Quality by design-based optimization and HP-TLC densitometric standardization of Theobroma cacao L. extract as a nutraceutical supplement

Atith Muppayyanamath ^1,2* Priyanka P Patil ^1,2 Vishal S Patil ¹ Vinayak Mastiholimath ^2* Harsha V Hegde ¹ HARISH DARASAGUPPE RAMACHANDRA ^1* Subarna Roy ¹

• ¹ National Institute of Traditional Medicine, Indian Council of Medical Research, Belgaum, India
• ² KLE College of Pharmacy, Belgaum, India

Background: Our previous studies identified the hydroalcoholic extract of defatted Theobroma cacao L. bean (CE) as a cancer-preventive and protective agent against chemotherapeutics like doxorubicin-induced organ toxicities, namely heart, liver, and kidney injuries. In this study, we developed an orally administrable nutraceutical tablet formulation from CE and evaluated its physico-chemical properties.Methods: An analytical method for phytochemical standardization was developed and acute oral toxicity was studied in female wistar rats following OECD 423 guidelines. Briefly, the CE was extracted using an 80:20 alcohol-water (% v/v) mixture through cold maceration. Subsequently, spray drying was employed to obtain powdered CE. Utilizing a Quality by Design (QbD) approach with Design Expert (DoE) software, we optimized CE tablets via direct compression. The central composite design (CCD) included 5 center points, with Avicel PH-101 and Croscarmellose sodium (CCS) as factors, measuring disintegration time, hardness, and friability.Results: Among the 13 formulations, batch F-9 emerged as the optimized one within the design space, containing 35% Avicel PH-101 and 5% CCS. The optimized formulation exhibited a disintegration time of 5.2 minutes, hardness of 4.2 kg/cm 2 , and friability of 0.34%. Importantly, no toxic effects were found at 2,000 mg/kg in the acute oral toxicity study. CE contains vital bioactive polyphenols, including (-)-Epigallocatechin-3-gallate (EGCG) and (+)-Catechin (CTN). We developed a marker-based HPTLC densitometric analysis using a mobile phase of 9:9:2 v/v [ethyl acetate: toluene: formic acid], which revealed CTN at Rf 0.49 and EGCG at Rf 0.23. This method was validated according to ICH requirements.In conclusion, novel validated HP-TLC method detects EGCG & CTN simultaneously in cocoa extract. Tablets formulated by direct compression are safe as nutraceuticals and hold promise as supplements in palliative cancer therapy.