Bacillus cereus was a Gram-positive bacterium that caused the majority of food poisoning cases.1 Food poisoning cases continued to increase, prompting various prevention and treatment efforts. The treatment of food poisoning was generally carried out using antibiotics, antidiarrheal agents, and supportive therapy. 2,3 The use of antibiotics, however, could lead to antibiotic- associated diarrhea and, in the long term, might cause resistance, adding complexity to managing food poisoning.4 Therefore, new treatment modalities with fewer side effects were needed. The utilization of natural materials that had been traditionally consumed for generations was also considered as a treatment option.5 One such natural product with potential was the white frangipani flower (Plumeria alba), commonly found in Indonesia, especially on the island of Bali. The leaves and flowers of Plumeria alba contained secondary metabolites that acted as antibacterial agents.6 These metabolites included compounds such as terpenoids, tannins, flavonoids, saponins, alkaloids, and steroids, which exhibited antibacterial properties and showed potential in inhibiting the growth of Bacillus cereus.6–8

Research on white frangipani flowers demonstrated antibacterial potential against both Gram-positive and Gram-negative bacteria. For instance, the petroleum ether extract of white frangipani flowers produced an inhibition zone of 15.2 mm against Bacillus cereus.6 Although research on white frangipani leaves was still limited, several studies indicated that leaf extracts of frangipani exhibited antibacterial activity against Staphylococcus aureus and showed potential as an antidiarrheal agent.9–11 Other studies revealed that extracts from the leaves and flowers of white frangipani exhibited varying antibacterial effects against Bacillus cereus, depending on the concentrations used.8,11 Based on these findings, further research on the antimicrobial potential of white frangipani leaves and flowers was crucial, particularly for addressing food poisoning caused by Bacillus cereus.

The type of research design conducted was experimental to prove the inhibitory effect of white frangipani (Plumeria alba) flower and leaf ethanol extracts on the

 growth of Bacillus cereus in vitro. The study employed a Post-Test Only Control Group Design. Antibacterial activity was tested using the Kirby-Bauer diffusion method. The white frangipani flowers and leaves used in the study were obtained from Denpasar, Bali, and harvested intact before being exposed to sunlight.

The samples were divided into two groups: the control group (K) and the experimental group (E). The control group consisted of a positive control, which used the antibiotic chloramphenicol, and a negative control, which used 70% ethanol. The experimental group was further divided into six subgroups: leaf extracts with concentrations of 25% (E1), 50% (E2), and 100% (E3), as well as flower extracts with concentrations of 25% (E4), 50% (E5), and 100% (E6). The research was conducted over six months from the issuance of the research permit and took place in the Microbiology Division of the Integrated Biomedical Laboratory, Faculty of Medicine, Udayana University.

Exctraction of Ethanol Extract

The extraction process was carried out using the maceration technique with 70% ethanol as the solvent. The process began by separating, cleaning, chopping, and drying the flowers and leaves at 40°C, followed by grinding them into a fine powder using a blender. Maceration was performed with a ratio of 1:5 between the simplicia and the solvent, where 200 grams of simplicia were placed into an Erlenmeyer flask and immersed in 1000 ml of 70% ethanol. The mixture was stirred for 15–30 minutes, sealed tightly with aluminum foil, and left overnight to allow sedimentation.

The residue was macerated again with 1000 ml of 70% ethanol overnight, and the maceration process was repeated until the filtrate became clear. The filtrate was then evaporated using a rotary evaporator at 70°C to obtain a thick extract. The 100% ethanol extracts of white frangipani flowers and leaves were diluted with 70% ethanol to prepare solutions with extract concentrations of 25%, 50%, and 100%. These solutions were stored in containers wrapped in aluminum foil to prevent exposure to direct light, which could affect the compounds in the extracts.

Agar Diffusion Inhibition Test

The inhibition test was conducted using the Kirby-Bauer method. A sample of Bacillus cereus (0.1 ml) with a concentration of 0.5 McFarland (1.5×10⁸ cells/ml) was added to a test tube containing liquid medium and incubated at 37°C for 24 hours. The extract was pipetted onto blank discs in a volume of 200 µl.In the first petri dish, ethanol leaf extracts with concentrations of 25%, 50%, and 100%, along with the positive control (K+) containing chloramphenicol and the negative control (K−) containing 70% ethanol, were used. The positive control (K+) was prepared by applying chloramphenicol to a blank disc. In the second petri dish, five blank discs were placed, each containing 200 µl of ethanol flower extracts with concentrations of 25%, 50%,   and 100%, along with       K(+) (chloramphenicol) and K(−) (70% ethanol). The bacterial suspension was incubated for 24 hours at 37°C. Inhibition zones formed around

 the discs were observed and measured using a caliper.

Data Analysis

The data were analyzed using SPSS. The Kruskal-Wallis test was used to determine differences in the inhibition zones. Subsequently, the Mann-Whitney U test was employed to assess differences between the inhibition zones for each tested concentration.

The results of the inhibition zone formed by the extract of leaves and white frangipani flowers (Plumeria alba) on Bacillus cereus are shown in Table 1. The inhibition zones formed by the leaf extract at various concentrations, namely 25%, 50%, and 100%, after five repetitions, were 0 mm. The inhibition zones produced by the flower extract showed varied results and increased with higher concentrations.

Tabel 1. Inhibition Zone of Ethanol Extract of Leaves and White Frangipani Flowers (Plumeria alba) on the Growth of Bacillus cereus

Tabel 2. Results of the Median and Range Test and Kruskal-Wallis Value on the Diameter of the Inhibition Zone of Bacillus cereus by Plumeria alba Flower Extract

In table 2, in study group out of 110, 67 subjects were male and 43 were female subjects. In control group 68 were male and 42 were female subjects.

For the flower extract with a concentration of 100%, the largest inhibition zone was obtained, with an average of 10 mm. The research results were continued with a distribution test using the Shapiro-Wilk test, and a value less than 0.05 (p<0.05) was obtained, indicating that the data was not normally distributed. The data was then tested for homogeneity using the Levene's test, and a

result less than 0.05 (p<0.05) was obtained, indicating that it was not homogeneously distributed with normal data. For data that was not normally distributed and not homogeneously distributed, a non-parametric test using the Kruskal-Wallis test was performed. The test results showed a value of

0.05 (p<0.05).

Tabel 3. Results of the Median and Range Test and Kruskal-Wallis Value on the Diameter of the Inhibition Zone of Bacillus cereus by Plumeria alba Flower Extract"

These results indicate that there is a significant difference in the test concentrations on the inhibition zones produced. The significant differences caused by the flower extract at various concentrations on Bacillus cereus were further analyzed using the Mann- Whitney U test to determine the significant differences between each test group. For the concentration groups of 25%-50%, 25%-

100%, and 50%-100%, the results obtained were p>0.05, indicating non-significant results. When each concentration was compared to both the positive and negative controls, the results obtained were p<0.05, indicating a significant difference between the test groups and the controls. 

The research results presented in Table 5.1 show that the 70% ethanol extract of white frangipani flowers has varying antibacterial effects at each tested concentration. The data also indicate that the inhibition zone level increases with the concentration of the extract. The test results of the white frangipani flower extract are consistent with studies that show similar results. In a study using a 70% ethanol extract of white frangipani flowers against Streptococcus pyogenes, which has similar properties to Bacillus cereus, an increase in the diameter of the inhibition zone was observed with increasing extract concentration.8

"The white frangipani leaf extract in the study showed an inhibition zone of 0 mm against Bacillus cereus, indicating no antibacterial effect from each test concentration over five repetitions. The difference in results compared to previous studies is suspected to be due to the white frangipani leaves used coming from different

growing regions, thereby affecting the phytochemical content.12,13 The use of different target bacteria in previous studies also contributes to the differences in results. Other diarrhea-causing bacteria such as Escherichia coli, or other bacteria like Streptococcus mutans, have different structures and characteristics.14

In a study on the phytochemical content of Plumeria alba using ethanol as a solvent, differences in secondary metabolite content were found between the leaves and flowers. Saponin content was found in the flower parts but not in the white frangipani leaves.15 Saponins function to lyse bacterial cells by lowering surface tension and then increasing cell permeability, allowing antibacterial substances to enter.16 Saponins bind to lipids and cause pore formation, which disrupts the bacterial plasma membrane and prevents the cross-linking of peptidoglycan side chains, leading to bacterial cell damage.17 Antibiotics, such as chloramphenicol, used against bacteria work directly by binding to the 50s ribosome and inhibiting the peptidoglycan transpeptidase enzyme, which is responsible for the cross-linking of bacterial peptidoglycan side chains.18 The aligned mechanisms of action in saponins and antibiotics demonstrate their suitability as modalities capable of destroying bacteria.

The absence of these secondary metabolites is suspected to be a determining factor in the difference in the inhibition zone produced against the bacteria. In the test of the essential oil composition of Plumeria alba leaves and flowers, a dominant linalool content was found in the flower parts, which was not found in the leaf parts.19 Linalool damages bacterial cell membranes by reducing membrane potential and causing the leakage of essential molecules such as DNA,

RNA, and proteins.20 This compound also inhibits energy pathways and key enzyme functions, leading to metabolic dysfunction and bacterial cell death. In a study on linalool nanoemulsion, it exhibited antibacterial activity that was twice as strong and 11.5% higher antibiofilm activity against Salmonella typhimurium, making it a potential natural antibacterial agent in the food industry.21 The absence of linalool is suspected to be a trigger for the lack of an inhibition zone, as the white frangipani leaves had an inhibitory effect on the growth of Bacillus cereus, whereas the leaf parts did not. The difference in effect is suspected to be due to the presence of natural agonists that inhibit antibacterial ability, namely the antagonistic and non-synergistic properties of the plant's secondary metabolites when used together at certain concentrations.22

There was a difference in inhibitory effect when using ethanol extracts of leaves compared to flowers of white frangipani (Plumeria alba) against the growth of Bacillus cereus, a cause of food poisoning. The ethanol extract of white frangipani flowers (Plumeria alba) showed a stronger inhibitory effect.

This research can be further developed with treatment determination tests. Exploration of concentrations, target bacteria, and the utilization of other parts of the Plumeria alba plant can be conducted to advance the research in providing further studies.

1. Griffiths MW, Schraft H. Bacillus cereus Food Poisoning. In: Foodborne Diseases: Third Edition [Internet]. Third Edit. Elsevier Inc.; 2017. p. 395–405. Available           from: http://dx.doi.org/10.1016/B978-0-12- 385007-2.00020-6
2. Anant JK, Inchulkar SR, Bhagat S. A Review Article on Food Poisoning. World Journal of Pharmaceutical and Life Sciences WJPLS. 2018;4(9):94– 9.
3. Fiedler G, Schneider C, Igbinosa EO, Kabisch J, Brinks E, Becker B, et al. Antibiotics resistance and toxin profiles of Bacillus cereus-group isolates from fresh vegetables from German retail markets. BMC Microbiol. 2019 Nov 9;19(1).
4. Abad CLR, Safdar N. A Review of Clostridioides difficile Infection and Antibiotic-Associated Diarrhea. Vol. 50, Gastroenterology Clinics of North America. W.B. Saunders; 2021. p. 323–40.
5. Sri Andila P, Warseno T, Syafitri W, Gede Tirta I. Ethnobotanical Study of Hindu Society in Tabanan Bali and The Conservation Efforts. Atlantis Press International BV [Internet]. 2022;22:590–7. Available from: http://www.theplantlist.org/
6. Sibi G, Venkategowda A, Gowda L. Isolation and Characterization of Antimicrobial Alkaloids from Plumeria alba Flowers against Food Borne Pathogens. American Journal of Life Sciences. 2014;2(6):1.
7. Suwitasari F, Istiqomah N. Aktivitas Antioksidan Ekstrak Etanol Kamboja Jepang (Adenium obesum) dan Kamboja Putih (Plumeria acuminata). Al-Kauniyah Jurnal Biologi. 2020;13(2):167–78.
8. Herdana K, Mardaningrat V, Aman I, Made Jawi I, Indrayani Aw. Uji Aktivitas Antibakteri Ekstrak Etanol Bunga Kamboja Putih (Plumeria Alba) Terhadap Streptococcus pyogenes. Jurnal Medika Udayana [Internet]. 2023;12(4):77–82. Available from: http://ojs.unud.ac.id/index.php/eum
9. Kumari S, Mazumder A, BhattacharyaS. Phytochemical Screening And Evaluation Of Anti-Diarrheal Potentiality Of Plumeria Alba Linn. Leaves. Int J Pharm Sci Res [Internet]. 2020;11(9):4458. Available from: http://dx.doi.org/10.13040/IJPSR.097 5-8232.11
10. Ningsih DR, Zusfahair, Purwati. Antibacterial Activity Cambodia Leaf Extract (Plumeria alba L.) to Staphylococcus aureus And Identification Of    Bioactive Compound Group Of Cambodia Leaf Extract. Molekul. 2014;9(2):101–9.
11. Hudatama Putra A, Corvianindya Y, Aris Wahyukundari M. Uji Aktivitas Antibakteri Ekstrak Etanol Daun Kamboja Putih (Plumeria acuminata) Terhadap Pertumbuhan Streptococcus mutans (Antibacterial Activity Of Etanol Extract Of White Frangipani leaf (Plumeria acuminata) Against The Growth Of Streptococcus mutans ). Abstrak e-Jurnal Pustaka Kesehatan. 2017;5(3):449–53.
12. Katuuk RHH, Wanget SA, Tumewu P. The Effect Of Differences In Site Height On The Content Of Secondary Metabolites Of Babadotan Weeds (Ageratum conyzoides L.). Cocos Bio. 2018;10(6):1–16.
13. Wardani YK, Betty E, Kristiani E, Sucahyo D. Korelasi Antara Aktivitas Antioksidan dengan Kandungan Senyawa Fenolik dan Lokasi Tumbuh Tanaman Celosia argentea Linn. Correlation Between Antioxidant Activity and Phenolic Compound Content and Plant Growth Locations of Celosia argentea Linn. Bioma. 2020;22(2):2598–2370.
14. Fleming E, Pabst V, Scholar Z, Xiong R, Voigt AY, Zhou W, et al. Cultivation of common bacterial species and strains from human skin, oral, and gut microbiota. BMC Microbiol. 2021 Dec 1;21(1).
15. Sibi G, Awasthi S, Dhananjaya K, Mallesha H, Ravikumar KR. Comparative Studies of Plumeria Species for their Phytochemical and Antifungal Properties Againts Citrus sinensis Pathogens. International Journal of Agricultural Research. 2012;7(6):324–31.
16. Hagaggi NSA, Mohamed AAA. Plant–bacterial endophyte secondary metabolite matching: a case study. Arch Microbiol [Internet]. 2020;202(10):2679–87. Available from: https://doi.org/10.1007/s00203- 020-01989-7
17. Dias MC, Pinto DCGA, Silva AMS. Plant Flavonoids: Chemical Characteristics and Biological Activity. Molecules [Internet]. 2021 Sep 4;26(17):5377–93. Available from: https://www.mdpi.com/1420- 3049/26/17/5377
18. Long KS, Porse BT. A conserved chloramphenicol binding site at the entrance to the ribosomal peptide exit tunnel. Nucleic Acids Res. 2003 Dec 15;31(24):7208–15.
19. Mary Mawumenyo Mamattah K, Kusiwaa Adomako A, Nketia Mensah C, Borquaye LS. Chemical Characterization, Antioxidant, Antimicrobial, and Antibiofilm Activities of Essential Oils of Plumeria alba (Forget-Me-Not). Biochem Res Int. 2023;2023:1–10.
20. Mączka W, Duda-Madej A, Grabarczyk M, Wińska K. Natural Compounds in the Battle against Microorganisms—Linalool. Vol. 27, Molecules. MDPI; 2022.
21. Prakash A, Vadivel V, Rubini D, Nithyanand P. Antibacterial and antibiofilm activities of linalool nanoemulsions against Salmonella Typhimurium. Food Biosci. 2019 Apr 1;28:57–65.
22. Arreneuz S, Agus Wibowo M, Hadari Nawawi JH. Aktivitas Antimikroba Ekstrak Daun Soma (Ploiarium Alternifolium Melch) Terhadap Jamur Malassezia Furfur Dan Bakteri Staphylococcus aureus. Jurnal Kimia Khatulistiwa. 2015;4(3):84–93.