Preliminary Characterisation of Rhizophora apiculata Fruit Carotenoids by TLC and Zeaxanthin–iNOS Docking

Baba K. Montolalu; Darus Saadah J.  Paransa; Desy M.H.  Mantiri; Jane M.  Mamuaja; Robert A.  Bara; Nickson J.  Kawung

doi:10.35800/0ekf3q25

Authors

Baba K. Montolalu Universitas Sam Ratulangi
Darus Saadah J. Paransa Universitas Sam Ratulangi
Desy M.H. Mantiri Universitas Sam Ratulangi
Jane M. Mamuaja Universitas Sam Ratulangi
Robert A. Bara Universitas Sam Ratulangi
Nickson J. Kawung Universitas Sam Ratulangi

DOI:

https://doi.org/10.35800/0ekf3q25

Keywords:

Rhizophora apiculata; carotenoids; thin-layer chromatography; iNOS; molecular docking

Abstract

Rhizophora apiculata fruit potentially contains carotenoid pigments that may contribute to the biological properties of mangrove plants. This study aimed to separate and preliminarily identify carotenoid pigments from R. apiculata fruit and to predict the molecular interaction of zeaxanthin with inducible nitric oxide synthase (iNOS). Pigments were extracted with ethanol, partitioned into an n-hexane phase, and separated by thin-layer chromatography on silica gel using acetone-petroleum ether (70:30) as the mobile phase. Pigments were characterized based on zone color, retardation factor (Rf), and UV-Vis absorption spectra. Molecular docking was performed against the human iNOS oxygenase domain (PDB ID: 3E7G) using Molegro Virtual Docker 6.0, with AT2 as the co-crystallized ligand for redocking validation. TLC separation produced seven zones with Rf values ranging from 0.62 to 1.00. TLC-derived fraction F1 was orange and exhibited absorption maxima at 425, 453, and 480 nm, supporting its preliminary identification as β-carotene. Fraction F3 was yellow, had an Rf value of 0.83, and showed a maximum absorption at 475 nm, supporting its preliminary identification as zeaxanthin. Redocking validation produced a mean RMSD of 0.9223 ± 0.0372 Å. Zeaxanthin yielded mean MolDock and Rerank Scores of −153.419 ± 19.994 and −120.843 ± 20.801, respectively, which were more negative than those obtained for AT2. Zeaxanthin interacted with several residues also recognized by AT2, including Trp372, Gly371, Trp194, Cys200, Met374, and Met434, with predominantly hydrophobic interactions. These findings provide preliminary evidence for the presence of β-carotene and zeaxanthin in R. apiculata fruit and support the predicted interaction of zeaxanthin within the iNOS binding pocket.

Keywords: Rhizophora apiculata; carotenoids; thin-layer chromatography; iNOS; molecular docking

ABSTRAK

Buah Rhizophora apiculata berpotensi mengandung pigmen karotenoid yang berkontribusi terhadap aktivitas biologis tumbuhan mangrove. Penelitian ini bertujuan memisahkan dan melakukan identifikasi awal pigmen karotenoid pada buah R. apiculata serta memprediksi interaksi molekuler zeaxanthin terhadap inducible nitric oxide synthase (iNOS). Pigmen diekstraksi menggunakan etanol dan dipartisi ke dalam fase n-heksana, kemudian dipisahkan menggunakan kromatografi lapis tipis pada pelat silika gel dengan fase gerak petroleum eter dan aseton (70:30). Karakterisasi pigmen dilakukan berdasarkan warna zona, nilai retardation factor (Rf), dan spektrum serapan UV-Vis. Penambatan molekuler dilakukan terhadap domain oksigenase iNOS manusia (PDB ID: 3E7G) menggunakan Molegro Virtual Docker 6.0, dengan AT2 sebagai ligan ko-kristal untuk validasi redocking. Pemisahan KLT menghasilkan tujuh zona dengan nilai Rf 0,62-1,00. Fraksi hasil KLT F1 berwarna oranye dengan serapan maksimum pada 425, 453, dan 480 nm sehingga diidentifikasi secara awal sebagai β-karoten, sedangkan F3 berwarna kuning dengan 0,83 dan serapan maksimum pada 475 nm sehingga diidentifikasi secara awal sebagai zeaxanthin. Validasi redocking menghasilkan RMSD rata-rata 0,9223 ± 0,0372 Å. Zeaxanthin memberikan MolDock Score dan Rerank Score rata-rata masing-masing sebesar −153,419 ± 19,994 dan −120,843 ± 20,801, yang lebih negatif dibandingkan AT2. Zeaxanthin berinteraksi dengan beberapa residu yang juga dikenali AT2, yaitu Trp372, Gly371, Trp194, Cys200, Met374, dan Met434, dengan pola interaksi yang didominasi kontak hidrofobik. Hasil penelitian menunjukkan keberadaan awal β-karoten dan zeaxanthin pada buah R. apiculata serta mendukung kemungkinan interaksi zeaxanthin pada kantong pengikatan iNOS.

Kata kunci: Rhizophora apiculata; karotenoid; kromatografi lapis tipis; iNOS; penambatan molekuler

References

Ashenafi, E. L., Nyman, M. C., Shelley, J. T., Mattson, N. S. 2023. Spectral Properties and Stability of Selected Carotenoid and Chlorophyll Compounds in Different Solvent Systems. Food Chemistry Advances, 2, 100178. https://doi.org/10.1016/j.focha.2022.100178

Bawias, M., Kemer, K., Mantiri, D. M., Kumampung, D. R., Paransa, D. S. J., Mantiri, R. 2018. Isolasi Pigmen Karotenoid pada Mikroalga Nannochloropsis sp. dengan Menggunakan Beda Pelarut. Jurnal Pesisir dan Laut Tropis, 6(2), 1–8. https://doi.org/10.35800/jplt.6.2.2018.20641

Bitencourt-Ferreira, G., de Azevedo, W. F. 2019. Molegro Virtual Docker for Docking. Methods in Molecular Biology, 2053, 149–167. https://doi.org/10.1007/978-1-4939-9752-7_10

Britton, G., Liaaen-Jensen, S., Pfander, H. (Eds.). 1995. Carotenoids: Volume 1B—Spectroscopy. Birkhäuser. p 360.

Das, S., Gupta, P., and De, B. 2017. Thin Layer Chromatographic Characterization of Carotenoid Isolates in Sugar Date Palm (Phoenix sylvestris) Fruit Epicarp and Inflorescence Axis. International Journal of Pharmacognosy and Phytochemical Research, 9(5), 680–684. https://doi.org/10.25258/phyto.v9i5.8148

Dong, J., Li, D., Kang, L., Luo, C., Wang, J. 2023. Insights into Human eNOS, nNOS and iNOS Structures and Medicinal Indications from Statistical Analyses of Their Interactions with Bound Compounds. Biophysics Reports, 9(3), 159–175. https://doi.org/10.52601/bpr.2023.210045

Garcin, E. D., Arvai, A. S., Rosenfeld, R. J., Kroeger, M. D., Crane, B. R., Andersson, G., Getzoff, E. D. 2008. Anchored Plasticity Opens Doors for Selective Inhibitor Design in Nitric Oxide Synthase. Nature Chemical Biology, 4(11), 700–707. https://doi.org/10.1038/nchembio.115

La Russa, D., Manni, G., Di Santo, C., Pieroni, B., Pellegrino, D., Barba, F. J., Bagetta, G., Fallarino, F., Montesano, D., Amantea, D. 2024. Zeaxanthin Exerts Anti-Inflammatory Effects In Vitro and Provides Significant Neuroprotection in Mice Subjected To Transient Middle Cerebral Artery Occlusion. PharmaNutrition, 27, 100368. https://doi.org/10.1016/j.phanu.2023.100368

Lin, W., Feng, Y., Li, D., and Lin, H. 2023. Bio-active Products from Mangrove Ecosystems. Marine Drugs, 21(4), 239. https://doi.org/10.3390/md21040239

Maoka, T. 2023. Carotenoids: Distribution, Function in Nature, and Analysis Using Lc-Photodiode Array Detector (DAD)-MS and MS/MS system. Mass Spectrometry, 12(1), A0133-A0133. https://doi.org/10.5702/massspectrometry.A0133

Morone, J., Hentschke, G., Pinto, E., Morais, J., Cruz, P., Vasconcelos, V., Martins, R., Lopes, G. 2024. Carotenoids from Cyanobacteria Modulate iNOS and Inhibit The Production of Inflammatory Mediators: Promising Agents for The Treatment of Inflammatory Conditions. Algal Research, 83, 103729. https://doi.org/10.1016/j.algal.2024.103729

Mokoginta, F. S., Paransa, D. S., Kemer, K., Paulus, J. J., Kawung, J., Manoppo, H. 2021. Analisis Jenis Pigmen Karotenoid pada Karapas Kepiting Jantan Grapsus albolineatus Latreille in Milbert 1812. Jurnal Ilmu Platax, 9(1), 132-137. https://doi.org/10.35800/jip.9.1.2021.34421.

Parthiban, A., Sivasankar, R., Sachithanandam, V., Khan, S. A., Jayshree, A., Murugan, K., Sridhar, R. 2022. An Integrative Review on Bioactive Compounds from Indian Mangroves for Future Drug Discovery. South African Journal of Botany, 149, 899–915. https://doi.org/10.1016/j.sajb.2021.10.004

Payne, T. D., Dixon, L. R., Schmidt, F. C., Blakeslee, J. J., Bennett, A. E., Schultz, Z. D. 2024. Identification and Quantification of Pigments in Plant Leaves Using Thin Layer Chromatography–Raman spectroscopy (TLC–Raman). Analytical Methods, 16(16), 2449–2455. https://doi.org/10.1039/D4AY00082J

Rodriguez-Amaya, D. B., Kimura, M. 2004. HarvestPlus Handbook for Carotenoid Analysis. HarvestPlus Technical Monograph 2. International Food Policy Research Institute. p 63.

Ryszkiewicz, P., Schlicker, E., Malinowska, B. 2025. Is Inducible Nitric Oxide Synthase (iNOS) Promising as A New Target Against Pulmonary Hypertension?. Antioxidants, 14(4), 377.

Saini, R. K., Prasad, P., Lokesh, V., Shang, X., Shin, J., Keum, Y.-S., Lee, J.-H. 2022. Carotenoids: Dietary Sources, Extraction, Encapsulation, Bioavailability, and Health Benefits—A Review of Recent Advancements. Antioxidants, 11(4), 795. https://doi.org/10.3390/antiox11040795

Salvanes, A. G. V., Devine, J., Jensen, K. H., Hestetun, J. T., Sjøtun, K., Glenner, H. (Eds.). 2018. Marine Ecological Field Methods: A Guide for Marine Biologists and Fisheries scientists. John Wiley & Sons. p 240. https://doi.org/10.1002/9781119184362

Shamsian, S., Sokouti, B., Dastmalchi, S. 2024. Benchmarking Different Docking Protocols for Predicting The Binding Poses of Ligands Complexed With Cyclooxygenase Enzymes and Screening Chemical Libraries. BioImpacts, 14(2), 29955. https://doi.org/10.34172/bi.2023.29955

Sianipar, H. F., Sinaga, M. P., Barat, W. O. B. 2025. The Potential Bioactive Compound of Rhizophora apiculata Mangrove Fruit Extract in Inhibiting The Growth of Salmonella typhi Bacteria. Jurnal Penelitian Pendidikan IPA, 11(8), 1068–1073. https://doi.org/10.29303/jppipa.v11i8.12344

Sun, T., Rao, S., Zhou, X., and Li, L. 2022. Plant Carotenoids: Recent Advances and Future Perspectives. Molecular Horticulture, 2, 3. https://doi.org/10.1186/s43897-022-00023-2

Warouw, F., Pertiwi, J. M., Mantiri, D. M., Paransa, D. S., Montolalu, R. I., Lumoindong, F., Kepel, R. C. 2024. Phytochemical Screening, Antioxidant and Antiinflammatory Activities of Green Macroalga Valonia aegagropila from Coastal Waters of Korakora, North Sulawesi, Indonesia. Aquaculture, Aquarium, Conservation & Legislation, 17(6), 2609-2619.