Identification of Chemical Compounds in Black Garlic Extract and Effect on Inhibiting Xanthine Oxidase Enzyme
DOI:
10.29303/jpm.v20i5.9292Published:
2025-07-16Issue:
Vol. 20 No. 5 (2025): July 2025Keywords:
Black Garlic, In Silico; Inhibitor; Kinetics; Xanthine Oxidase EnzymeArticles
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Abstract
Xanthine oxidase is an enzyme involved in the catalysis of the oxidation reaction that converts hypoxanthine into xanthine and subsequently into uric acid. Elevated uric acid levels can pose various health risks. Gout treatment can be achieved by inhibiting the activity of the xanthine oxidase enzyme using black garlic. This study aims to identify the chemical compounds in black garlic methanol extract, assess the inhibitory effect of this extract on the xanthine oxidase enzyme using in silico methods, and determine the type of inhibition based on enzyme kinetics. The in silico analysis was conducted to evaluate the binding affinity of flavonoid compounds in black garlic extract with xanthine oxidase. The in vitro analysis tested the inhibition of the xanthine oxidase enzyme by black garlic using the UV-Vis spectrophotometry method at a wavelength of 291.7 nm, based on a decrease in uric acid concentration as an indicator of reduced enzyme activity. The type of inhibition mechanism was determined through enzyme kinetics using the Michaelis-Menten equation, which was transformed into the Lineweaver-Burk equation in a double reciprocal form. Black garlic methanol extract contains 133 chemical compounds, including 22 flavonoid compounds that are thought to inhibit xanthine oxidase. According to in silico studies, quercetin-3-O-malonylglucoside exhibits the lowest binding affinity (-9.2 kcal/mol) with the xanthine oxidase enzyme compared to the xanthine substrate (-5.2 kcal/mol) and allopurinol (-5.3 kcal/mol). Inhibition of the xanthine oxidase enzyme by black garlic demonstrated the highest inhibition of 76.352% at a concentration of 10 ppm of black garlic extract. The inhibition type of the xanthine oxidase enzyme by black garlic methanol extract showed a competitive inhibition mechanism, evidenced by an increase in the KM value from 0.014 to 0.134 without a significant change in the Vmax value. Thus, it can be concluded that black garlic extract has the potential to be a natural inhibitor of the enzyme xanthine oxidase that can be used to treat gout or hyperuricemia.
References
N. Liu et al., “The Role of Oxidative Stress in Hyperuricemia and Xanthine Oxidoreductase (XOR) Inhibitors,” Oxidative Medicine and Cellular Longevity, vol. 2021, no. 1, p. 1470380, Jan. 2021, doi: 10.1155/2021/1470380.
G. B. Pangala, A. N. Mahendra, I. M. Jawi, and N. W. S. Dewi, “Ekstrak Etanol Bawang Hitam (Allium sativum L.) Menurunkan Kadar Asam Urat Mencit Jantan Model Hiperurisemia,” Jurnal Medika Udayana, vol. 11, no. 8, pp. 89–93, 2022, doi: doi:10.24843.MU.2022.V11.i8.P17.
Q. Wang, X. Wen, and J. Kong, “Recent Progress on Uric Acid Detection: A Review,” Critical Reviews in Analytical Chemistry, vol. 50, no. 4, pp. 359–375, Jul. 2020, doi: 10.1080/10408347.2019.1637711.
D. Raharjo, T. A. Lisyani, and N. I. Wulandari, “Efektivitas Penghambatan Enzim Xanthin Oksidase Fraksi Etanol, Etil Asetat dan N-Heksane dari Ekstrak Etanol Daun Bakau Kurap (Rhizophora Stylosa Griff.),” PHARMASIPHA, vol. 8, no. 2, pp. 83–90, 2024, doi: 10.21111/pharmasipha.v8i2.
L. Strilchuk, F. Fogacci, and A. F. Cicero, “Safety and tolerability of available urate-lowering drugs: a critical review,” Expert Opinion on Drug Safety, vol. 18, no. 4, pp. 261–271, Apr. 2019, doi: 10.1080/14740338.2019.1594771.
K. Najman, A. Sadowska, and E. Hallmann, “Influence of Thermal Processing on the Bioactive, Antioxidant, and Physicochemical Properties of Conventional and Organic Agriculture Black Garlic (Allium sativum L.),” Applied Sciences, vol. 10, no. 23, p. 8638, Dec. 2020, doi: 10.3390/app10238638.
Z. Qiu, Z. Zheng, B. Zhang, D. Sun‐Waterhouse, and X. Qiao, “Formation, Nutritional Value, and Enhancement of Characteristic Components in Black Garlic: A Review for Maximizing the Goodness to Humans,” Comp Rev Food Sci Food Safe, vol. 19, no. 2, pp. 801–834, Mar. 2020, doi: 10.1111/1541-4337.12529.
B. I. Sari et al., “Isolation, Fractionation, and Characterization of Xanthine Oxidase from Goat’s Milk,” in AIP Conference Proceedings 2638, Mataram, Indonesia, 2022, pp. 070009-1-070009–5. doi: 10.1063/5.0104306.
H. Xue, M. Xu, D. Gong, and G. Zhang, “Mechanism of Flavonoids Inhibiting Xanthine Oxidase and Alleviating Hyperuricemia from Structure–Activity Relationship and Animal Experiments: A Review,” Food Frontiers, vol. 4, no. 4, pp. 1643–1665, Dec. 2023, doi: 10.1002/fft2.287.
G. Litwack, “Enzymes,” in Human Biochemistry, Elsevier, 2018, pp. 95–129. doi: 10.1016/B978-0-12-383864-3.00005-3.
Y.-C. Chen, T.-H. Kao, C.-Y. Tseng, W.-T. Chang, and C.-L. Hsu, “Methanolic Extract of Black Garlic Ameliorates Diet-induced Obesity via Regulating Adipogenesis, Adipokine Biosynthesis, and Lipolysis,” Journal of Functional Foods, vol. 9, pp. 98–108, Jul. 2014, doi: 10.1016/j.jff.2014.02.019.
A. A. Setiawan, S. Kumala, D. R. L., N. D. Yuliana, Rochimah, and S. N. Rangkuti, “Uji Efektivitas Ekstrak Metanol 80% Black Garlic Tunggal Sebagai Antihiperurisemia Terhadap Tikus,” MS, vol. 8, no. 2, pp. 625–632, May 2023, doi: 10.37874/ms.v8i2.755.
R. D. Martha, D. Danar, Y. D. Safitri, and H. Parbuntari, “Identifikasi Senyawa Flavonoid Daun Jinten (Plectranthus amboinicus) Menggunakan Liquid Chromatography-Mass Spectrometry (LC-MS) dan Potensinya sebagai Antikanker Secara In-Vitro,” Sainteks, vol. 21, no. 1, p. 67, Apr. 2024, doi: 10.30595/sainteks.v21i1.21171.
T. Oktaviani, O. Hermansyah, I. Ikhsan, S. Rahmawati, and N. Wirahmi, “Docking Molecular Senyawa pada Tanaman Famili Apiaceae sebagai Penghambat Enzim Xanthine Oxidase,” Higea, vol. 16, no. 1, p. 34, Mar. 2024, doi: 10.52689/higea.v16i1.593.
Tukiran, A. R. Setiawan, I. C. Constaty, and F. N. Safitri, “The Potency of Java Apple (Syzygium samarangense) AS α-Glucosidase and α-Amylase Inhibitor: An In-Silico Approach,” TJNPR, vol. 7, no. 8, pp. 3741–3746, Aug. 2023, doi: 10.26538/tjnpr/v7i8.26.
T. Dhammaraj et al., “In Vitro Investigation of Xanthine Oxidase Inhibitory and Antioxidant Activities of 3,4,5-trihydroxycinnamic acid.,” vol. 13, no. 3, 2024.
H. Malikhah and N. Herdyastuti, “Effect of Kaffir Lime (Cytrus hystrix) Leaf Extract on Xanthine Oxidase Inhibition,” World J. Adv. Res. Rev., vol. 17, no. 1, pp. 1069–1078, Jan. 2023, doi: 10.30574/wjarr.2023.17.1.0100.
K. S. Artini, D. Raharjo, and E. Wijayanti, “Efek Penghambatan Enzim Xantin Oxidase oleh Ekstrak Etanol Biji Pepaya (Carica papaya Linn.) secara In Vitro.,” in Prosiding Seminar Informasi Kesehatan Nasional, in SiKensNas 2021. Surakarta: Universitas Duta Bangsa Surakarta, 2021, pp. 163–167. doi: https://doi.org/10.47701/sikenas.v0i0.1249.
A. R. Y. Eff, S. T. Rahayu, and R. D. Syachfitri, “Uji Aktivitas Penghambatan Xantin Oksidase secara In-Vitro oleh Isolat 6,4’-Dihidroksi-4-Metoksibenzofenon-2-O-B-D Glukopiranosida(C20H22O10) yang Diisolasi dari Mahkota Dewa (Phaleria macrocarpa (Scheff.) Boerl),” Pharmaceutical Sciences and Research (PSR), vol. 3, no. 1, pp. 1–11, 2016.
R. Mardiansyah and E. Sulistyowati, “Inhibisi Ion Logam Cu2+ terhadap Kinetika Enzim Tripsin,” Jurnal Kimia Dasar, vol. 7, no. 2, pp. 50–58, 2018.
R. A. Alfauzi, L. Hartati, D. Suhendra, T. P. Rahayu, and Nur Hidayah, “Ekstraksi Senyawa Bioaktif Kulit Jengkol (Archidendron jiringa) dengan Konsentrasi Pelarut Metanol Berbeda sebagai Pakan Tambahan Ternak Ruminansia: Extraction of Jengkol (Archidendron jiringa) Peel Bioactive Compounds with Different Concentrations of Methanol Solvents as Supplementary Feed for Ruminants,” JINTP, vol. 20, no. 3, pp. 95–103, Dec. 2022, doi: 10.29244/jintp.20.3.95-103.
C. Zhang, R. Wang, G. Zhang, and D. Gong, “Mechanistic Insights into the Inhibition of Quercetin on Xanthine Oxidase,” International Journal of Biological Macromolecules, vol. 112, pp. 405–412, Jun. 2018, doi: 10.1016/j.ijbiomac.2018.01.190.
J. M. Pauff and R. Hille, “Inhibition Studies of Bovine Xanthine Oxidase by Luteolin, Silibinin, Quercetin, and Curcumin,” J. Nat. Prod., vol. 72, no. 4, pp. 725–731, Apr. 2009, doi: 10.1021/np8007123.
Y. Wang, G. Zhang, J. Pan, and D. Gong, “Novel Insights into the Inhibitory Mechanism of Kaempferol on Xanthine Oxidase,” J. Agric. Food Chem., vol. 63, no. 2, pp. 526–534, Jan. 2015, doi: 10.1021/jf505584m.
J. Yan, G. Zhang, Y. Hu, and Y. Ma, “Effect of Luteolin on Xanthine Oxidase: Inhibition Kinetics and Interaction Mechanism Merging with Docking Simulation,” Food Chemistry, vol. 141, no. 4, pp. 3766–3773, Dec. 2013, doi: 10.1016/j.foodchem.2013.06.092.
S. Lin, G. Zhang, Y. Liao, J. Pan, and D. Gong, “Dietary Flavonoids as Xanthine Oxidase Inhibitors: Structure–Affinity and Structure–Activity Relationships,” J. Agric. Food Chem., vol. 63, no. 35, pp. 7784–7794, Sep. 2015, doi: 10.1021/acs.jafc.5b03386.
D. W. Dari, Andika, and Miajunnisa, “Uji Potensi Senyawa Metabolit Sekunder Tanaman Putri Malu (Mimosa pudica L.) Sebagai Inhibitor Xanthine Oxidase Secara In Silico,” Jurnal Kesehatan Kefarmasian, vol. 3, no. 2, pp. 171–183, 2022.
A. Abdul, A. A. Winih Kinasih, and F. Qonitah, “Analisis In Silico Interaksi Senyawa Kurkuminoid terhadap Enzim Main Protease 6lu7 Dari SARS-COV-2,” DJP, vol. 3, no. 1, pp. 1–7, Jun. 2023, doi: 10.47701/djp.v3i1.2904.
M. Safithri, S. Miantika, and L. Ambarsari, “Analisis In Silico Senyawa Aktif Sirih Merah (Piper crocatum) sebagai Inhibitor Xantin Oksidase,” Curr. Biochem., vol. 9, no. 2, pp. 51–62, Dec. 2022, doi: 10.29244/cb.9.2.1.
A. R. Lestari, I. Batubara, S. T. Wahyudi, and A. Ilmiawati, “Phenolic Compound in Garlic (Allium sativum) and Black Garlic Potency as Antigout Using Molecular Docking Approach,” J. Kim. Sains Apl., vol. 25, no. 7, pp. 253–263, Aug. 2022, doi: 10.14710/jksa.25.7.253-263.
A. A. Setiawan, S. Kumala, D. R. Laksmitawati, N. D. Yuliana, and A. Yuniarto, “Methanol Extract of Black Garlic Protects Against Hyperuricemia by Xanthine Oxidase Inhibitory Activity In-Vitro,” JMPAS, vol. 13, no. 4, pp. 6663–6668, Aug. 2024, doi: 10.55522/jmpas.v13i4.6622.
H. Ouyang, K. Hou, W. Peng, Z. Liu, and H. Deng, “Antioxidant and Xanthine Oxidase Inhibitory Activities of Total Polyphenols from Onion,” Saudi Journal of Biological Sciences, vol. 25, no. 7, pp. 1509–1513, Nov. 2018, doi: 10.1016/j.sjbs.2017.08.005.
Y. Farida and R. A. Firmansyah, “Aktivitas Penghambatan Xanthine Oxidase Ekstrak Etanol dan Air Dari Herba Suruhan (Peperomia pellucida L.),” Proc. Mul. Pharm. Conf., vol. 3, pp. 482–487, Apr. 2016, doi: 10.25026/mpc.v3i2.149.
D. Istia’nah, U. Utami, and A. Barizi, “Karakterisasi Enzim Amilase dari Bakteri Bacillus megaterium pada Variasi Suhu, pH dan Konsentrasi Substrat,” JRBA, vol. 2, no. 1, pp. 11–17, Mar. 2020, doi: 10.26740/jrba.v2n1.p11-17.
S. Nagar, U. A. Argikar, and D. Tweedie, Eds., Enzyme Kinetics in Drug Metabolism: Fundamentals and Applications, vol. 2342. in Methods in Molecular Biology, vol. 2342. New York, NY: Springer US, 2021. doi: 10.1007/978-1-0716-1554-6.
Author Biographies
Mahrunisa Nur Afifah, Department of Chemistry, Faculty of Mathematics and Natural Science, State University of Surabaya
Nuniek Herdyastuti, Department of Chemistry, Faculty of Mathematics and Natural Science, State University of Surabaya
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