Intratumoral administration of Hibiscus sabdariffa- derived anthocyanins exerts potent antitumor effects in murine cancer models

Abstract

Introduction: Cancer remains the leading cause of death worldwide, with increasing incidence rates. Natural compounds have gained attention as potential therapeutic agents due to their bioactive properties. Anthocyanins, particularly delphinidin-3-sambubioside (Dp-3-sam) and cyanidin-3-sambubioside (Cn-3-sam), are flavonoids with antioxidant and potential antitumor properties. This study investigates the antitumor effects of anthocyanins extracted from Hibiscus sabdariffa L. (H. sabdariffa), administered intratumorally, and their potential as adjuvants to chemotherapy.

Methods: Anthocyanins were extracted from H. sabdariffa and characterized using high-performance liquid chromatography (HPLC). The total phenolic content was determined using the Folin-Ciocalteu method. Antioxidant activity was assessed through DPPH, ABTS, and FRAP assays. The antiproliferative effects of Dp-3-sam and Cn-3-sam were evaluated in vitro using MCA-205 fibrosarcoma and CT26 colon carcinoma cell lines. In vivo studies were conducted on mouse tumor models to assess tumor growth inhibition following intratumoral administration of anthocyanins alone or in combination with doxorubicin. The impact on angiogenesis, immune cell recruitment, and long-term immune memory was also analyzed.

Results: HPLC analysis confirmed the presence of Dp-3-sam and Cn-3-sam in the H. sabdariffa extract. The anthocyanins exhibited significant antioxidant activity in all assays. In vitro studies demonstrated dose-dependent inhibition of cancer cell proliferation. In vivo, intratumoral administration of anthocyanins led to a significant reduction in tumor growth. The combination of anthocyanins with doxorubicin further enhanced tumor suppression. Mechanistically, Dp-3-sam and Cn-3-sam reduced angiogenesis and promoted immune cell recruitment but did not elicit an effective antitumor immune response alone. However, co-administration with doxorubicin reversed this limitation, leading to increased immune activation and resistance to tumor rechallenge, suggesting the induction of long-term immune memory.

Discussion: These findings highlight the potential of H. sabdariffa-derived anthocyanins as adjuvants in cancer therapy. When administered intratumorally, they enhance chemotherapy efficacy and immunogenicity. However, further studies are needed to optimize dosing strategies, evaluate long-term safety, and assess clinical applicability.

Keywords: Hibiscus sabdariffa L.; anthocyanins; antitumor activity; immune modulation; intratumoral therapy.

Figure 1

Anthocyanin characterization and antioxidant activity of Hibiscus sabdariffa L. extracts. (A) RP-HPLC chromatogram of H. Sabdariffa showing anthocyanidin peaks for delphinidin-3-sambubioside (Dp3-sam) and cyanidin-3-sambubioside (Cn3-sam). (B) RP-HPLC chromatogram of purified anthocyanins after XAD-7HP column chromatography. (C, D) Quantification of delphinidin and cyanidin in the ethanolic extract (EtcH) and purified extract (EeaH) via RP-HPLC. (E) Antioxidant activity of ethanolic (EtcH) and purified extracts (EeaH) determined via DPPH, ABTS, and FRAP assays. Significant differences in antioxidant capacity were noted between the extracts. The data are expressed as the means ± SDs.

Figure 2

Antiproliferative effects of Hibiscus sabdariffa L. extracts on MCA-205 and CT26 cancer cells. (A) Thymidine incorporation assay in MCA-205 fibrosarcoma and CT26 colon carcinoma cell lines. A total of 1 × 10⁵ cells were incubated with various concentrations of anthocyanins for 24 and 48 hours. (B) Real-time analysis of cell adhesion via xCELLigence of 3.5 × 10⁴ MCA-205 and CT26 cells with different concentrations of H. Sabdariffa anthocyanins and doxorubicin. The data were analyzed via one-way ANOVA with the Kruskal−Wallis test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, ns, nonsignificant.

Figure 3

Gene expression analysis of the tumor response to anthocyanin treatment. RNA sequencing analysis in which 5 x 10⁵ MCA-205 cells were stimulated with Dp3-sam/Cn3-sam and analyzed vs. control cells. (A) Volcano plot showing DEGs in MCA-205 tumors treated with anthocyanins. (B) Gene set enrichment analysis (GSEA) identifying key pathways involved in the immune response, oxidative stress, and apoptosis.

Figure 4

In vivo antitumor effects of Hibiscus sabdariffa L. extracts on the CT26 tumor model. (A) Graphical abstract of the assay. A total of 5 × 10⁵ CT26 cells were injected into the mice, which were then intratumorally injected with Dp3-sam/Cn3-sam, H. Sabdariffa, doxorubicin, or the combination. (B) Tumor volume measurements in BALB/c mice bearing CT26 colon carcinoma after treatment with H. Sabdariffa, Dp3-sam/Cn3-sam, doxorubicin and combo. (C) Tumor growth curves showing individual responses in each treatment group, including the number of fully cured mice. The data were analyzed via one-way ANOVA with Sidak’s multiple comparisons test. *p ≤ 0.05, ns, non-significant.

Figure 5

In vivo antitumor effects of Hibiscus sabdariffa L. extracts on MCA-205 tumor model. (A) Graphical abstract showing 5 × 10⁵ MCA-205 cells inoculated to mice and intratumorally injected with H. Sabdariffa, Dp3-sam/Cn3-sam, doxorubicin or the combination. (B) Tumor volume measurements in C57BL/6 mice bearing MCA-205 fibrosarcomas after treatment with H-Sabdariffa, Dp3-sam/Cn3-sam, doxorubicin and combo. (C) Tumor growth curves showing individual responses in each treatment group, including the number of fully cured mice. (D) Mice previously cured of MCA-205 tumors were rechallenged with the same tumor cells to assess the long-term immune response. Tumor volume over time was measured in previously treated and naïve control mice. Kaplan−Meier curve of mouse survival. The data were analyzed via one-way ANOVA with Sidak’s multiple comparisons test and the log rank test. *p ≤ 0.05, **p ≤ 0.01, ns, non-significant.

Figure 6

Mechanism of action of anthocyanin treatment in the MCA-205 tumor model. Ten days after tumor implantation, sterile Dp3-sam/Cn3-sam extract was administered intratumorally. Mice were euthanized 6 hours after injection, and tumors were resected for analysis. (A) Volcano plot showing the upregulated and downregulated genes in tumors treated with purified anthocyanins. (B) GSEA plot illustrating significant pathway activation following anthocyanin treatment, highlighting oxidative stress and immune-related pathways. (C) Representative images and quantification of CD3⁺ T-cell infiltration in tumors treated with H. Sabdariffa anthocyanins. (D) CD31⁺ endothelial cell density, showing reduced vascularization in treated tumors. The data were analyzed via one-way ANOVA with Tukey’s multiple comparisons test. ***p ≤ 0.001, ns, non-significant.