Identification of α-Amylase Inhibitory Peptides from Tryptic Hydrolysate of  Caulerpa racemosa  Green Algae Protein

Izatul Husna; Ahmad Habibie; Endang Astuti; Tri Joko Raharjo

doi:10.22146/ijc.89147

Identification of α-Amylase Inhibitory Peptides from Tryptic Hydrolysate of Caulerpa racemosa Green Algae Protein

https://doi.org/10.22146/ijc.89147

Izatul Husna⁽¹⁾, Ahmad Habibie⁽²⁾, Endang Astuti⁽³⁾, Tri Joko Raharjo^(4*)

(1) Department of Biotechnology, Graduate School, Universitas Gadjah Mada, Sekip Utara, Yogyakarta 55281, Indonesia
(2) Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Gadjah Mada, Sekip Utara, Yogyakarta 55281, Indonesia
(3) Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Gadjah Mada, Sekip Utara, Yogyakarta 55281, Indonesia
(4) Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Gadjah Mada, Sekip Utara, Yogyakarta 55281, Indonesia
(*) Corresponding Author

Abstract

Peptides have been reported as a potential alternative for antidiabetic therapy by inhibiting α-amylase, one of the key enzymes involved in carbohydrate metabolism. This study aimed to identify α-amylase inhibitory peptides derived from trypsin hydrolysate of Caulerpa racemosa protein. The protein was extracted using trichloroacetic acid (TCA)/acetone. The resulting peptides were fractionated using a strong cationic exchanger-solid phase extraction (SCX-SPE) column. The peptide fractions were tested for α-amylase inhibitory activity, and the peptides in the most active fraction were identified using liquid chromatography-high resolution mass spectrometry (LC-HRMS). Seven fractions were obtained from SCX-SPE elution at pH 3 to 9. The fraction eluted at pH 5 exhibited the highest α-amylase inhibitory activity, with an IC₅₀ value of 43.70 µg/mL. Four peptides with the sequences VQKEKR, MNFYCISSK, DLCDYIHNK, and ISICYEK were identified from this fraction. Molecular docking studies revealed that the peptides with sequences MNFYCISSK and VQKEKR had the strongest interactions with α-amylase. The peptide binding affinity energies of MNFYCISSK and VQKEKR were −10.3 and −9.4 kcal/mol, respectively, with RMSD values of 0.3 ± 0.0 and 0.3 ± 0.2 Å. It can be concluded that the peptides with sequences MNFYCISSK and VQKEKR could be proposed as potential antidiabetic peptides with an α-amylase inhibition mechanism.

Keywords

peptide; α-amylase; inhibitor; Culerpa racemosa; antidiabetic

Full Text:

Full Text PDF

References

[1] Cho, N.H., Shaw, J.E., Karuranga, S., Huang, Y., da Rocha Fernandes, J.D., Ohlrogge, A.W., and Malanda, B., 2018, IDF Diabetes Atlas: Global estimates of diabetes prevalence for 2017 and projections for 2045, Diabetes Res. Clin. Pract., 138, 271–281.

[2] Ministry of Health of the Republic of Indonesia, 2022, Infodatin: Tetap Produktif, Cegah, dan Atasi Diabetes Melitus, https://lib.pkr.ac.id/home/detail/24427659/610.5+Kes+t, accessed on July 8, 2025.

[3] Cheng, H.T., Xu, X., Lim, P.S., and Hung, K.Y., 2021, Worldwide epidemiology of diabetes-related end-stage renal disease, 2000–2015, Diabetes Care, 44 (1), 89–97.

[4] Erener, S., 2020, Diabetes, infection risk and COVID-19, Mol. Metab., 39, 101044.

[5] Sumlang, G.H.P., Handoyo, R.L., Laksitorini, M.D., Astuti, E., and Raharjo, T.J., 2025, Goat milk casein peptides as potential α-amylase inhibitors: A computational and experimental approach, Indones. J. Chem., 25 (3), 770–786.

[6] Fan, S., Liu, Q., Du, Q., Zeng, X., Wu, Z., Pan, D., and Tu, M., 2024, Multiple roles of food-derived bioactive peptides in the management of T2DM and commercial solutions: A review, Int. J. Biol. Macromol., 279, 134993.

[7] Treacy, J., Williams, A., Bais, R., Willson, K., Worthley, C., Reece, J., Bessell, J., and Thomas, D., 2001, Evaluation of amylase and lipase in the diagnosis of acute pancreatitis, ANZ J. Surg., 71 (10), 577–582.

[8] Dhevika, S., and Deivasigamani, B., 2018, Phytochemical profiling and GC-MS analysis of Caulerpa racemosa, Res. J. Life Sci., Bioinf., Pharm. Chem. Sci., 4 (5), 155–165.

[9] Hao, H., Fu, M., Yan, R., He, B., Li, M., Liu, Q., Cai, Y., Zhang, X., and Huang, R., 2019, Chemical composition and immunostimulatory properties of green alga Caulerpa racemosa var peltata, Food Agric. Immunol., 30 (1), 937–954.

[10] Huda, M.B., Astuti, E., and Raharjo, T.J., 2021, Synthesis of mono-ketone curcumin analogs from 3-benzyloxybenzaldehyde and their activity assay as inhibitor of α-amylase, Key Eng. Mater., 884, 304–311.

[11] Niu, L., Zhang, H., Wu, Z., Wang, Y., Liu, H., Wu, X., and Wang, W., 2018, Modified TCA/acetone precipitation of plant proteins for proteomic analysis, PLoS One, 13 (12), e0202238.

[12] Cruz-Casas, D.E., Aguilar, C.N., Ascacio-Valdés, J.A., Rodríguez-Herrera, R., Chávez-González, M.L., and Flores-Gallegos, A.C., 2021, Enzymatic hydrolysis and microbial fermentation: The most favorable biotechnological methods for the release of bioactive peptides, Food Chem.: Mol. Sci., 3, 100047.

[13] Kumar, M., Tomar, M., Potkule, J., Verma, R., Punia, S., Mahapatra, A., Belwal, T., Dahuja, A., Joshi, S., Berwal, M.K., Satankar, V., Bhoite, A.G., Amarowicz, R., Kaur, C., and Kennedy, J.F., 2021, Advances in the plant protein extraction: Mechanism and recommendations, Food Hydrocolloids, 115, 106595.

[14] Prasad, B.J., Sharavanan, P.S., and Sivaraj, R., 2019, Efficiency of Oryza punctata extract on glucose regulation: Inhibition of α-amylase and α-glucosidase activities, Grain Oil Sci. Technol., 2 (2), 44–48.

[15] Pishdad, R., Pishdad, P., and Pishdad, G.R., 2020, Acarbose versus repaglinide in diabetes treatment: A new appraisal of two old rivals, Am. J. Med. Sci., 359 (4), 212–217.

[16] Svensson, B., Fukuda, K., Nielsen, P.K., and Bønsager, B.C., 2004, Proteinaceous α-amylase inhibitors, Biochim. Biophys. Acta, Proteins Proteomics, 1696 (2), 145–156.

[17] Wang, J., Wu, T., Fang, L., Liu, C., Liu, X., Li, H., Shi, J., Li, M., and Min, W., 2020, Anti-diabetic effect by walnut (Juglans mandshurica Maxim.)-derived peptide LPLLR through inhibiting α-glucosidase and α-amylase, and alleviating insulin resistance of hepatic HepG2 cells, J. Funct. Foods, 69, 103944.

[18] Zhou, H., Safdar, B., Li, H., Yang, L., Ying, Z., and Liu, X., 2023, Identification of a novel α-amylase inhibitory activity peptide from quinoa protein hydrolysate, Food Chem., 403, 134434.

[19] Van Zundert, G.C.P., Rodrigues, J.P.G.L.M., Trellet, M., Schmitz, C., Kastritis, P.L., Karaca, E., Melquiond, A.S.J., van Dijk, M., de Vries, S.J., and Bonvin, A.M.J.J., 2016, The HADDOCK2.2 web server: User-friendly integrative modeling of biomolecular complexes, J. Mol. Biol., 428 (4), 720–725.

[20] Lensink, M.F., and Wodak, S.J., 2013, Docking, scoring, and affinity prediction in CAPRI, Proteins: Struct., Funct., Bioinf., 81 (12), 2082–2095.

[21] Bernaldez, M.J.A., Billones, J.B., and Magpantay, A., 2018, In silico analysis of binding interactions between GSK983 and human DHODH through docking and molecular dynamics, AIP Conf. Proc., 2045 (1), 20073.

[22] Liang, Z., and Li, Q.X., 2018, π-Cation interactions in molecular recognition: Perspectives on pharmaceuticals and pesticides, J. Agric. Food. Chem., 66 (13), 3315–3323.

DOI: https://doi.org/10.22146/ijc.89147