Abstract

Histopathological staining is essential for the assessment of tissue morphology, with hematoxylin–eosin (H&E) staining being the most widely used technique. However, eosin poses several limitations, including carcinogenic potential, tissue damage, flammability, and environmental hazards. Natural pigments derived from Rhodomyrtus tomentosa (karamunting), Melastoma malabathricum (senduduk), and Daemonorops draco (jernang) fruits represent promising safer alternatives due to their richness in chromogenic compounds. This study aimed to evaluate the potential of these fruit extracts as substitutes for eosin in H&E histopathological staining. A true experimental design was employed, comprising qualitative phytochemical screening, gas chromatography–mass spectrometry (GC–MS) profiling, extract pH analysis, and histopathological staining of pancreatic tissue obtained from diabetic white rats (Rattus norvegicus). Staining performance was assessed using extract concentrations of 70%, 80%, and 90%, with conventional H&E staining as a positive control and unstained sections as a negative control. Phytochemical analysis revealed that senduduk extracts contained flavonoids, anthocyanins, and tannins; karamunting extracts contained flavonoids, anthocyanins, alkaloids, saponins, and tannins; and jernang extracts contained flavonoids, anthocyanins, and tannins. GC–MS analysis identified 19 metabolites in senduduk, 27 in karamunting, and 4 in jernang extracts. All extracts exhibited acidic pH values. Among the tested conditions, the 90% karamunting extract produced the most satisfactory staining, yielding a distinct reddish coloration of the cytoplasm and connective tissue with adequate contrast to delineate pathological alterations, whereas other extracts and concentrations resulted in suboptimal staining quality. To our knowledge, this is the first study to comparatively evaluate the staining performance of these three natural fruit extracts on pancreatic tissue from diabetic rats using an integrated phytochemical, metabolomic, and pH profiling approach. The findings indicate that a 90% karamunting fruit extract has potential as a natural substitute for eosin in H&E histopathological staining.

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