Sambucus nigra L. (European elderberry) is distinguished among medicinal and edible plants by its exceptionally high cyanidin-3-O-glucoside (C3G) content, which markedly exceeds that of common berries. This unique phytochemical profile establishes C3G as the principal bioactive constituent underlying the antitumor activity of S. nigra. While numerous reviews on elderberry have been published, none has systematically integrated C3G biosynthesis, transcriptional regulation, in vivo metabolism, and anti-tumor mechanisms specifically in S. nigra—a critical research gap that this review fills for the first time. Herein, we systematically examine the chemical structure and content distribution of C3G in S. nigra, elucidate the phenylpropanoid–flavonoid biosynthetic pathway and the regulatory patterns of the MYB-bHLH-WD40 (MBW) transcriptional complex, and highlight the current research gap regarding the cloning and functional characterization of core MBW factors in this species. We further reveal the absorption and distribution characteristics of C3G in the human body, the gut microbiota-mediated biotransformation process, and the synergistic antitumor effects of its primary metabolite, protocatechuic acid. The molecular mechanisms through which C3G exerts antitumor activity, including the induction of tumor cell apoptosis, cell cycle arrest, inhibition of epithelial–mesenchymal transition, and modulation of key signaling pathways, such as NF-κB, PI3K/AKT/mTOR, and JNK, are systematically elaborated. This is the first review to construct a comprehensive “biosynthetic regulation–in vivo metabolism–antitumor function” framework for C3G in S. nigra, thereby addressing critical research gaps and providing a theoretical foundation for the germplasm breeding of high-C3G cultivars, functional product development, and clinical adjuvant cancer therapy.

Reverse transcription quantitative PCR (RT-qPCR) is one of the most widely used techniques for gene expression analysis in molecular biology. However, the accuracy of relative gene expression quantification largely depends on the stability of the reference genes used for normalization. Sambucus nigra L. (elderberry) is a valuable medicinal and edible plant rich in anthocyanins and other bioactive compounds. Despite its increasing research and application value, no reference genes have been validated for this species. In this study, we applied RT-qPCR alongside four algorithms (GeNorm, NormFinder, BestKeeper, and RefFinder) to assess the expression stability of nine candidate reference genes across ten samples representing five tissue types (including stems, flowers, leaves, roots, fruits) at different developmental stages. The results showed that VAMP and Pol were the most suitable reference gene combination for normalization across different tissues of S. nigra. For studies involving only vegetative tissues (leaves and stems), our results recommend that RPB2 and RPB5 are the most stable and suitable reference genes. This study provides reliable reference genes for accurate RT-qPCR-based gene expression analysis in S. nigra and establishes a useful methodological basis for future functional genomics and molecular breeding studies in this species.