**4. Conclusions**

From the methanolic extract of the Red Sea red algae *Hypnea musciformis*, two new ceramides and another first reported one were isolated. Moreover, other chemical metabolites were detected by using the LC-ESI-HRMS technique. Both new metabolites, ceramides **A** (**1**) and **B** (**2**), exhibited significant in vitro cytotoxic activity against the human breast adenocarcinoma (MCF-7) cell line. The activity of ceramides **A** (**1**) and **B** (**2**) was investigated in an EAC mouse model, where both ceramides at doses of 1 and 2 mg/kg significantly decreased the tumor size, serum levels of VEGF-B and TNF-<sup>α</sup>, and expression of the biomarker midkine growth factor in the tumor tissue, with significant upregulation of the apoptotic factors p53, Bax, and caspase-3. Ceramide **B** (**2**), at a therapeutic dose of 2 mg/kg, showed the most potent antiangiogenic activity and the highest expression of the investigated apoptotic factors. Molecular docking suggested the interaction of these ceramides with the p53 transcription factor, leading to its accumulation and activation of the downstream apoptotic pathways. The current study introduced two potentially effective anti-cancer ceramides isolated from the Red Sea algae *Hypnea musciformis*, which exhibited antiangiogenic and apoptotic effects in the experimentally induced mammary tumor. The major limitation of this work is that the effects of the isolated ceramides were not investigated on healthy cell lines, suggesting that further research is required to evaluate the toxicity of those compounds and to determine their therapeutic indices.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/md20010063/s1, Figures S1 and S2: LC-ESI-HRMS chromatogram of crude extract of *Hypnea musciformis* (positive and negative modes); Figure S3: ESI-HRMS chromatogram of ceramide **A** (**1**); Figures S4 and S5: 1H NMR and 13C NMR spectra of ceramide **A** (**1**); Figure S6: ESI-HRMS chromatogram of fatty acid methyl ester of ceramide **A** (**1**); Figure S7: ESI-HRMS chromatogram of ceramide **B** (**2**); Figures S8 and S9: 1H NMR and 13C NMR spectra of ceramide **B** (**2**); Figure S10: ESI-HRMS chromatogram of fatty acid methyl ester of ceramide **B** (**2**); Figure S11: GC-MS analysis of fatty acid methyl ester of ceramide **B** (**2**); Figure S12: ESI-HRMS chromatogram of ceramide **C** (**3**); Figures S13 and S14: 1H NMR and 13C NMR spectra of ceramide **C** (**3**); Figure S15: ESI-HRMS chromatogram of fatty acid methyl ester of ceramide **C** (**3**); Figure S16: ESI-HRMS chromatogram of compound **4**; Figures S17 and S18: 1H NMR and 13C NMR spectra of compound **4**; Figure S19: ESI-HRMS chromatogram of compound **5**; Figures S20 and S21: 1H NMR and 13C NMR spectra of

compound **5**; Figure S22: ESI-HRMS chromatogram of compound **6**; Figure S23: 1H NMR spectra of compound **6**; Table S1: Liver enzymes and kidney markers in the study groups.

**Author Contributions:** Conceptualization, R.F.A.A., S.A.A., S.S.E., E.E.E. and M.S.G.; methodology, R.F.A.A., E.E.E., R.M.H., E.T.M., M.S.G., E.S.H. and S.S.E.; software, M.S.G. and M.A.H.; validation, K.M.H., E.E.E., A.K.I. and M.S.G.; formal analysis, K.M.H., E.E.E., R.M.H., E.T.M., S.S.E., M.S.G. and A.K.I.; investigation, R.F.A.A., S.S.E., A.K.I., E.E.E., E.S.H., M.S.G., R.M.H., E.T.M. and U.R.A.; resources, K.M.H., S.S.E., R.M.D. and S.A.A.; funding acquisition, R.M.D., S.S.E., K.M.H. and S.A.A.; data curation, M.S.G., E.E.E., A.K.I., H.A.H., S.S.E. and M.A.H.; writing—original draft preparation, R.F.A.A., E.S.H., S.S.E., E.E.E., R.M.H., E.T.M. and M.S.G.; writing—review and editing, R.F.A.A., S.A.A., E.E.E., E.S.H., U.R.A. and S.S.E.; supervision, S.A.A., R.F.A.A., E.E.E., H.A.H., E.S.H. and A.K.I. All authors have read and agreed to the published version of the manuscript.

**Funding:** The Deanship of Scientific Research (DSR) at King Abdulaziz University in Jeddah, Saudi Arabia funded this project under gran<sup>t</sup> no. D-029-166-1440.

**Institutional Review Board Statement:** The study protocol was approved by the ethical committee of the Faculty of Pharmacy at Suez Canal University (201605PHDA1).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data are contained within the article and the Supplementary Materials.

**Acknowledgments:** This work was supported by the Deanship of Scientific Research (DSR) at King Abdulaziz University in Jeddah, Saudi Arabia under gran<sup>t</sup> no. D-029-166-1440. The authors, therefore, gratefully acknowledge the DSR for technical and financial support. The authors are grateful to Tarek A. Temraz of the Marine Science Department in the Faculty of Science at Suez Canal University for his taxonomical identification of the Red Sea red algae *Hypnea musciformis*.

**Conflicts of Interest:** The authors declare no conflict of interest.
