• Comparative Evaluation of Barberry-Derived Honey and Commercial Honey on HT-29 Colorectal Cancer Cells: In Vitro Anticancer Effects
  • Nafiseh Erfanian,1 Sahbasadat Khatami,2 Homayoun Zahedipour,3 Asghar Zarban,4 Hamid Kabiri-rad,5 Saeed Nasseri,6,*
    1. Cellular and Molecular Research Center, Birjand University of Medical Sciences, Birjand, Iran
    2. Cellular and Molecular Research Center, Birjand University of Medical Sciences, Birjand, Iran
    3. Student Research Committee, Birjand University of Medical Sciences, Birjand, Iran
    4. Clinical Biochemistry Department, Faculty of Medicine, Birjand University of Medical Sciences, Birjand, Iran
    5. Cellular and Molecular Research Center, Birjand University of Medical Sciences, Birjand, Iran
    6. Cellular and Molecular Research Center, Birjand University of Medical Sciences, Birjand, Iran


  • Introduction: Colorectal cancer (CRC) remains one of the leading causes of cancer-related mortality worldwide. Despite notable progress in chemotherapy, targeted therapies, and immunotherapy, the clinical outcomes for many patients are still unsatisfactory, often due to limited efficacy and considerable side effects. These challenges underscore the need for safer, complementary therapeutic approaches. Among natural substances, honey has attracted scientific interest due to its diverse composition of bioactive compounds—particularly phenolics, proteins, and antioxidants—which are associated with anti-inflammatory, antimicrobial, and anticancer activities. In this study, we introduce and apply a novel biochemical evaluation system—referred to as the PAD score (Phenolic–Protein–Antioxidant–Diastase)—designed to comprehensively assess the therapeutic quality of honey. The PAD score integrates four critical indicators: total phenolic content (mg gallic acid equivalent), antioxidant capacity (mg Trolox equivalent), protein concentration (mg), and diastase enzymatic activity (measured in diastase number, DN). To facilitate interpretation, honey samples were classified based on PAD scores into six quality tiers: very poor (<100), poor (100–199), moderate (200–299), good (300–399), very good (400–499), and excellent (>500). While these individual biochemical components have been studied separately, to our knowledge, this is the first study to propose a unified scoring system and explore its association with direct anticancer efficacy in a CRC model.
  • Methods: Two honeys with markedly different PAD scores were selected for comparison: a high PAD score honey (HPH), derived from Berberis vulgaris (barberry) flowers, with a PAD score of 543 (classified as excellent), and a low PAD score commercial honey (LPH), with a PAD score below 74 (very poor). Spectrophotometric and enzymatic assays were used to biochemically characterize the honeys, confirming significant differences in their total phenolic content, antioxidant capacity, protein content, and diastase activity. To evaluate anticancer properties, HT-29 colorectal cancer cells were treated with varying concentrations of HPH and LPH over 24, 48, and 72 hours. Cell viability was assessed using MTT assays. Cell migration was examined using a scratch (wound healing) assay. Apoptosis-related gene expression levels (Bax, Bcl-2, Caspase-3) were measured using RT-qPCR. Additionally, flow cytometry analysis was conducted to assess changes in cell cycle distribution and potential cytostatic effects.
  • Results: Biochemical assays confirmed that HPH had significantly higher phenolic content, antioxidant power, protein concentration, and diastase activity than LPH. MTT results revealed that HPH caused a notable reduction in HT-29 cell viability in a dose- and time-dependent manner, with a significantly lower IC50 value compared to LPH. Scratch assays showed that HPH markedly inhibited cell migration; after 72 hours, more than 60% of the wound area remained unhealed in the HPH group, compared to less than 30% in control and LPH-treated cells. Gene expression analysis via RT-qPCR demonstrated that HPH significantly upregulated pro-apoptotic genes (Bax and Caspase-3) while downregulating the anti-apoptotic gene Bcl-2, indicating activation of apoptotic pathways. Flow cytometry data further confirmed that HPH induced cell cycle arrest at the G0/G1 phase, suggesting a combined cytotoxic and cytostatic effect. In contrast, LPH showed only mild or non-significant changes across all assays.
  • Conclusion: Our findings underscore the pivotal role of honey’s biochemical richness in determining its therapeutic efficacy. HPH displayed superior antiproliferative, anti-migratory, and pro-apoptotic effects against HT-29 colorectal cancer cells, mediated through apoptosis induction and cell cycle arrest. These effects were not observed in LPH, reinforcing the importance of biochemical quality in honey-based therapeutic interventions. This study not only introduces the PAD score as a practical and informative index for classifying the medicinal potential of honey but also provides compelling evidence for its relevance in oncology research. We propose the PAD scoring system as a valuable tool for identifying high-quality honeys suitable for use as adjunctive agents in cancer therapy. Further in vivo and clinical studies are recommended to validate these promising findings and support the integration of PAD-based honey evaluation in evidence-based complementary medicine.
  • Keywords: Colorectal cancer, HT-29, Honey, PAD score, Apoptosis, Natural therapy