraction. Flavonoid compounds cause damage to cell membranes and the release of intracellular bacterial compounds (Samputri et al., 2020). Besides flavonoids, tannins may also contribute to the antibacterial activity of the ethyl acetate fraction by complexing with bacterial proteins, disrupting cell wall integrity, and interfering with microbial adhesion, thereby inhibiting the growth of Streptococcus sanguinis. The increase in extract content is directly proportional to the increase in inhibitory diameter. The higher the concentration of a substance, the greater the number of microorganisms that can be inhibited (Fatriadi et al., 2018)(Setyaningsih et al., 2024).

Antibacterial activity test on ethanol extract, ethyl acetate fraction, and n-hexane fraction using disc diffusion showed that the ethyl acetate fraction of Berebat had the best antibacterial activity. Among the tested samples, the ethyl acetate fraction exhibited the strongest antibacterial activity against Streptococcus sanguinis and showed significantly higher overall inhibitory effects than the other fractions based on Kruskal–Wallis analysis.

The author would like to thank the Faculty of Pharmacy, National Institute of Science and Technology (ISTN) for supporting research facilities.

EPS and RA conducted the experiment, SB and RA conducted an antibacterial activity test, and EPS and SB wrote and revised the manuscript. All authors agreed on the final version of this manuscript.

The authors state that they have no conflict of interest with respect to the work described in this manuscript.

The authors state that they have no ethical issues.