2.4. Antioxidant Activities2.4.1. D

2.4. Antioxidant Activities
2.4.1. DPPH Radical Scavenging Assay

The DPPH radical scavenging activities of methanol extract, EAF, BF, and WF were tested according to Yamaguchi et al. [27]. Briefly, 0.2 mL of the sample solutions of different concentrations was added to 1 mL of 0.1 mM of freshly prepared DPPH solution. The reaction mixtures were shaken vigorously and absorbance at 517 nm was determined after 20 min at room temperature. Control sample was prepared containing the same volume without test compounds or reference antioxidants, while DMSO was used as blank. The reference antioxidant BHT was used as the positive control in all the assays. The radical scavenging activity was measured as a decrease in the absorbance of DPPH• and calculated as follows: where is the absorbance of the control and is the absorbance of the extract or fractions or standard.
2.4.2. Superoxide Radical-Scavenging Assay

The superoxide radical-scavenging effect was determined by the method of Nishikimi et al. [28]. The reaction mixture with NBT (1 mM) in phosphate buffer (0.1 M, pH 7.4), NADH (1 mM) with or without samples, and PMS (0.1 mM) was incubated at room temperature for 5 min and the absorbance was recorded at 560 nm. The inhibition percentage was calculated against a control without the samples. The scavenging ability was calculated using the equation as described for DPPH assay.
2.4.3. Hydroxyl Radical Scavenging Assay

The capacity of the extract and compound to reduce hydroxyl radical-mediated peroxidation was carried out by the method of Hinneburg et al. [29]. Briefly, 0.5 mL of 5.6 mM 2-deoxy-D-ribose in KH2PO4−NaOH buffer (50 mM, pH 7.4), 0.2 mL of 100 μM FeCl3, and 104 mM EDTA (1 : 1  v/v) solution were added to 0.1 mL of different concentrations of test samples, followed by 100 μL of 1.0 mM H2O2 and 0.1 mL of 1.0 mM aqueous BHT. The reaction mixtures were shaken vigorously and incubated at 50°C for 30 min. Subsequently, 1 mL of 2.8% TCA and 1 mL of 1.0% TBA were added to each tube containing reaction mixture and samples were mixed well again and boiled in a water bath at 50°C for 30 min. The absorbance of solution was read at 532 nm. The hydroxyl radical scavenging ability was calculated using the formula as described for DPPH assay and the values are presented as means of triplicate analyses.
2.4.4. FRAP (Ferric Reducing Antioxidant Power) Assay

The FRAP assay was determined by the method of Benzie and Strain [30] with minor modifications. It depends on the ability of the sample to reduce the ferric tripyridyltriazine (Fe (III)-TPTZ) complex to ferrous tripyridyltriazine (Fe (II)-TPTZ) at low pH. Fe (II)-TPTZ has an intensive blue color which can be read at 593 nm. The stock solutions consist of 300 mM acetate buffer (pH 3.6), 10 mM 2, 4, 6 tripyridyl S triazine (TPTZ) in 40 mM of HCl, and 20 mM ferric chloride solution. The fresh working solution was prepared by mixing 25 mL of acetate buffer, 2.5 mL of TPTZ, and 2.5 mL of FeCl3·6H2O and the temperature was maintained to 37°C before use. The various concentrations of extract, compound, and BHT (10–50 μg/mL) were allowed to react with 2 mL of the FRAP solution for 30 min in the dark condition. The absorbance was recorded at 593 nm. The results are expressed in μM Fe (II)/g and were estimated using aqueous FeSO4·7H2O (20–100 μM) as standard for calibration.
2.4.5. Nitric Oxide Radical Scavenging Assay

At physiological pH, nitric oxide generated from sodium nitroprusside in aqueous solution interacts with oxygen to produce nitrite ions, which were measured by the Griess reaction [31]. Briefly, 3 mL of the reaction mixture containing 10 mM sodium nitroprusside and the test samples (10–50 μg/mL) in phosphate buffer were incubated for 150 min at 25°C. After incubation, 0.5 mL of the reaction mixture was mixed with 1 mL of sulfanilic acid reagent (0.33% in 20% glacial acetic acid) and allowed to stand for 5 min for complete diazotization. Then, 1 mL of naphthyl ethylene diamine dihydrochloride (0.1% w/v) was added and the mixture was allowed to stand for 30 min at 25°C. A pink colored chromophore is generated and the absorbance was measured spectrophotometrically at 540 nm against a blank sample. The nitric oxide radical scavenging activity of the extract and compound is reported as % inhibition and was calculated using the formula as described for DPPH assay.
2.4.6. Lipid Peroxidation Assay

Lipid peroxidation (LPO) assay was performed according to the protocol described by Damien et al. [32] to measure the lipid peroxide formed, using egg yolk homogenates as lipid-rich media. Briefly, 0.5 mL of egg homogenate (10% v/v prepared in 1.15% w/v KCl) was added to 0.1 mL of each test samples (10–50 μg/mL) taken in a test tube and made up to 1 mL with double distilled water. Thereafter, 0.05 mL of FeSO4 (0.07 M) was added to induce lipid peroxidation, and the mixture was incubated for 30 min at room temperature. Then, 1.5 mL of 3.5 M acetic acid (pH adjusted to 3.5) w

Results (Indonesian) 1:

2.4. aktivitas antioksidan2.4.1. DPPH radikal pemulungan AssayEkstrak DPPH radikal scavenging kegiatan metanol, EAF, BF dan WF diuji menurut Yamaguchi et al. [27]. Secara singkat, 0.2 mL larutan sampel konsentrasi yang berbeda telah ditambahkan ke 1 mL 0,1 mM larutan DPPH baru disiapkan. Reaksi campuran yang terguncang dengan penuh semangat dan daya serap di 517 nm ditentukan setelah 20 menit pada suhu kamar. Contoh kontrol disiapkan yang mengandung volume yang sama tanpa pengujian senyawa atau referensi antioksidan, sementara DMSO digunakan sebagai kosong. Referensi antioksidan BHT digunakan sebagai kontrol positif di semua tes. Kegiatan scavenging radikal diukur sebagai penurunan absorbansi DPPH• dan dihitung sebagai berikut: mana absorbansi kontrol dan daya serap ekstrak atau pecahan atau standar.2.4.2. superoksida radikal pemulungan AssayEfek radikal pemulungan superoksida ditentukan dengan metode Nishikimi et al. [28]. Campuran reaksi dengan Netbios (1 mM) di buffer fosfat (0,1 M, pH 7,4), NADH (1 mM) dengan atau tanpa sampel, dan PMS (0.1 mM) diinkubasi pada suhu kamar selama 5 menit dan absorbansi tercatat pada 560 nm. Persentase inhibisi dihitung terhadap kontrol tanpa sampel. Kemampuan scavenging dihitung menggunakan persamaan seperti yang dijelaskan untuk DPPH assay.2.4.3. hidroksil radikal pemulungan AssayThe capacity of the extract and compound to reduce hydroxyl radical-mediated peroxidation was carried out by the method of Hinneburg et al. [29]. Briefly, 0.5 mL of 5.6 mM 2-deoxy-D-ribose in KH2PO4−NaOH buffer (50 mM, pH 7.4), 0.2 mL of 100 μM FeCl3, and 104 mM EDTA (1 : 1 v/v) solution were added to 0.1 mL of different concentrations of test samples, followed by 100 μL of 1.0 mM H2O2 and 0.1 mL of 1.0 mM aqueous BHT. The reaction mixtures were shaken vigorously and incubated at 50°C for 30 min. Subsequently, 1 mL of 2.8% TCA and 1 mL of 1.0% TBA were added to each tube containing reaction mixture and samples were mixed well again and boiled in a water bath at 50°C for 30 min. The absorbance of solution was read at 532 nm. The hydroxyl radical scavenging ability was calculated using the formula as described for DPPH assay and the values are presented as means of triplicate analyses.2.4.4. FRAP (Ferric Reducing Antioxidant Power) AssayFRAP assay ditentukan dengan metode Benzie dan ketegangan [30] dengan modifikasi kecil. Hal ini tergantung pada kemampuan sampel untuk mengurangi tripyridyltriazine besi (Fe (III)-TPTZ) kompleks tripyridyltriazine besi (Fe (II)-TPTZ) pada pH rendah. FE (II)-TPTZ memiliki warna biru yang intensif yang dapat dibaca di 593 nm. Solusi saham terdiri dari 300 mM asetat penyangga (pH 3.6), 10 mM 2, 4, 6 tripyridyl S triazine (TPTZ) di 40 mM HCl, dan 20 mM atau feri klorida solusi. Solusinya bekerja segar disiapkan oleh pencampuran 25 mL asetat buffer, 2,5 mL TPTZ, dan 2,5 mL FeCl3·6H2O dan suhu dipertahankan sampai 37° C sebelum digunakan. Konsentrasi berbagai ekstrak, senyawa dan BHT (10-50 μg/mL) diizinkan untuk bereaksi dengan 2 mL larutan FRAP selama 30 menit dalam kondisi gelap. Absorbansi tercatat pada 593 nm. Hasil dinyatakan dalam μM Fe (II) /g dan diperkirakan menggunakan larutan FeSO4·7H2O (20-100 μM) sebagai standar untuk kalibrasi.2.4.5. oksida nitrat radikal pemulungan AssayPada fisiologis pH, oksida nitrat dihasilkan dari nitroprusside natrium dalam larutan berinteraksi dengan oksigen untuk menghasilkan nitrit ion, yang diukur oleh reaksi Griess [31]. Secara singkat, 3 mL campuran reaksi yang berisi 10 mM natrium nitroprusside dan sampel uji (10-50 μg/mL) dalam buffer fosfat yang diinkubasi untuk 150menit pada 25° C. Setelah inkubasi, 0.5 mL campuran reaksi dicampur dengan 1 mL pereaksi asam sulfanilic (0,33% dalam 20% glacial acetic acid) dan memungkinkan untuk berdiri selama 5 menit untuk diazotization lengkap. Kemudian, 1 mL naphthyl etilen diamina dihydrochloride (0,1% w/v) ditambahkan dan campuran diperbolehkan untuk berdiri selama 30 menit di 25° C. Kromofor berwarna pink yang dihasilkan dan absorbansi diukur spectrophotometrically di 540 nm terhadap sampel kosong. Oksida nitrat radikal scavenging aktivitas ekstrak dan senyawa dilaporkan sebagai % penghambatan dan dihitung dengan menggunakan formula seperti yang dijelaskan untuk DPPH assay.2.4.6. lipid Peroxidation AssayLipid peroxidation (LPO) assay dilakukan menurut protokol yang dijelaskan oleh Damien et al. [32] untuk mengukur peroksida lipid yang terbentuk, menggunakan telur kuning telur homogenates sebagai lipid-media yang kaya. Secara singkat, 0.5 mL telur homogenate (10% v/v disiapkan di 1,15% w/v KCl) telah ditambahkan ke 0, 1 mL setiap pengujian sampel (10-50 μg/mL) diambil dalam tabung dan membuat hingga 1 mL dengan air suling ganda. Setelah itu, 0.05 mL FeSO4 (0.07 M) telah ditambahkan untuk menginduksi peroxidation lemak, dan campuran diinkubasi selama 30 menit pada suhu kamar. Kemudian, 1.5 mL asam asetat 3.5 M (pH disesuaikan kepada 3,5) w