Evaluation of Cytoplasmic and Stromal Components in Selected Skin Diseases with Annatto Seed-pigment Crude Extract as Counterstain
Oraedu Felix Ifeanyichukwu *
Department of Histopathology/Cytology, Faculty of Medical Laboratory Science, Rivers State University, Port Harcourt, Nigeria.
Ndubuka Gideon Ihebuzo
Departments of Biomedical Technology and Anatomy, Federal University of Technology Owerri, Nigeria.
Godam Elvis Tam
Department of Human Anatomy, Rivers State University, Port Harcourt, Nigeria.
Charles Confidence Nkesichi
Department of Histopathology/Cytology, Rivers State University, Port Harcourt, Nigeria.
*Author to whom correspondence should be addressed.
Abstract
Background: Interest in natural dyes has increased because of their biodegradability, low toxicity, and widespread availability. Certain components of natural dyes, such as the polyphenols present in Bixa orellana (annatto), exhibit a high affinity for denatured and inflammatory proteins, a characteristic that enhances their binding to diseased tissues.
Aim: This study aimed to evaluate the cytoplasmic and stromal components of diseased skin tissues using crude annatto seed-pigment extract.
Study Design: This was an experimental study. Crude extracts prepared using 95% ethanol and acetone as solvents were compared with the conventional eosin counterstain. The extracts were applied to skin tissue sections with three established histopathological diagnoses: chronic granulomatous inflammation, lichen planus, and chronic spongiotic dermatitis.
Methodology: Pigment from Bixa orellana was extracted with 95% ethanol and acetone using the maceration method. One hundred grams of seeds were soaked in 200 mL of 95% ethanol, and another 100 g were soaked in 200 mL of acetone at 25 °C for 72 hours. A total of 90 archived skin tissue blocks were used: 30 each of chronic granulomatous inflammation, lichen planus, and chronic spongiotic dermatitis. The phytochemicals of interest were determined using a UV-visible spectrophotometer, and the stability of the extracts was evaluated. The extracts were used to stain the tissues and were compared with the standard eosin counterstain.
Results: Spectrophotometric screening revealed polyphenols (~67 mg/mL) and carotenoids (~27 mg/mL). Both extracts remained stable over eight weeks, with minor fluctuations in absorbance. The 95% ethanol extract consistently outperformed the acetone extract in cytoplasmic and stromal definition (P < 0.05). However, standard haematoxylin and eosin provided significantly superior nuclear clarity, contrast, and overall morphological preservation compared with both extracts (P < 0.001).
Conclusion: The study showed that annatto seed-pigment extract can demonstrate cytoplasmic and stromal components in selected diseased skin tissues when used with Mayer’s haematoxylin.
Keywords: Bixa orellana, natural counterstain, cytoplasmic staining, stromal staining, haematoxylin, eosin, histopathology, skin diseases, plant-derived dyes