An in vitro Adipogenic Potential and Glucose Uptake Stimulatory Effect of Betulinic Acid and Stigmasterol Isolated from Tetracera indica in 3T3-L1 Cell Line

(Potensi Adipogenik in vitro dan Kesan Rangsangan Pengambilan Glukosa Asid Betulinik dan Stigmasterol Dipencilkan daripada Tetracera indica dalam Titisan Sel 3T3-L1)

MD. MAHMUDUL HASAN^1,6, QAMAR UDDIN AHMED^1,2,*, JALIFAH LATIP³, SITI ZAITON MAT SOAD², MUHAMMAD TAHER⁴, AWIS SUKARNI MOHMAD SABERE² & ZAINUL AMIRUDDIN ZAKARIA⁵

¹Drug Discovery and Synthetic Chemistry Research Group, Department of Pharmaceutical Chemistry, Kulliyyah of Pharmacy, International Islamic University Malaysia, 25200 Kuantan, Pahang Darul Makmur, Malaysia

²Pharmacognosy Research Group, Department of Pharmaceutical Chemistry, Kulliyyah of Pharmacy, International Islamic University Malaysia, 25200 Kuantan, Pahang Darul Makmur, Malaysia

³Department of Chemical Sciences, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Darul Ehsan, Malaysia

⁴Department of Pharmaceutical Technology, Kulliyyah of Pharmacy, International Islamic University Malaysia, 25200 Kuantan, Pahang Darul Makmur, Malaysia

⁵Borneo Research for Algesia, Inflammation and Neurodegeneration (BRAIN) Group, Faculty of Medicine and Health Sciences, Universiti Malaysia Sabah, Kota Kinabalu 88400, Sabah, Malaysia

⁶School of Biosciences, Faculty of Health and Medical Sciences, Taylor’s University, 47500 Subang Jaya, Selangor Darul Ehsan, Malaysia

Received: 17 February 2022/Accepted: 16 December 2022

Abstract

Aerial parts of Tetracera indica Merr. (Dilleniaceae) are rich in betulinic acid and stigmasterol and traditionally used to treat diabetes. This study was aimed to evaluate an in vitro antidiabetic potential of betulinicacid and stigmasterol to ascertain whether they may contribute antidiabetic effect to T. indica. Initially, betulinicacid and stigmasterol were isolated from the most effective subfraction (ethyl acetate) and subjected to an in vitro antidiabetic investigation through adipogenesis and fluorescence glucose (2-NBDG) uptake assays using 3T3-L1 fibroblast. MTT viability assay was performed at 0.78 to 100 µg/mL for 48 h to determine the safe concentration. Both compounds were subjected to 2-NBDG uptake test on the differentiated adipocytes. The cells were treated in safe concentrations (25-100 µg/mL) as well as in different adipogenic cocktails, which were modified by the addition of compounds to be investigated and in the presence or absence of insulin (10 µM). Rosiglitazone (10 µM) was used as standard. Stems ethanol extract and its fractions (hexane and ethyl acetate), betulinic acid and stigmasterol were found safe at their highest concentration (100 µg/mL) by inhibiting cells well below their IC₅₀ values viz. 18.60, 35.27, 21.40, 28.86 and 33.06%, respectively. Both betulinic acid and stigmasterol at the highest safe concentration (100 µg/mL) significantly (p <0.05) induced adipogenesis like insulin, enhanced adipogenesis like rosiglitazone and exhibited glucose uptake activity. The present study demonstrates that both betulinic acid and stigmasterol possess an in vitro antidiabetic potential. However, in vivo antiglycemic study on these compounds and their chemical analogs are still warranted to ensure their therapeutic potential as safe antidiabetic agents.

Keywords: Adipogenesis; betulinic acid; insulin like activity; insulin sensitizing activity; stigmasterol; 2-NBDG uptake activity; 3T3-L1 preadipocyte cells

Abstrak

Bahagian udara Tetracera indica Merr. (Dilleniaceae) kaya dengan asid betulinik dan stigmasterol serta digunakan secara tradisi untuk merawat diabetes. Kajian ini dijalankan untuk menilai potensi antidiabetik asid betulinik dan stigmasterol secara in vitro untuk memastikan sama ada kedua-dua sebatian ini menyumbang kepada kesan antidiabetik oleh T. indica. Asid betulinik dan stigmasterol diasingkan daripada subfraksi (etil asetat) yang paling berkesan. Kesan antidiabetik in vitro dikaji melalui asai adipogenesis dan asai pengambilan glukosa berpendaflour (2-NBDG) menggunakan selfibroblas 3T3-L1. Asai kebolehhidupan MTT dijalankan pada kepekatan antara 0.78 hingga 100 µg/mL selama 48 jam bagi menentukan kepekatan yang selamat. Akhir sekali, kedua-dua sebatian diuji dengan asai 2-NBDG ke atas sel adiposit terbeza. Sel tersebut dirawat pada julat kepekatan selamat (25-100 µg/mL) dengan koktel adipogenik yang berbeza dengan pengubahsuaian adalah pada penambahan sebatian kajian dan dalam kehadiran (10 µM) atau tanpa insulin. Rosiglitazon (10 µM) digunakan sebagai sebatian piawai. Ekstrak etanol batang, fraksi (heksana dan etil asetat), asid betulinik dan stigmasterol dikenal pasti selamat pada kepekatan tertinggi (100 µg/mL), dengan merencat pertumbuhan sel di bawah nilai IC₅₀ masing-masing iaitu 18.60, 35.27, 21.40, 28.86 dan 33.06%. Asid betulinik dan stigmasterol, kedua-duanya pada kepekatan selamat tertinggi (100 µg/mL), secara signifikan (p <0.05) mengaruh adipogenesis seperti insulin, meningkatkan adipogenesis seperti rosiglitazon dan mempamerkan aktiviti pengambilan glukosa. Kajian ini menunjukkan kedua-dua asid betulinik dan stigmasterol berpotensi sebagai antidiabetik in vitro. Walau bagaimanapun, kajian antiglisemik in vivo terhadap kedua-dua sebatian dan terbitannya masih diperlukan untuk memastikan potensi terapeutik sebatian sebagai agen antidiabetik yang selamat.

Kata kunci: Adipogenesis; aktiviti pengambilan 2-NBDG; aktiviti pensensitifan insulin; aktiviti seperti insulin; asidbetulinik; stigmasterol; sel preadiposit 3T3-L1

REFERENCES

Ahmed, Q.U., Ali, A.H.M., Mukhtar, S., Meshari, A.A., Parveen, H., Sabere, A.S.M., Nawi, M.S.M., Khatib, A., Siddiqui, M.J., Umar, A. & Alhassan, A.M. 2020. Medicinal potential of isoflavonoids: Polyphenols that may cure diabetes. Molecules 25(23): 5491.

Ahmed, Q.U., Umar, A., Taher, M., Susanti, D., Amiroudine, M.Z.A.M. & Latip, J. 2014. Phytochemical investigation of the leaves of Tetracera scandens Linn. and in vitro antidiabetic activity of hypoletin. Proceedings of the International Conference on Science, Technology and Social Sciences (ICSTSS) 2012. pp. 591-608.

Ahmed, Q.U., Dogarai, B.B.S., Amiroudine, M.Z.A.M., Taher, M., Latip, J., Umar, A. & Muhammad, B.Y. 2012. Antidiabetic activity of the leaves of Tetracera indica Merr. (Dilleniaceae) in vivo and in vitro. Journal of Medicinal Plants Research 6: 5912-5922.

Alhassan, A.M., Ahmed, Q.U., Latip, J. & Shah, S.A.A. 2019. A new sulphated flavone and other phytoconstituents from the leaves of Tetracera indica Merr. and their alpha-glucosidase inhibitory activity. Natural Product Research 33(1): 1-8.

Choi, J.H., Banks, A.S., Kamenecka, T.M., Busby, S.A., Chalmers, M.J., Kumar, N., Kuruvilla, D.S., Shin, Y., He, Y., Bruning, J.B. & Marciano, D.P. 2011. Anti-diabetic actions of a non-agonist PPARγ ligand blocking Cdk5-mediated phosphorylation. Nature 477: 477-481.

Choi, J.Y., Na, M., Hyun, H.I., Ho, L.S., Young, B.E., Yeon, K.B. & Seog, A.J. 2009. Isolation of betulinic acid, its methyl ester and guaiane sesquiterpenoids with protein tyrosine phosphatase 1B inhibitory activity from the roots of Saussurea lappa C.B. Clarke. Molecules 14(1): 266-272.

de Melo, C.L., Queiroz, M.G., Arruda, F.A.C., Rodrigues, A.M., de Sousa, D.F., Almeida, J.G., Pessoa, O.D., Silveira, E.R., Menezes, D.B., Melo, T.S., Santos, F.A. & Rao, V.S. 2009. Betulinic acid, a natural pentacyclic triterpenoid, prevents abdominal fat accumulation in mice fed a high-fat diet. Journal of Agricultural and Food Chemistry 57: 8776-8781.

Drzewoski, J. & Hanefeld, M. 2021. The current and potential therapeutic use of metformin-the good old drug. Pharmaceuticals 14(2): 122.

Fazakerley, D.J., Koumanov, F. & Holman, G.D. 2022. GLUT4 on the move. Biochemical Journal 479(3): 445-462.

Hasan, M.M., Ahmed, Q.U., Soad, S.Z.M., Latip, J., Taher, M., Syafiq, T.M.F., Sarian, M.N., Alhassan, A.M. & Zakaria, Z.A. 2017. Flavonoids from Tetracera indica Merr. induce adipogenesis and exert glucose uptake activities in 3T3-L1 adipocyte cells. BMC Complementary Medicine & Therapies 17(1): 431-444.

Jin, T., Yu, H. & Huang, X.F. 2016. Selective binding modes and allosteric inhibitory effects of lupane triterpenes on protein tyrosine phosphatase 1B. Scientific Reports 6: 20766.

Kooti, W. 2016. The role of medicinal plants in the treatment of diabetes: A systematic review. Electron Physician 8(1): 1832-1842.

Li, S., Eguchi, N., Lau, H. & Ichii, H. 2020. The role of the Nrf2 signaling in obesity and insulin resistance. International Journal of Molecular Sciences 21(18): 6973.

Ma, J.Z., Yang, X.W., Zhang, J.J., Liu, X., Deng, L.L., Shen, X.L. & Xu, G. 2014. Sterols and terpenoids from Viburnum odoratissimum. Natural Products and Bioprospecting 4: 175-180.

Moseti, D., Regassa, A. & Kim, W.K. 2016. Molecular regulation of adipogenesis and potential anti-adipogenic bioactive molecules. International Journal of Molecular Sciences 17(1): 124.

Nazaruk, J. & Borzym-Kluczyk, M. 2015. The role of triterpenes in the management of diabetes mellitus and its complications. Phytochemistry Reviews 14: 675-690.

Ogurtsova, K. 2017. IDF Diabetes Atlas: Global estimates for the prevalence of diabetes for 2015 and 2040. Diabetes Research and Clinical Practice 128(1): 40-50.

Panda, S., Jafri, M., Kar, A. & Meheta, B.K. 2009. Thyroid inhibitory, antiperoxidative and hypoglycemic effects of stigmasterol isolated from Butea monosperma. Fitoterapia 80: 123-126.

Park, J.H., Kim, R.Y. & Park, E. 2012. Antidiabetic activity of fruits and vegetables commonly consumed in Korea: Inhibitory potential against α-glucosidase and insulin-like action in vitro. Food Science and Biotechnology 21: 1187-1193.

Roheem, F.O., Ahmed, Q.U., Mat So'ad, S.Z., Shah, S.A.A., Latip, J., Alhassan, A.M. & Syed Mohammad, S.N.A. 2020. Assessment of free radical scavenging and digestive enzyme inhibitory activities of extract, fractions and isolated compounds from Tetracera macrophylla leaves. Journal of Herbal Medicine 22: 100351.

Ruiz-Ojeda, F.J., Rupérez, A.I., Gomez-Llorente, C., Gil, A. & Aguilera, C.M. 2016. Cell models and their application for studying adipogenic differentiation in relation to obesity: A review. International Journal of Molecular Sciences 17(7): 1040.

Singab, A.N., El-Beshbishy, H.A., Yonekawa, M., Nomura, T. & Fukai, T. 2005. Hypoglycemic effect of Egyptian Morus alba root bark extract: Effect on diabetes and lipid peroxidation of streptozotocin-induced diabetic rats. Journal of Ethnopharmacology 100: 333-338.

Wang, J., Huang, M., Yang, J., Ma, X., Zheng, S., Deng, S., Huang, Y., Yang, X. & Zhao, P. 2017. Anti-diabetic activity of stigmasterol from soybean oil by targeting the GLUT4 glucose transporter. Food & Nutrition Research 61(1): 1364117.

Wang, T., Wang, J., Hu, X., Huang, X.J. & Chen, G.X. 2020. Current understanding of glucose transporter 4 expression and functional mechanisms. World Journal of Biological Chemistry 11(3): 76-98.

World Health Organisation. 2020. Expert panel endorses protocol for COVID-19 herbal medicine clinical trials. https://www.afro.who.int/news/expert-panel-endorses-protocol-covid-19-herbal-medicine-clinical-trials. Accessed on April 14, 2021.

Zimmet, P.Z. 2017. Diabetes and its drivers: The largest epidemic in human history? Clinical Diabetes and Endocrinology 3(1): 1.

^*Corresponding author; email: quahmed@iium.edu.my

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