Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2025 Jun 11;26(12):5605.
doi: 10.3390/ijms26125605.

Neuroprotective Potential of Betulin and Its Drug Formulation with Cyclodextrin-In Vitro Assessment

Agnieszka Zakrzeska  1 , Agnieszka Kołodziejczyk  2   3 , Piotr Komorowski  2 , Paulina Sokołowska  4 , Bożena Rokita  3 , Radosław A Wach  3 , Małgorzata Siatkowska  2   3
Affiliations

Neuroprotective Potential of Betulin and Its Drug Formulation with Cyclodextrin-In Vitro Assessment

Agnieszka Zakrzeska et al. Int J Mol Sci. .

Abstract

Central nervous system disorders, such as Alzheimer's disease, Parkinson's disease, and multiple sclerosis, are associated with complex pathophysiological mechanisms involving oxidative stress, inflammation, and protein misfolding. According to the literature, betulin, a natural compound derived from the bark of birch trees, demonstrates promising neuroprotective effects. This study investigates the neuroprotective potential of betulin and its complex with cyclodextrin, referred to as Betula Forte, using an in vitro model of differentiated SH-SY5Y neuroblastoma cells. Specifically, the study explores the antioxidant and antiapoptotic properties of these compounds under oxidative stress induced by hydrogen peroxide (H2O2). Our results indicated that Betula Forte exhibited lower cytotoxicity compared to betulin alone. Both substances enhanced cell viability in pre-incubation and co-incubation models, with Betula Forte showing superior efficacy under severe oxidative stress. Additionally, both substances exhibited protective effects against H2O2-induced oxidative stress and apoptosis, as evidenced by reduced levels of reactive oxygen species (ROS) and a lower number of apoptotic cells compared with the H2O2-treated cells. These findings suggest that Betula Forte, due to lower cytotoxicity, may offer a more effective neuroprotective strategy than betulin alone, highlighting its potential as a therapeutic agent for neurodegenerative diseases.

Keywords: ROS; SH-SY5Y; apoptosis; betulin; differentiated neuroblastoma cells; neuroprotection.

PubMed Disclaimer

Conflict of interest statement

Authors: Małgorzata Siatkowska, Agnieszka Kołodziejczyk, Bożena Rokita, and Radosław A. Wach were employed by the company BioMatGel, Pienista 41F/11, 94-109 Lodz, Poland. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Cell viability after 24 h of incubation of differentiated SH-SY5Y neuroblastoma cells with (A) betulin, (B) Betula Forte, and (C) H2O2 at various concentrations. Data are presented as means ± standard deviations (SD). The number of independent experiments was as follows: (A) n = 4, (B) n = 4, and (C) n = 5 (six independent measurements were analyzed in each experimental repetition). * indicates significant differences between the negative control (NC) and the tested compounds; * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001.
Figure 2
Figure 2
Cell viability after 24 h of incubation of differentiated SH-SY5Y neuroblastoma cells with (A,C) betulin and (B,D) Betula Forte in the pre-incubation variant. Data are presented as means ± standard deviations (SD). The number of independent experiments was as follows: (A–C) n = 4, (D) n = 5 (six independent measurements were analyzed in each experimental repetition). # indicates significant differences between the positive control (PC), i.e., H2O2 at IC 50 or IC80, and the tested concentrations of betulin or Betula Forte (# p < 0.05, ### p < 0.005, #### p < 0.001). * indicates significant differences between two different concentrations of the tested compound (* p < 0.05).
Figure 3
Figure 3
Cell viability after 24 h of incubation of differentiated SH-SY5Y neuroblastoma cells with (A,C) betulin and (B,D) Betula Forte in the co-incubation variant. Data are presented as means ± standard deviations (SD). The number of independent experiments was as follows: (A) n = 4, (B) n = 4, (C) n = 7, (D) n = 5 (six independent measurements were analyzed in each experimental repetition). # indicates significant differences between the positive control (PC), i.e., H2O2 at IC 50 or IC80, and the tested concentrations of betulin or Betula Forte (### p < 0.005, #### p < 0.001). * indicates significant differences between two different concentrations of the tested compound (* p < 0.05, *** p < 0.005, **** p < 0.001).
Figure 4
Figure 4
ROS detection in differentiated SH-SY5Y neuroblastoma cells after exposure to (A) betulin and (B) Betula Forte at various concentrations and during co-incubation with H2O2 at IC50. Data are presented as means ± standard deviations (SD). The number of independent experiments was as follows: (A,B) n = 3 (six independent measurements were analyzed in each experimental repetition). # indicates significant differences between the ROS generating agent (H2O2 at IC 50) and the tested compound added simultaneously to the culture (## p < 0.01, ### p < 0.005). * indicates significant differences between the negative control (NC) and the tested compounds (* p < 0.05, *** p < 0.005, **** p < 0.001).
Figure 5
Figure 5
Apoptosis detection in differentiated SH-SY5Y neuroblastoma cells after exposure to (A) betulin and (B) Betula Forte at various concentrations and during co-incubation with H2O2 at IC50. (C) Fluorescence images of cells stained with Annexin V and Hoechst (scale bar is 100 µm). Data are presented as means ± standard deviations (SD). The number of independent experiments was as follows: (A,B) n = 4 (six independent measurements were analyzed in each experimental repetition). # indicates significant differences between the apoptosis generating agent (H2O2 at IC 50) and the tested compound added simultaneously to the culture (# p < 0.05, ## p < 0.01). * indicates significant differences between the negative control (NC) and the tested compounds (** p < 0.01, **** p < 0.001).
Figure 6
Figure 6
Schematic representation of pre-incubation and co-incubation experimental design for the SH-SY5Y cell line, along with sample nomenclature.
See this image and copyright information in PMC

References

    1. Sealfon S.C., Motiwala R., Stacy C.B., editors. Mount Sinai Expert Guides. Neurology. John Wiley & Sons Inc.; Chichester, UK: Hoboken, NJ, USA: 2016.
    1. Jha N.K., Chen W.-C., Kumar S., Dubey R., Tsai L.-W., Kar R., Jha S.K., Gupta P.K., Sharma A., Gundamaraju R., et al. Molecular Mechanisms of Developmental Pathways in Neurological Disorders: A Pharmacological and Therapeutic Review. Open Biol. 2022;12:210289. doi: 10.1098/rsob.210289. - DOI - PMC - PubMed
    1. Adepoju F.O., Duru K.C., Li E., Kovaleva E.G., Tsurkan M.V. Pharmacological Potential of Betulin as a Multitarget Compound. Biomolecules. 2023;13:1105. doi: 10.3390/biom13071105. - DOI - PMC - PubMed
    1. Park J.S., Rehman I.U., Choe K., Ahmad R., Lee H.J., Kim M.O. A Triterpenoid Lupeol as an Antioxidant and Anti-Neuroinflammatory Agent: Impacts on Oxidative Stress in Alzheimer’s Disease. Nutrients. 2023;15:3059. doi: 10.3390/nu15133059. - DOI - PMC - PubMed
    1. Gonzalez G., Hodoň J., Kazakova A., D’Acunto C.W., Kaňovský P., Urban M., Strnad M. Novel Pentacyclic Triterpenes Exhibiting Strong Neuroprotective Activity in SH-SY5Y Cells in Salsolinol- and Glutamate-Induced Neurodegeneration Models. Eur. J. Med. Chem. 2021;213:113168. doi: 10.1016/j.ejmech.2021.113168. - DOI - PubMed

LinkOut - more resources