Antimicrobic Activity of IPPU Padang (Ammannia octandra L.f.) Leaves Ethanol Extract against Skin Pathogenic Microbials Muharni Saputri*, Vriezka Mierza, Nirwana Putri Department of Pharmacy, Faculty of Pharmacy, Universitas Tjut Nyak Dhien, Jl. Gatot Subroto, Gg. Rasmi, Medan, North Sumatra 20123, Indonesia Abstract Ippu Padang plant (Ammannia octandra L.f.) belongs to the family of Lythraceae, a hardy plant that can grow to a height of 50 cm. According to previous research, Ippu Padang leaves contain glycosides, alkaloids, flavonoids and tannins. The presence of alkaloid compounds, flavonoids and tannins is predicted to have potential as an antifungal agent against fungi and antibacterial agent. This research used an experimental method. The steps included collecting plant material, plant identification, processing plants into Simplicia powder, phytochemical screening of Simplicia powder, extracting Simplicia by maceration method using ethanol solvent, antifungal and antibacterial activity test using agar diffusion method and determination of inhibition diameter of leaf ethanol extract. The results of phytochemical screening showed that ippu Padang leaves contained secondary metabolites, namely alkaloids, flavonoids, glycosides, anthraquinone glycosides, tannins, saponins and steroids. The results also revealed that the concentration with the largest inhibitory diameter was 400 mg/ml, namely 12.4 mm against the Candida albicans, 17.46 mm against the Dermacoccus nishinomiyaensis bacteria, 18.53 mm against the Micrococcus luteus bacteria, 19.38 mm against the Pseudomonas aeruginosa bacteria, and 17.71 mm against Staphylococcus epidermidis bacteria. It was concluded that the ethanol extract of ippu Padang leaves could inhibit the growth of the Candida albicans, the bacteria Dermacoccus nishinomiyaensis, Micrococcus luteus, Pseudomonas aeruginosa and Staphylococcus epidermidis. Keywords: Ammannia octandra L.f.; Candida albicans; Dermacoccus nishinomiyaensis; Micrococcus luteus; Pseudomonas aeruginosa; Staphylococcus epidermidis Data of article Received Reviewed Accepted : : : 28 Dec 2021 17 Feb 2022 21 Feb 2022 DOI 10.18196/jfaps.v2i1.13505 Type of article: Research INTRODUCTION Skin is the largest organ that covers the entire surface of the body. It is often in contact with safe and unsafe objects that can cause skin diseases. Skin disease is a * Corresponding author, e-mail: muharnisaputri@gmail.com skin disorder caused by interactions between fungi, bacteria, viruses, and others. This disease is common in society due to several factors such as climate, environment, unclean living habits, allergies and others.1 Journal of Fundamental and Applied Pharmaceutical Science, 3(1), August 2022 2 The Candida albicans fungus, as the cause of candidiasis, is commonly found in the oral cavity, digestive tract, reproductive tract and skin.2 The Dermacoccus nishinomiyaensis bacteria, previously known as Micrococcus nishinomiyaensis, is a gram-positive bacterium that exhibits the implication of peritonitis associated with peritoneal dialysis, catheter-use bacteremia, polymicrobial infections of the skin and urinary tract.3 Micrococcus luteus is an opportunistic pathogenic bacterium or bacteria that will not cause disease if another disease does not precede it. This bacterium is one of the gram-positive bacteria that can cause complex dermatitis.4 The Pseudomonas aeruginosa bacterium will become a pathogen if it is in a place with abnormal resistance, such as damaged skin due to tissue damage. These bacteria include gram-negative bacteria that can cause secondary infections in wounds, burns and soft tissue infections.5 Staphylococcus epidermidis bacteria mostly live as normal flora on human skin. These bacteria include gram-positive bacteria that can cause acne when the amount is excessive on the skin6. Diseases caused by fungi and bacteria are very common and can be treated with antifungals and antibacterials. However, excessive antifungal and antibacterial drugs and inappropriate antifungals and antibiotics can lead to resistance. Resistance occurs when microorganisms change to turn infection drugs to become ineffective. Resistance causes the microbe to fail to respond to the given drug, leading to the prolongation of the disease.7 Ippu Padang leaves have secondary metabolites of glycosides, alkaloids, flavonoids, and tannins secondary metabolites. Secondary metabolites which are estimated to inhibit microbial growth are flavonoid alkaloids and tannins,8 but it is known that there has been no research on the activity of ethanol extract of ippu Padang leaf extract against fungi and pathogenic skin bacteria; therefore, researchers are interested in conducting this research. METHOD An antimicrobial activity test with the agar diffusion method was used to determine the activity of antimicrobial agents. A petri dish containing an antimicrobial agent was placed on an agar medium where microorganisms grew, which would diffuse into the agar medium. It was later incubated for 48 hours at a temperature of 25 ± 2ºC for fungi and incubated for 24 hours at a temperature of 35 ± 2ºC for bacteria. The clear zone indicated the growth inhibition of microorganisms on the surface of the agar medium.9 RESULTS AND DISCUSSION Determining the water content in the simplicia leaves of ippu Padang showed 5.5%. Fulfilling the requirements for simplicia content was carried out as the water content was not more than 10%. Simplicia with excess water content stored for a long time will produce enzymes that can affect the content of chemical compounds and turn them into other products that may not have a pharmacological effect like the original compound.10 The results of phytochemical screening showed that the ethanol extract of ippu Padang leaves contained secondary metabolites, which were positive for alkaloids, flavonoids, glycosides, anthraquinone glycosides, tannins, saponins and steroids. Table 1 shows the Muharni Saputri, Vriezka Mierza, & Nirwana Putri | Antimicrobic Activity of IPPU Padang (Ammannia octandra L.f.) Leaves Ethanol Extract against Skin Pathogenic Microbials 3 results of phytochemical screening on ippu Padang leaves. Table 1. Phytochemical Screening No Screening Reagent Observation Results 1 Alkaloids Dragendorf Chocolate precipitate + Bouchardat Chocolate precipitate Mayer Chocolate precipitate 2 Flavonoids Zn + HCl Red + Mg + HCl Red 3 Glycosides: + Sugar Molisch White precipitate Fehling A+ B Brick red precipitate Non-Sugar Acetic acid anhydride + Sulfuric acid Purple 4 Anthraquinone Glycosides NaOH Red + 5 Tannins FeCl3 5% Blackish green + 6 Saponins Hot water, shaken + HCl 1.5 cm high foam + 7 Cyanogenic glycosides Na. Picrate Yellow - 8 Steroids- Triterpenoids Acetic acid anhydride + Sulfuric acid Blackish green + Description: (+) Contains the test compound (-) Does not contain the test compound An antimicrobial activity test was conducted to measure the response of microbial growth to antimicrobial materials. One of the uses of antimicrobial activity testing is to obtain an effective and efficient treatment system. The effectiveness of an antibacterial agent in inhibiting microbial growth depends on the nature of the test bacteria, concentration and length of contact time. Measurement of antimicrobial activity was carried out using the agar well diffusion method, characterized by the formation of a clear part around the well (inhibitory diameter) if the tested extract could inhibit microbial growth. The area of the clear part was then measured in diameter.11 The diameter of the inhibition was divided into several categories based on the diameter of the clear part around the well, including weak (<5 mm), medium (5-10 mm), strong (10-20 mm) and very strong (>20 mm).12 Journal of Fundamental and Applied Pharmaceutical Science, 3(1), August 2022 4 Figure 1. Antimicrobial activity test results Based on Figure 1, B- (Negative blank) does not have a diameter of inhibition for testing all microbes. The ethanol extract of ippu Padang leaves tended to have an inhibitory diameter at a certain concentration even though it had no inhibitory diameter of 12.5 mg/ml. It indicated that the B- used DMSO: ethanol (3:4) could not inhibit microbial growth. The concentration of 400 mg/ml had the largest inhibitory diameter among all the tests carried out where the diameter of inhibition was almost the same as the diameter of the inhibitory B+ (positive blank) used, namely nystatin for fungi and chloramphenicol for bacteria. Based on Figure 1, it can be observed that the higher the concentration is, the larger the diameter of the formed inhibition will be. In line with it, according to the statement of Surjowardojo et al. (2015), the greater the concentration is, the greater the interaction of the extract with the tested microbes will be. It causes a larger diameter of the inhibition as the extract with a large concentration contains a large number of chemical compounds that affect microbial growth.13 Furthermore, alkaloids in ippu Padang leaves can damage proteins that can harm enzyme activity and cause death in microbial cells. Alkaloids can also arrange microbial cell walls so that they cannot be formed completely and cause death in these microbial cells, leading to the formation of an inhibitory diameter.14 Flavonoids, as antifungals, work by damaging proteins, inhibiting the enzyme system, interfering with the formation of the end of hyphae, and constricting the cell wall so that the fungal cell wall died.15 Flavonoids as antibacterial work by inhibiting the synthesis of nucleic acids, inhibiting cell membranes' function and inhibiting the metabolic energy of the bacteria.14 The mechanism of action of tannins as an antimicrobial is to inhibit the activation of the enzyme and disrupt transport protein in the cell's lining so that the antimicrobial cell cannot be formed. In addition, the antimicrobial effect of tannins also can be 0 5 10 15 20 25 30 12.5 25 50 100 200 300 400 B+ B- In h ib it io n d ia m e te r Consentration mg/ml Antimicrobial activity test results Candida albicans Dermacoccus nishinomiyaensis Micrococcus luteus Pseudomonas aeruginosa Staphylococcus epidermidis Muharni Saputri, Vriezka Mierza, & Nirwana Putri | Antimicrobic Activity of IPPU Padang (Ammannia octandra L.f.) Leaves Ethanol Extract against Skin Pathogenic Microbials 5 through reaction with cell membranes and inactivation of the function of the genetic material of.14 CONCLUSION Based on the results of this research, it can be concluded that the results of phytochemical screening of ippu Padang leaf Simplicia powder showed the presence of alkaloids, flavonoids, glycosides, anthraquinone glycosides, tannins, saponins and steroids. Ippu Padang leaf ethanol extract had antimicrobial activity against the Candida albicans, the Dermacoccus nishinomiyaensis, Micrococcus luteus, Pseudomonas aeruginosa and Staphylococcus epidermidis bacteria ACKNOWLEDGMENT The authors would like to thank Tjut Nyak Dhien University for their guidance and assistance in conducting this research. REFERENCES 1. Putri, D. D., Furwan, M. T., Perdana, R. S. Klasifikasi Penyakit Kulit pada Manusia Menggunakan Metode Binary Decision Tree Support Vector Machine (BDTSM). Jurnal Pengembangan Teknologi Informasi dan Ilmu Komputer. 2018; 2(5): 1912- 1920. 2. Alioes, Y., Kartika, A., Zain, E. A., Azzura, V. Uji Potensi Antijamur Candida albicans Ekstrak Daun Gelinggang (Cassia alata L.) Dibandingkan dengan Sediaan Daun Sirih yang Beredar di Pasaran Secara In Vitro. Jurnal Kimia Riset. 2018; 3(2): 1-6. https://doi.org/10.20473/jkr.v3i2.12040 3. Bolo, N. R., Diamos, M. J. C., Su, G. L. S., Ocampo, M. A. B., Suyom, L. M. Isolation, Identification, and Evaluation of Polyethylene Glycol and Low-Density Polyethylene-Degrading Bacteria from Payatas Dumpsite, Quezon City, Philippines. Philippine Journal of Health Research and Development. 2015; 19(1): 50-59. 4. Boro, S. E. E., Suartha, I. N., Sudimartini, L. M. Efektivitas Ekstrak Daun Mimba terhadap Micrococcus luteus yang Diisolasi dari Anjing Penderita Dermatitis Kompleks. Indonesia Medicus Veterinus. 2018; 7(5):588-596. https://doi.org/10.24843/bulvet.2019. v11.i01.p05 5. Aggraini, D., Yulindra, U. G., Savira, M., Djojosugito, F. A., Hidayat, N. Prevalensi dan Pola Sensitivitas, Antimikroba Multidrug Resistant Pseudomonas aeruginosa di RSUD Arifin Achmad. Majalah Kedokteran Bandung. 2018; 50(1): 6-12. https://doi.org/10.15395/mkb.v50n1.1150 6. Nugrahani, A. R., Gunawan, F., Khumaidi, A. Aktivitas Antibakteri Ekstrak Etanol Daun Kapas (Gossypium barbadense L.) terhadap Staphylococcus epidermidis dan Propionibacterium acnes. Jurnal Farmasi Udayana. 2020; 9(1): 52-61. https://doi.org/10.24843/JFU.2020.v0 9.i01.p08 7. Humaida, R. Strategy to Handle Resistance of Antibiotics. J Majority. 2014; 3(7): 113-119. 8. Kavitha, G., Sivakkumar, T., Abraham, E. Phytochemical Screening anf Anti-oxidant Activity of https://doi.org/10.20473/jkr.v3i2.12040 https://doi.org/10.24843/bulvet.2019.v11.i01.p05 https://doi.org/10.24843/bulvet.2019.v11.i01.p05 https://doi.org/10.15395/mkb.v50n1.1150 https://doi.org/10.24843/JFU.2020.v09.i01.p08 https://doi.org/10.24843/JFU.2020.v09.i01.p08 Journal of Fundamental and Applied Pharmaceutical Science, 3(1), August 2022 6 Ammannia octandra L.f. JK Welfare & Pharmascope Foundation. 2019; 10(3): 2470-2476. https://doi.org/10.26452/ijrps.v10i3.1496 9. Mierza, V., Rosidah., Haro, G., Suryanto, D. Influence of Variant Extraction Methods (Clasical Prosedure) for Antibacterial Activity of Rarugadong (Dioscorea pyrifolia Kunth.) Tuber. Journal of Inovation in Applied Pharmaceutical Science (JIAPS). 2019; 4.(1): 1-6. 10. Siswati. Analisa Kadar Air dan Kadar Abu Pada Simplisia Temu Giring (Curcumae heyneana) dan Simplisia Kunyit (Curcumae domestica) di Balai Riset Standarisasi Industri Medan. Tugas Akhir. Fakultas Farmasi Universitas Sumatera Utara. 2020. 11. Trisia, A., Philyris, R., Toemon, A. N. Uji Aktivitas Antibakteri Ekstrak Etanol Daun Kalanduyung (Guazuma ulmifolia Lam.) terhadap Pertumbuhan Staphylococcus aureus dengan Metode Difusi Cakram (Kirby- Bauer). Anterior Jurnal. 2018; 17(2): 136-143. https://doi.org/10.33084/anterior.v17i2.12 12. Badaring, D. R., Fiqriansyah, M., Bahri, A. Identifikasi Morfologi Mikroba pada Ruangan Water Closet Jurusan Biologi Universitas Negeri Makassar. Prosiding Seminar Nasional Biologi FMIPA UNM. Makassar: Program Studi Biologi, Universitas Negeri Makassar. 2020:161-168. 13. Surjowardoyo, P., Susilorini, T. E., Sirait, G. R. B. Daya Hambat Dekok Kulit Apel Manalagi (Malus sylvestrs Mill.) terhadap Pertumbuhan Staphylococcus aureus dan Pseudomonas sp. Penyebab Mastitis pada Sapi Perah. J. Ternak Tropika. 2015; 16(2): 40-48. https://doi.org/10.21776/ub.jtapro.20 15.016.02.6 14. Rijayanti, R. P. Uji Aktivitas Antibakteri Ekstrak Etanol Daun Mangga Bacang (Mangifera feotida L.) terhadap Staphylococcus aureus secara In Vitro. Naskah Publikasi. Fakultas kedokteran, Universitas Tanjung Pura. 2014. 15. Yanti, N., Samingan., Mudatsir. Uji Aktivitas Antifungi Ekstrak Etanol Gal Manjakani (Quercus infectoria) terhadap Candida abicans. Jurnal Ilmiah Mahasiswa Pendidikan Biologi. 2016; 1(1): 1-9 . https://doi.org/10.26452/ijrps.v10i3.1496 https://doi.org/10.33084/anterior.v17i2.12 https://doi.org/10.21776/ub.jtapro.2015.016.02.6 https://doi.org/10.21776/ub.jtapro.2015.016.02.6