Biology, Medicine, & Natural Product Chemistry ISSN 2089-6514 (paper) Volume 14, Number 2, October 2025 | Pages: 777-781 | DOI: 10.14421/biomedich.2025.142.777-781 ISSN 2540-9328 (online) Antioxidant Activity Test of Bride's Tears (Antigonon leptopus) Leaves Using the DPPH Method Nailil Wafiq Azizah, Majida Ramadhan*, Faisal, Devana Rahma Aldina, Ramadina Novia Putri, Nur Laila Anggraini, Sabilatul Mufarrohah, Satria Gemilang, Firadika Fitriansyah, Hosiyah, Nuvia Harini Wulan Suci, Nihlatus Sofiya, Terra Januarista, Ramizard Rafsanjani Biology Department; Faculty of Mathematics and Natural Science, Universitas Islam Malang, Jl. MT Haryono 193 Gedung Utsman bin Affan, Tel. +620341551932, Indonesia. Corresponding author* majida.ramadhan@unisma.ac.id Manuscript received: 10 June, 2025. Revision accepted: 18 September, 2025. Published: 01 October, 2025. Abstract Bridal tear leaves can be used to treat various health and beauty problems because they contain various active compounds, such as flavonoids, saponins, and tannins. These compounds have antioxidant, anti-inflammatory, and antibacterial properties. This flower contains antioxidants that can help protect the skin from damage caused by free radicals. This research aims to see the effectiveness of antioxidants from bridal tear leaves. The research method involves leaf extraction using 96% ethanol and testing antioxidant activity using the DPPH method. The research results showed that the extract sample from bride's tear leaves was (IC50 – 69.7146 ug/mL). This shows that the bride's tear leaf extract is quite strong in inhibiting free radical reactions. Keywords: Antioxidant; Etanol Extract; DPPH. INTRODUCTION Indonesia has a relatively high number of diabetes cases, based on data obtained in 2018 Basic Health Research (RISKESDAS) results, showing the prevalence of Diabetes Mellitus in Indonesia based on doctor's diagnosis in the population aged ≥15 years by province in 2013 by 1.5%, increasing in 2018 by 2.0%. The diversity of plant species in Indonesia has various benefits. The benefits of plants are obtained from the compounds in plants. Active plant compounds are obtained by extraction. One of the benefits of plant extracts is that they can withstand or block the occurrence of free radicals. Compounds with the ability to ward off free radicals are called antioxidants. Antioxidants are compounds that can inhibit and prevent oxidation (Simanjuntak, 2012). The way antioxidants work is by stopping free radical reactions from metabolism in the body or from the environment (Meigaria et al., 2016). Free radicals are molecules with one or more unpaired electrons. Free radicals in the body cause cancer, stroke, heart disease, and premature aging (Rahman et al., 2016). One of the plants that can act as an antioxidant and prevent free radicals is the leaves of bridal tears (Antigonon leptopus). Bride's tears is a type of creeping herb that is also often found wrapped around tree trunks. This plant has a twisting tool that functions as a flower support. Bride's tears flowers can grow well in conditions exposed to full and direct sunlight or also in a slightly shaded place. A suitable place for planting this flower is between 1 - 1800 m above sea level (Meidita, 2023). The content of the bride's tears leaves includes Alkaloids, saponins, tannins, sterols and / or unsaturated triterpenes, flavonoids, glycosides, coumarins, phytosterols, triterpenoidal sapogenins, phenols, amino acids, steroids, xanthoproteins, carboxylic acids, quinines, resins, n- hentriacontane, ferulic acid, 4-hydroxycinnamic acid, quercetin-3-rhamnoside, kaempherol-3-glucoside along with ß-sitosterol, ß-sitosterol-glucoside, and d-mannitol (Suryowinito, 1997). In research conducted by Lestari (2021), the content in bride's tears leaves has properties antioxidant and can inhibit diabetes. Testing the antioxidant content is needed to find out how strong the bride's tears leaves are as antioxidants using the DPPH test. The DPPH method is a method that can be used to determine the antioxidant activity in the sample to be tested by looking at its ability to counteract DPPH free radicals, the use of the DPPH test is used because it has several advantages including a simple, easy, fast, sensitive method, and requires a small amount of sample (Cavalcante, 2018). Based on the description that has been presented, it is necessary to test https://doi.org/10.14421/biomedich.2025.142.777-781 778 Biology, Medicine, & Natural Product Chemistry 14 (2), 2025: 777-781 antioxidants in bride's tears leaves using the DPPH method (Camero et al, 2018). MATERIALS AND METHODS Tools and Materials The tools used in this study include analytical scales, blender, piper, micropipette, measuring cup, Durham tube, oven, test tube and rack, DPPH kit, sieve, aluminum foil, spectrophotometer, shaker, filter paper, erlenmayer, glass funnel, evaporator flask and rotary evaporator. The materials used include bride's tears leaves, methanol, DPPH solution and quercetin. Procedures A. Sample Preparation Sample preparation begins with collecting or picking the bride's tears leaves, which is carried out in Kebonagung village, Pakisaji District, Malang Regency. B. Sample Preparation Sample preparation begins with collecting or picking bride's tears leaves carried out in Kebonagung village, Pakisaji District, Malang Regency. C. Making simplisia Fresh bride's tears leaves are first cleaned and sorted then dried using an oven at 50 degrees Celsius. After drying the sample is crushed using a blender until smooth, the bride's eye powder is then sifted to get the finest powder and check the water content. Water content in simplisia must be below 10%, which aims to avoid the rapid growth of fungi in the extract. D. Extraction Simplisia was weighed as much as 30 grams and then added 96% ethanol solvent in a mass-to-mass ratio with a volume ratio of 1:10. The sample was heated using a hot plate at a temperature of 40 ° C - 50 ° C for 2 hours with 130 rpm. simplisia powder was put into a maceration container and ethanol was added and then allowed to stand for 24 hours. After 24 hours continued with evaporation using a Rotary Evaporator with an rpm of 50 at 70 ° C for 1 hour to get a paste-shaped extract. The paste obtained was weighed and the result was 3.515 grams. E. Calculation of yield The yield is obtained by comparing the weight of the red pomegranate leaf extract and the weight of the simplisia before extraction. According to Kurniawan (2016) the calculation of yield can be calculated with the following equation: 𝑌𝑖𝑒𝑙𝑑 (%) = 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝐸𝑥𝑡𝑟𝑎𝑐𝑡 (𝑔) 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑆𝑖𝑚𝑝𝑙𝑖𝑠𝑖𝑎 (𝑔) 𝑥 100 F. Preparation of DPPH DPPH powder was weighed as much as 0.007 grams and then dissolved with 50 mL of ethanol, vortexed until dissolved. Furthermore, the DPPH solution was taken 1 mL then ethanol was added to 5 mL and allowed to stand for 30 minutes. Then a master solution was made with 50 mg of extract and 50 ml of methanol, made a solution with a concentration of 200, 100, 50, 12 and 5 ppm. Each solution was taken 1 mil and added DPPH as much as 3 ml and incubated for 30 minutes. G. Maximum wavelength measurement The maximum wavelength was calculated using a spectrophotometer using a comparison solution, namely quercetin. Whether it is lower or higher than the comparison solution, or even has the same antioxidant activity. H. Antioxidant activity test of bride's tears leaf extract The results of the maximum wavelength calculation from the spectrophotometer were analyzed using Excell to determine the sample’s IC50. RESULTS AND DISCUSSION Extraction and Yield Calculation Extraction of bride's tears leaf extract simplisia using maceration method obtained the yield value in Table I. Table 1. Extraction Results and Calculation of the Yield of Bride's Tear leaves (Antigonon leptopus). Simplisia Weight Bride's tears leaf Extract Caracteristic Extract Weight Yield Weight Shape, colour, and smell 30 gram 3,515 gram 11,71 gram thick, green leafy odor The yield value shows the bioactive value contained in plants, the higher the yield value, the higher the bioactive value of plants (Budiyanto, 2015). According to the Indonesian Ministry of Health, the yield requirement can be used if the yield value is more than 10%. Table 1 shows that the yield can be used with a value of 11, 71%. Azizah et al. – Antioxidant Activity Test of Bride's Tears (Antigonon leptopus) Leaves … 779 Qualitative Test Figure 1. The result of DPPH addition to Bride's Tear Leaf Extract (Antigonon leptopus). The clearer the color resulting from the addition of DPPH to the extract, the higher the antioxidant content in the extract. In Figure 1, it is known that the clearest color is at a concentration of 12, 5 ppm and the darkest at a concentration of 200 ppm. This color change indicates the antioxidant activity that is happening. If DPPH is reduced, it is indicated by a decrease in purplish color to yellow color due to the antioxidant activity of the extract (Miksusanti, 2012). Antioxidant activity test Measurement of the ethanol extract of bride's tears leaves (Antigonon leptopus) was carried out at various concentrations, namely 12.5, 50, 100, and 200 µg/mL. The results of the antioxidant activity test by looking at the results of the calculation of absorbance or absorbance carried out on a spectrophotometer were analyzed to determine the percentage of inbibition and IC50 on the leaves of the bride's tears, can be seen in Table 2. Table 2. Analysis of activity test and IC50 on bride's tear leaves (Antigonon leptopus). The IC50 test results on the sample have a value of 69.7146 ug/mL as presented in table 2. Table 2 shows that bride's tears leaves with the maceration method have antioxidant activity. The results of the linear regression equation can be seen in Figure 2. shows that the equation between %inbibition with extract concentration and obtained the standard equation curve y = ax + b, with a value of y = 0.0025x + 0.5752, y with a value of 50 obtained from IC50. The high and low antioxidant activity in the sample is seen from the Efficient Concentration (EC50) or Inhibition Concentration (IC50) value, which is the value of 50% DPPH losing its free radical properties. The smaller the IC50 value, the higher the antioxidant activity of the sample. According to (Sepriyani et al., 2020) a compound is said to be a very strong antioxidant if the IC50 value is less than 50 µg/mL, strong if the IC50 value is between 50-100 µg/mL, moderate if the IC50 value is 100-150 µg/mL, weak if the IC50 value ranges from 150-200 µg/mL. Based on this statement, bride's tears leaves (Antikodon leptopus) are classified in the strong category. Figure 2. Linear regression of bride's tears (Antikodon leptopus) leaf antioxidant assay The test results of the comparison solution in the form of quercetin have an IC50 value of 35.189 can be seen in table 3. The IC50 value of the test solution is smaller than the IC50 value of the bride's tears leaf sample. Although the IC50 value of the comparison solution is smaller, the antioxidant activity of the comparison solution is included in the strong category. The smaller the IC50 price, the stronger the antioxidant in counteracting free radicals or can be said to have stronger antioxidant activity (Bujang, 2014). Table 3. Analysis of activity test and IC50 on bride's tear leaves (Antigonon leptopus). Concentration Review Absorbance Range Inbibition (%) IC50 1 0,737 2 0,568 3 0,555 1 0,557 2 0,523 3 0,483 1 0,351 2 0,221 3 0,483 1 0,087 2 0,176 3 0,122 61,89 67,97 83,65 92,11 69,7146 12,5 ppm 50 ppm 100 ppm 200 ppm 0,128 0,266 0,521 0,620 Concentration Review Absorbance Range Inbibition (%) IC50 1 1,243 2 1,167 3 1,207 1 1,023 2 1,147 3 1,07 1 0,908 2 0,902 3 0,842 1 0,768 2 0,638 3 0.623 1 0,503 2 0,513 3 0,404 30 0,473 70,9096 35,189 33,6202 25 0,884 45,6771 27,5 0,676 58,4306 20 1,207 25,7939 22,5 1,08 780 Biology, Medicine, & Natural Product Chemistry 14 (2), 2025: 777-781 The results of the linear regression equation can be seen in Figure 3. shows that the equation between %inbibition and the concentration of the comparator solution obtained a standard equation curve y = ax + b, with a value of y = 4.6017x - 68.156. Figure 3. Linear regression of the antioxidant assay of the comparison solution (quercetin). An antioxidant activity test in a plant is very important to know whether the plant has been proven to have binding activity against free radicals. Bride's tear leaves can be used to treat various health and beauty problems because they contain various active compounds, such as flavonoids, saponins, and tannins. These compounds have antioxidant, anti-inflammatory, and antibacterial properties. In this study, the samples used were extracts of bride's tears leaves that had been macerated using 96% ethanol and quercetin as a comparator, Quercetin is the main flavonoid compound included in the flavonol group (Materska, 2008). The selection of 96% ethanol as a solvent is preferred because it presents a better amount of poly phenols compared to water extracts, is more efficient in penetrating cell walls and pulling polyphenols out of cells, and is preservative against microorganisms (Lapornik et al., 2005). DPPH compound is a molecule containing unstable nitrogen free radical compounds that can bind hydrogen ions so it is used for testing antioxidant activity (Permana, 2003). The presence of antioxidant activity in the test solution of bride's tears leaf extract in various concentrations is proven qualitatively by the gradual color change to a pale color at various concentrations of the test solution. It can be seen that the bride's tear leaf extract test solution with ethanol solvent at a concentration of 125 ppm still has a purplish color while at the largest concentration of 100 ppm the color of the solution is more purple, this change shows an indication of the activity of antioxidant content and shows that almost all free electrons in DPPH have bonded with antioxidant hydrogen atoms in the sample so as to convert DPPH to DPPH-H which has lost its free radical properties (Juniarti, 2018), The greater the color change to a pale color in the solution indicates that the greater the concentration of antioxidants in the solution. CONCLUSIONS From the results, it can be concluded that the antioxidant activity of the bride's tears leaf extract sample (Antigonon leptopus) is in the strong category with an IC59 value (69.71463 ug/mL). Acknowledgements: The author would like to express sincere appreciation to Universitas Islam Malang (UNISMA) for the academic support and resources provided throughout the research process. Gratitude is also extended to the Faculty of Islamic Studies, whose guidance and encouragement have been instrumental in completing this work. Authors’ Contributions: Majidah Ramadan1 & Faisal designed the study. Terra Januarista & Ramizard Rafsanjan carried out the laboratory work. Nailil Wafiq Azizah, Devana Rahma Aldina & Ramadina Novia Putri analyzed the data. Nur Laila Anggraini, Sabilatul Mufarrohah, Satria Gemilang, Firadika Fitriansyah, Hosiyah, Nuvia Harini Wulan Suci, Nihlatus Sofiya wrote the manuscript. All authors read and approved the final version of the manuscript Competing Interests: The authors declare that there are no competing interests. REFERENCES Budiyanto, M. S. A. (2015). Potensi Antioksidan, Inhibitor Tirosinase, dan Nilai Toksisitas dari Beberapa Spesies Tanaman Mangrove di Indonesia.Cavalcante GM, da Silva Cabral AE, Silva CC. 2018. Aktivitas Leishmanicide Flavonoid Alami dan Sintetis: Tinjauan Mini. Cetakan J. Pharm. medis. Sains. 2018; 7 :25–34. Bujang, A. (2014). Changes on Amino Acid Content In Soybeans, Garbanzo beans and Groundnut During Pretreatment and Tempe Making, Sain Malaysian 43 (4): 551 - 557 Departemen Kesehatan Republik Indonesia. (2017). Farmakope Hebal Indonesia Edisi II. In Farmakope Herbal Indonesia.Juniarti et al.. Kandungan Senyawa Kimia, Uji Toksisitas (Brine Shrimp Lethality Test) dan antioksidan (1,1- diphenyl-2-pikrilhydrazy dari Ekstrak Daun Saga (Abrus precatorius L.). Makara, Sains. (2009) 13(1) 50-54. Tersedia dari: doi:https://doi.org/10.7454/mss.v1311.378. Kurniawati, I., and Maftuch, H. A. (2016). Penentuan pelarut dan lama ekstraksi terbaik pada teknik maserasi Gracilaria sp. serta pengaruhnya terhadap kadar air dan rendemen. Jurnal Ilmu Perikanan. 2016. 7(2), 72-77. Meidita, B. (2023). Teknik Budidaya Tanaman Air Mata Pengantin (Antigonon leptopus) Secara Generatif Dalam Mendukung Ketersediaan Pakan Lebah Kelulut (Trigona Spp.). UNIVERSITAS LAMBUNG MANGKURAT. Meigaria, Komang Mirah, I Wayan Mudianta, and Ni Wayan Martiningsih. (2016). Skrining Fitokimia Dan Uji Aktivitas Antioksidan Ekstrak Aseton Daun Kelor (Moringa Oleifera). Jurnal Wahana Matematika dan Sains 10(2): 1–11. Azizah et al. – Antioxidant Activity Test of Bride's Tears (Antigonon leptopus) Leaves … 781 Miksusanti, Elvita dan Hotdelina. S. (2012). Aktivitas Antioksidan dan Sifat Kestabilan Warna Campuran Ekstrak Etil Asetat Kulit Buah Manggis (Garcinia mangostana L.) dan Kayu Secang (Caesalpinia sappan L.). Junal Penelitian Sains. Universitas Sriwijaya, Sumatra Utara. Pratimasari, D. (2009). Uji Aktivitas Penangkap Radikal Buah Carica papaya L. Dengan Metode DPPH dan Penetapan Kadar Fenolik Serta Flavonoid Totalnya, Fakultas Farmasi Universitas Muhammadiyah Surakarta. Surakarta. Rahman, Arif, Abd Malik, and Aktsar Roskiana Ahmad. (2016). Skrining Fitokimia Dan Uji Aktivitas Antioksidan Ekstrak Etanolik Buah Buni (Antidesma Bunius (L.) Spreng). Jurnal Fitofarmaka Indonesia 3(2): 159–63. Sepriyani, H., Devitria, R., Surya, A., & Sari, S. (2020). Aktivitas Antioksidan Ekstrak Metanol Daun Pepaya (Carica papaya L) Dengan Metode DPPH. Jurnal Penelitian Farmasi Indonesia, 9(1), DPPH. 10,863-867. Simanjuntak, Kristina. (2012). Peran Antioksidan Flavonoid Dalam Meningkatkan Kesehatan. FK UPN Veteran Jakarta 3. Suhaling, S. (2010). Uji Aktivitas Antioksidan Ekstrak Metanol Kacang Merah (Phaseolus vulgaris L.) dengan Metode DPPH. Skripsi. Universitas Islam Alauddin. Makasar. Suryowinoto. S.M. (1997). Flora Eksotika, Tanaman Hias Berbunga. Penerbit Kanisius, Yogyakarta. WHO. 2016. Media Centre Diabetes Mellitus. Retrieved Juni 03, 2024. Zhao L., Yuan X., Wang J., Feng Y., Ji F., Li Z., Bian J. (2019). Tinjauan tentang flavon yang menargetkan protein kinase serin/treonin untuk obat antikanker potensial. Obat Bioorganik. kimia; 27 :677–685. THIS PAGE INTENTIONALLY LEFT BLANK