Method Development for Determination of Cyanidin-3-O-Glucoside Level in Combination of Roselle and Butterfly Pea Flower Extracts by TLC-Densitometry and its Correlation with Antidiabetic and Antioxidant Activities

Background: Roselle flowers (Hibiscus sabdariffa L.) and butterfly pea flowers (Clitoria ternatea L.) are known for their antioxidant and antidiabetic activities. The combination of these flowers has the potential to be developed into a phytopharmaceutical. Cyanidin-3-O-glucoside (Cy3G's), a key compound in both flowers, is believed to play a significant role in these biological activities. However, the development of a reliable analytical method to detect this compound is essential for ensuring the quality of raw materials. Objective: This study aims to develop and validate a TLC-Densitometry method for determining Cy3G's levels in a combination of roselle and butterfly pea flower extracts, and to correlate the results with their antioxidant and antidiabetic activities. Methods: The research was conducted in three main stages: 1) Extract preparation using the maceration method, 2) Development and validation of a TLC-Densitometry analytical method for Cy3G's identification and quantification, and 3) Antioxidant activity testing using the DPPH method and antidiabetic activity testing based on the inhibition of the alpha-glucosidase enzyme. Results: The developed TLC-Densitometry method, using a mobile phase of n-butanol, acetic acid, and water (4:1:5 v/v/v), was validated and met the required parameters of specificity, linearity, accuracy, and precision. Results showed that butterfly pea flower extract had the highest Cy3G's content, followed by the combination extract and roselle extract. The strongest antioxidant activity was observed in butterfly pea flower extract (IC50 0,0979 mg/mL), categorized as strong, while the combination extract showed moderate antioxidant activity (IC50 0,1101 mg/mL). However, antidiabetic activity in all samples was weak. Conclusion: The developed TLC-Densitometry method can be used for determining Cy3G's levels in a combination of roselle and butterfly pea flower extracts. Butterfly pea flower extract demonstrated the greatest antioxidant potential, while antidiabetic activity was relatively weak across all samples.