Biology, Medicine, & Natural Product Chemistry ISSN 2089-6514 (paper) Volume 14, Number 2, October 2025 | Pages: 1257-1263 | DOI: 10.14421/biomedich.2025.142.1257-1263 ISSN 2540-9328 (online) Analysis of the Antioxidant Activity of Young and Mature Bidara Leaves Using 2,2-Diphenyl-1-Picrylhydrazyl (DPPH) and Phytochemical Screening Test Alvelin Bunga Kurniawati1, Gatra Ervi Jayanti1*, Majida Ramadhan1, Dian Novita Wulandari2, Ike Widyaningrum2 1Study Program of Biology, Faculty of Mathematics and Natural Sciences, Universitas Islam Malang, 2Study Program of Pharmacy, Faculty of Medicine, Universitas Islam Malang. Jl. Jalan Mayjen Haryono No. 193, Malang 65144, Tel. +62-341-551932, Fax. +62-341-552249, Indonesia. Corresponding author* gatra.ervi@unisma.ac.id Abstract This study aimed to compare the antioxidant activity of young and mature bidara leaves using the DPPH method and to identify the content of secondary metabolites through phytochemical testing. Antioxidant activity was measured using the IC50 parameter, which represents the concentration of the extract that can capture 50% of DPPH free radicals. The test results showed that mature bidara leaves had an IC50 value of 34.984 ppm, indicating vigorous antioxidant activity, while young leaves had an IC50 value of 100.327 ppm, classified as moderate activity. Phytochemical testing revealed that both types of leaves contained alkaloids, saponins, and tannins, while flavonoids and triterpenoids were only found in old leaves. These results suggest that variations in secondary metabolite content, influenced by leaf age, contribute to differences in antioxidant activity. Thus, older bidara leaves are more potent as an antioxidant source compared to younger leaves. This study supports the utilization of bidara plants in the pharmaceutical industry and the development of traditional medicine based on natural ingredients. Keywords: Antioxidant; Bidara (Ziziphus mauritiana); DPPH; Phytochemical; Secondary metabolite. INTRODUCTION Indonesia has thousands of plant species spread across various regions. This biodiversity can be utilized as raw materials for modern and traditional medicines. Indonesian society has long been familiar with and used traditional medicine to treat various illnesses. The rising cost of modern medicine in the market is one reason for revisiting the use of conventional medicine. Many medicinal plants in Indonesia have been utilized as raw materials for medicinal purposes, and some of these plant species have undergone clinical testing to assess their phytochemical content, efficacy, and safety of use (Kusumawati, 2018). Bidara leaves (Ziziphus mauritiana) have long been used in traditional medicine due to their various bioactive compounds. The bidara plant is more widely known for its health benefits, including anti-inflammatory, antibacterial, and antioxidant properties. The phytochemicals present in bidara leaves, including flavonoids, alkaloids, saponins, and tannins, play a crucial role in producing their pharmacological effects. Some phytochemical compounds that contribute to pharmacological effects include flavonoids, which are believed to have antibacterial activity by disrupting protein bonds in bacterial cell membranes, and saponins, which can form complexes with bacterial proteins and cell walls, leading to cell wall damage (Ardinimia et al., 2023). Additionally, triterpenoids function as antioxidants due to their ability to scavenge DPPH radicals (Samirana et al., 2017). The natural complex compounds in these plants can be used as free radical scavengers. These complex compounds are used without isolating specific components to obtain a single compound. All components in the plant are used to form complex compounds. According to Jayanti and Rahayu (2023), complex antioxidants have more electrons than single antioxidants, making them more efficient. Antioxidants are compounds that, in specific amounts, can inhibit or counteract the adverse effects of oxidation. Natural antioxidants are often preferred over synthetic ones because some synthetic antioxidants, such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), have recently been suspected of being carcinogenic (Kesuma, 2015). Manuscript received: 16 September, 2025. Revision accepted: 04 December, 2025. Published: 13 December, 2025. https://doi.org/10.14421/biomedich.2025.142.1257-1263 1258 Biology, Medicine, & Natural Product Chemistry 14 (2), 2025: 1257-1263 The leaves and branches of the jujube (Ziziphus jujuba), synonymous with Ziziphus mauritiana Lamk, are known to contain various active compounds. Among others, 14 compounds, including coumarin, rutin, saponin, tannin, and tartaric acid, are found in the leaves. In comparison, three main compounds, namely betulinic acid, ceanothic acid, and leucopelargonidine, are found in the branches. Based on analysis using Duke's Phytochemical and Ethnobotanical Database, these compounds have antioxidant potential because their Pa values are higher than their Pi values, with coumarin and rutin from the leaves and alpha from the branches showing Pa values >0.7, which indicates vigorous antioxidant activity and is estimated to be not much different from laboratory test results. Additionally, the DPPH test also demonstrated antioxidant activity, as indicated by a change in the colour of the solution, with the combination of leaves and branches showing higher activity than the single samples (Nisa et al., 2023). This study tested antioxidant activity using the 2,2- diphenyl-1-picrylhydrazyl (DPPH) method by spectrophotometry. DPPH is a relatively stable free radical compound. This compound is suitable for use as a reagent for testing compounds that have a free radical scavenging effect (antioxidant compounds). The parameter used for the DPPH radical scavenging test is IC50, which is the concentration of the extract or test fraction required to capture 50% of the DPPH radicals (Taufik, 2016). A comparative test was also conducted, namely phytochemical screening, to determine the compound content in the sample. Based on several studies conducted, bidara leaves are one of the plants tested for their effectiveness as an antibacterial agent. This is due to the content of several secondary metabolites in bidara leaves that can function as antibacterial agents. According to Muharrami et al. (2019), the results of phytochemical screening of bidara leaves extracted using the maceration method with 96% ethanol solvent revealed the presence of several secondary compounds, including phenolic compounds, tannins, and saponins. On the other hand, research by Ardinimia et al. (2023) explains that the flavonoids and saponins present in Bidara leaves exhibit significant antibacterial activity. However, this study did not further investigate the antioxidant activity or the potential for development as a raw material for phytopharmaceuticals. The purpose of this study was to compare the antioxidant activity of young and mature bidara eaves using the DPPH method and to identify their secondary metabolites through phytochemical testing. Therefore, this study was conducted to support the updating of data that can be used for further studies related to the antioxidant activity found in old and young bidara leaves. MATERIALS AND METHODS Study area This research was conducted from June to August 2025 at the Biochemistry Laboratory, Terpadu Laboratory, Universitas Islam Malang, East Java, Indonesia. Procedures Sample Preparation Mature and young bidara leaves were obtained from the Al-Qur'an Garden of the Universitas Islam Malang. The initial process involved sorting old and young leaves, washing them, drying them in the sun, and then sorting them by hand. Then, the leaves were dried in an oven at 60°C until they were scorched. After drying, the samples were blended into a powder and sieved using a 40-mesh sieve to increase the surface area, thereby facilitating the extraction process (Wijaya & Noviana, 2022). Subsequently, the dry weight of the sample was measured, and the moisture content was calculated to ensure it was <10%. Extraction The bidara leaves were extracted using the maceration method. A total of 100 grams of crude drug was weighed and placed in a dark glass bottle (reagent bottle), then 700 mL of 96% pro-analytical ethanol was added. The mixture was homogenized repeatedly and left to stand for 3 days and 2 nights. The first maceration filtrate was then filtered. Next, 300 mL of 96% pro-analytical ethanol was added to the residue in the dark glass bottle from the first maceration, homogenized, and filtered again into an Erlenmeyer flask. The filtrates from the first and second macerations were combined and evaporated using a rotary evaporator at a speed of 30 rpm and a temperature of 65°C to produce a concentrated extract or paste (Salamah et al., 2024). Yield Calculation Yield is the ratio between the dry weight obtained and the weight of the raw material used. In extraction, yield is calculated by comparing the final weight of the extract obtained with the initial weight of the cell biomass used, then multiplying by 100% (i) (Mahyantika et al., 2025): Yield (%) = weight of extract (g) weight of raw material (g) x 100% (i) Antioxidant Testing Using the DPPH Method ▪ Determination of the Maximum Wavelength of 0.1 mM DPPH Solution A 0.1 mM DPPH solution was prepared by weighing 2 mg of DPPH powder and dissolving it in pro- analysis ethanol in a 50 mL volumetric flask, bringing it up to the mark. Then, 3 mL of the DPPH solution was pipetted into a cuvette and measured using a UV- Vis spectrophotometer in the wavelength range of 400–600 nm. After the maximum wavelength was Kurniawati et al – Phytochemical and Antioxidant Bidara Leaves 1259 determined, absorbance measurements were performed by mixing 3 mL of DPPH solution with 1 mL of analytical-grade ethanol, and absorbance was measured at the determined wavelength (Theafelicia & Narsito Wulan, 2023). ▪ Preparation of Standard Solution as Reference The standard reference solution was prepared by weighing 1 mg of quercetin and dissolving it in pro- analysis ethanol in a 10 mL volumetric flask, bringing it up to the mark. The solution was homogenized using a vortex and then divided into several concentrations (3.13 ppm, 6.25 ppm, 12.5 ppm, 25 ppm, and 50 ppm). Each concentration is pipetted into a reaction tube and mixed with 3 mL of 0.1 mM DPPH solution. The mixture is vortexed again and incubated in the dark for 30 minutes, then measured using a UV-Visible spectrophotometer at a wavelength of 517 nm (Theafelicia & Narsito Wulan, 2023). ▪ Preparation of Test Solutions from Old and Young Bidara Leaf Extracts Samples of old and young Bidara leaf extracts were prepared by weighing 10 mg of extract and dissolving it in pro-analysis ethanol in a 10 mL measuring flask, then bringing the volume up to the mark. The sample solution was homogenized using a vortex and then divided into several concentrations (20 ppm, 40 ppm, 60 ppm, 80 ppm, and 100 ppm). Each concentration was pipetted into a reaction tube and mixed with 3 mL of 0.1 mM DPPH solution. The mixture was vortexed again and incubated for 30 minutes, then measured using a UV-Vis spectrophotometer at a wavelength of 517 nm (Theafelicia & Wulan, 2023). ▪ DPPH Free Radical Scavenging Activity Antioxidant activity was determined through inhibition values using a UV-Vis spectrophotometer. The absorbance data obtained were used to calculate the percentage of DPPH free radical inhibition. Antioxidant capacity in capturing free radicals was expressed using the following formula (ii) (Khalil et al., 2020): DPPH Free Radical Inhibition (%) = 𝐴0−𝐴1 𝐴0 × 100% (ii) Information: A0 = Control Absorbance A1 = Sample/Standard Absorbance The control absorbance is the absorbance data of DPPH mixed with ethanol for analysis. In contrast, the sample/standard absorbance is the absorbance data of the standard/sample solution mixed with DPPH (Khalil et al., 2020). Antioxidant Testing Using Phytochemical Screening Methods The phytochemical screening test method refers to Ramadhan et al. (2019) conducted qualitatively with color tests using reagents to determine the secondary metabolites contained in plants: ▪ Alkaloid Test a) Mayer’s Test The liquid extract of mature and young bidara leaves was placed in a test tube. Then, 4 drops of Mayer’s reagent were added drop by drop into the test tube, and the tube was shaken gently. The formation of a white precipitate accompanied by a yellowish color change indicated a positive result for alkaloids. b) Wagner’s Test The liquid extract of mature and young bidara leaves was placed in a test tube. Then, 2 drops of Wagner’s reagent were added drop by drop into the test tube, and the tube was shaken gently. The formation of a reddish-brown precipitate indicated a positive result for alkaloids. c) Dragendorff’s Test The liquid extract of mature and young bidara leaves was placed in a test tube. Then, 2 drops of Dragendorff’s reagent were added drop by drop into the test tube, and the tube was shaken gently. The formation of an orange or yellow precipitate indicated a positive result for alkaloids. d) Bouchardat Test The liquid extract of mature and young bidara leaves was placed in a test tube. Then, 2 drops of Bouchardat reagent were added drop by drop into the test tube, and the tube was shaken gently. The formation of a brown precipitate indicated a positive result for alkaloids. ▪ Flavonoid Test a) Shinoda Test The liquid extract was placed in a test tube. Then, 4 drops of absolute ethanol and 2 drops of concentrated hydrochloric acid were added to the test tube, and the mixture was shaken gently. A color change to red indicated the presence of aurone and chalcone. If no colour change occurred, a small spatula of magnesium was added to the test tube containing the extract, and the mixture was gently shaken. A color change from pink to red, such as orange, red, or magenta, indicated the presence of flavones and flavonols. b) 10% Sodium Hydroxide Test The liquid extract of mature and young bidara leaves was placed in a test tube. Then, 2 drops of 10% NaOH solution were added into the test tube, and the mixture was shaken gently. The color changes to reddish yellow, dark orange, reddish purple, or blue indicate the presence of anthocyanins, flavones, flavonols, or chalcones. 1260 Biology, Medicine, & Natural Product Chemistry 14 (2), 2025: 1257-1263 ▪ Saponin Test a) Foam Test The liquid extract of mature and young bidara leaves was placed in a test tube. Then, 10 mL of distilled water was added to the test tube, and the mixture was shaken continuously and gently. The formation of a stable foam layer about 1 cm high indicated a positive result for saponins. ▪ Tanin Test a) Braymer’s Test The liquid extract of mature and young bidara leaves was placed in a test tube. Then, 2 drops of Braymer’s reagent were added drop by drop into the test tube, and the tube was shaken gently. The formation of a greenish-black precipitate indicated a positive result for tannins. b) Base Solution Test The liquid extract of old and young bidara leaves was placed in a test tube. Then, 2 drops of 10% ammonium hydroxide solution were added to the test tube, and the mixture was shaken gently. The formation of a fluorescent yellow color indicated a positive result for tannins. ▪ Steroid/Triterpenoid Test a) Salkowski Test The liquid extract of mature and young bidara leaves was placed in a test tube. Then, two drops of Salkowski reagent were added drop by drop into the test tube, and the mixture was shaken gently. The formation of a brown ring in the center of the solution indicated the presence of steroids. b) Lieberman Bourchard Test The liquid extract of mature and young bidara leaves was placed in a test tube. Then, two drops of Lieberman-Bourchard reagent were added slowly to the test tube, and the mixture was shaken gently. After incubation for 5 minutes, the appearance of a blue- green color indicated the presence of sterols, while a pink to purplish-red color indicated the presence of terpenoids. Data analysis The research data were analyzed both descriptively and quantitatively, as well as qualitatively. Phytochemical screening activity was analyzed using tables and figures. In contrast, antioxidant activity was analyzed using Microsoft Excel with data presented in graphs and expressed as IC50 values obtained from the linear regression equation y = ax + b (Werdyani et al., 2019). The higher the IC50 value, the lower the antioxidant activity of the sample; conversely, the lower the IC50 value, the higher the antioxidant activity of the sample. Meanwhile, to compare the differences between the two treatment group means, a T-test was conducted using PAST 4.03. RESULTS AND DISCUSSION Quercetin Reference Solution Test Table 1. Results of Quercetin Antioxidant Activity Measurements. Concentration (ppm) Absorbance % Inhibition IC50 3,125 0,731 28,655 2,283 (Very Strong) 6,25 0,601 41,343 12,5 0,381 62,815 25 0,159 84,482 50 0,023 97,755 Figure 1. Linear regression curve of the antioxidant activity of quercetin reference solution. Antioxidant Activity Test Using the DPPH Method Table 2. Results of Antioxidant Activity Measurements of mature Bidara Leaf Samples. Concentration (ppm) Absorbance % Inhibition IC50 20 0,365 36,65 34,984 (Very Strong) 40 0,268 53,52 60 0,161 72,02 80 0,092 84,06 100 0,048 91,60 Table 3. Results of Antioxidant Activity Measurements of Young Bidara Leaf Samples Concentration (ppm) Absorbance % Inhibition IC50 20 0,130 55,90 100,327 (Currently) 40 0,076 74,22 60 0,097 67,21 80 0,122 58,50 100 0,148 49,80 28,655 41,343 62,815 84,482 97,755 y = 18,134x + 8,6082 R² = 0,9911 0,000 20,000 40,000 60,000 80,000 100,000 120,000 0,00 1,00 2,00 3,00 4,00 5,00 6,00 A b so rb an ce Concentration (ppm) Kurniawati et al – Phytochemical and Antioxidant Bidara Leaves 1261 T Test Figure 2. Results of statistical analysis comparing young bidara leaves (YBL) and mature bidara leaves (OBL). Phytochemical Screening Test Table 4. Chemical Compounds in Mature and Young Bidara Leaves. Test Reagents mature Bidara Leaves Young Bidara Leaves Alkaloid Mayer’s + + Wagner’s + + Dragendorff’s + + Bouchardat + + Flavonoid Shinoda - - NaOH 10% + + Saponin Busa + + Tanin Braymer’s + + Larutan Basa + + Triterpenoid Salkowski - - Lieberman bouchad - - Discussion The determination of antioxidant activity began with the preparation of extracts from mature and young jujube leaves (Ziziphus mauritiana L.) using pro-analytical ethanol as a solvent in the maceration method. Ethanol was chosen because it can extract both polar and non- polar soluble components, enabling the extraction of all chemical compounds present in jujube leaves (Dianda & Suharti, 2023). The extracts of mature and young jujube leaves obtained were then tested using the (DPPH) method with quercetin as the standard solution. The DPPH method was used because it is simple, fast, sensitive, and widely used to evaluate the ability of chemical compounds in samples to scavenge free radicals (Putri, 2023). Additionally, a phytochemical screening test was conducted to determine the content of compounds present in the old and young bidara leaves. The antioxidant activity test of the reference solution was conducted at concentrations of 3.13 ppm, 6.25 ppm, 12.5 ppm, 25 ppm, and 50 ppm. Quercetin was reacted with the DPPH reagent and then measured for absorbance using a UV-Vis spectrophotometer at a wavelength of 517 nm. The absorbance values and inhibition percentages of the standard solutions are presented in Table 1, demonstrating extreme free radical scavenging activity with an IC₅₀ value of 2.283 ppm, which falls into the powerful category (<50 ppm). This is evidenced by the increase in inhibition percentage with increasing concentration, where at the lowest concentration of 3.125 ppm, it was already able to inhibit by 28.655%, and continued to increase to 97.755% at a concentration of 50 ppm. The inhibition percentage is a parameter reflecting the effectiveness of antioxidants in inhibiting free radicals. The parameter used to measure the antioxidant capacity of a compound is IC50. The IC50 value indicates the concentration of the antioxidant compound required to capture 50% of DPPH free radicals (Pratiwi et al., 2023). The inhibition concentration was calculated using a linear regression equation to determine the relationship between concentration (ppm) (x) and absorbance (y) (Figure 1). From the curve, a linear regression equation, y = 18.134x + 8.6082, with an R² value of 0.9911, was obtained, indicating a robust and linear relationship between concentration and free radical inhibition activity. Antioxidant activity testing of mature and young bidara leaves using the DPPH method yielded IC50 values of 34.984 for mature bidara leaves, indicating extreme antioxidant activity. In contrast, young bidara leaves had an IC50 value of 100.327, categorized as moderate. The smaller the IC50 value obtained, the higher the antioxidant activity of a compound, and conversely, the larger the IC50 value, the weaker the antioxidant activity (Putri, 2023). This is presented in Tables 2 and 3, which show a significant difference in antioxidant activity between mature and young bidara leaves, where mature bidara leaves exhibit a clear and consistent inhibitory pattern that increases with rising concentration. Based on the graph, the average value of the variable measured in young bidara leaves (YBL) was 0.1146, and in old bidara leaves (OBL), it was 0.1866. Although the mean value of DBT is higher than that of DBM, the statistical test results indicate that there is no significant difference between the two (p > 0.05), as indicated by the same letter (“a”) on both treatments. This indicates that the measured activity levels are relatively comparable between young and mature leaves. Variations may influence this difference in mean values in the content of secondary metabolites such as flavonoids, tannins, and saponins, which play a role in biological activity (Wulansari, 2022). The phytochemical testing process in this study was conducted to determine the chemical compounds present in the water extract samples of bidara leaves. The phytochemical tests used included tests for alkaloids, flavonoids, tannins, saponins, and triterpenoids. The results of the phytochemical tests, as shown in Table 4, indicate that both young and mature bidara leaf extracts contain alkaloids, saponins, and tannins. However, flavonoids and triterpenoids were only detected in mature a a 0 0,05 0,1 0,15 0,2 0,25 Young Bidara Leaves Old Bidara Leaves Mean 0,1146 0,1866 M ea su ra b le V ar ia b le s Leaf Category 1262 Biology, Medicine, & Natural Product Chemistry 14 (2), 2025: 1257-1263 leaves. This suggests that leaf maturity influences the content of secondary metabolites. These results are consistent with Wulansari's (2022) study, which reported that ethanol extracts of bidara leaves contain alkaloids, saponins, tannins, and flavonoids, but no triterpenoids were detected. These differences may be due to differences in leaf age, solvent, or extraction method. The results of this study align with those of Hafiz et al. (2025) and Javed et al. (2022), which demonstrate that bidara leaf extract exhibits high antioxidant activity and contains several active compounds, including tannins, saponins, alkaloids, and flavonoids. Hafiz et al. (2025) explored three species of Ziziphus (Ziziphus mauritiana, Ziziphus spina-christi, and Ziziphus jujuba). They found that Ziziphus mauritiana leaves had the highest antioxidant capacity, as determined by DPPH assays, consistent with their high total phenolic content (6.534 g GAE/100 g) and total flavonoid content (2.025 g QE/100 g). This present study also identified the main flavonoid compounds, including rutin, quercetin, kaempferol, and apigenin, via HPLC, and demonstrated a strong correlation between flavonoid content and antioxidant activity. However, a fundamental difference from the study by Hafiz et al. (2025) is that their study compared antioxidant activity between species and plant parts (leaves, fruits, seeds), while this study focused on comparing antioxidant activity between old and young bidara leaves within a single species, Ziziphus mauritiana. Meanwhile, the study conducted by Javed et al. (2022) on the phytochemistry and antioxidant activity of Ziziphus mauritiana, a species within the genus Ziziphus, revealed the presence of compounds such as tannins, saponins, alkaloids, and flavonoids in the methanol extracts of its leaves and fruits. Both studies support the idea that plants of the Ziziphus genus, including Bidara, are rich in secondary metabolites that function as both antioxidants and antimicrobials. Differences in antioxidant activity between different parts of the plant also indicate that the age and part of the plant used significantly influence their chemical composition and bioactive potential. CONCLUSIONS The extract of mature bidara leaves showed more potent antioxidant activity (IC₅₀ = 34.984 ppm, powerful category) compared to young leaves (IC₅₀ = 100.327 ppm, moderate category). This indicates that the maturity level of the leaves affects their antioxidant capacity. Phytochemical screening results also confirm that both young and old leaves contain alkaloids, tannins, and saponins, but flavonoids are only detected in old leaves. These differences in secondary metabolite content contribute to the higher antioxidant activity of old bidara leaves. Therefore, mature bidara leaves have greater potential to be developed as a natural source of antioxidants compared to young leaves. Acknowledgements: The author would like to express gratitude to the UNISMA Institutional Grant (HI-ma) in accordance with the Research Grant Implementation Agreement Number: 057/G164/U.LPPM/K/B.07/III/2025 dated March 3, 2025, for the financial support provided from the initial stage to the completion of this final project. Competing Interests: The authors declare that there are no competing interests. REFERENCES Ardinimia, S. D., Putri, A. F., Ramanda, Y. M., Putri, N., Dzakiyyah, H., Gianni, P., Surabaya, U. N., & Ketintang, J. (2023). 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