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.
Conclusion:
In conclusion, the novel, validated HP-TLC method simultaneously detects EGCG and CTN in the cocoa extract. Tablets formulated by direct compression are safe as nutraceuticals and hold promise as supplements in palliative cancer therapy.
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1 Introduction
A nutraceutical is defined as a substance derived from food or its components that is used to treat or prevent disease. In 1989, Stephen De-Felice combined the words “nutrition” and “pharmaceutical” to form the word “nutraceutical.” “Nutraceutical is a food, food ingredients, or any dietary supplement that have specific health and medical benefits,” says De-Felice (1, 2). Nutraceuticals have been demonstrated to have antioxidant, anti-inflammatory, anticancer, and lipid-lowering effects (3–5). Theobroma cacao L., widely recognized as cocoa, is not only valued for its culinary uses but also regarded as a medicinal plant; traditionally, the term “cacao” is used to describe the raw components of the Theobroma cacao L. fruit, whereas “cocoa” generally refers to its processed forms, and it boasts an array of secondary bio-actives, including flavonoids, terpenes, alkaloids, and polyphenols (6, 7). Among the natural sources of bioactive compounds, cocoa stands out, containing approximately 12–18% polyphenols by dry weight (8, 9). These polyphenols can be categorized into three groups: flavan-3-ols (37%) (including catechins and epicatechins), anthocyanins (4%), and procyanidins (58%) (10). Remarkably, these compounds contribute to the total antioxidant composition in raw, unfermented cocoa beans (11). Beyond its delightful taste, cocoa harbors two key methylxanthines: theobromine (3.7%) and caffeine (0.2%) (12). These compounds were produced through in vitro fermentation under controlled conditions to make them fat-free (on a fat-free basis) and to avoid any potential incompatibilities. One study evaluated the protective and anticancer potential of cocoa bean extract (CE) in doxorubicin (DOX)-treated mice with Ehrlich ascites carcinoma (EAC). The results showed that CE selectively targets cancer cells, enhances DOX efficacy, reduces organ damage, and improves antioxidant defenses in mice (13, 14).
Among its various applications, the oral route emerges as the preferred method of drug delivery due to its simplicity, patient compliance, and flexible dose forms (15, 16). Tablets, as unit doses containing active components and excipients, dominate conventional medication administration. However, their rapid breakdown in the digestive tract often leads to elevated plasma drug concentrations. Quality by Design (QbD), guided by ICH Q8-(R2) principles, revolutionizes pharmaceutical development. By emphasizing product and process comprehension, risk management, and continuous improvement, QbD proactively prevents issues rather than reacting to them (17, 18). Key principles include sound scientific risk management, design space establishment, and control strategy implementation. Response surface design (RSD), a powerful QbD tool, offers three variants: central composite design (CCD), Box–Behnken design, and optimal design. CCD, widely employed, features cube, center, and axial points for testing and analysis (19, 20). Its orthogonal blocks facilitate independent variable analysis, minimizing regression coefficient variance. In addition, rotational blocks ensure consistent variance prediction for equidistant points from the center. Statistical models with polynomial or quadratic relationships further explore variables and responses. In this study, we explored methods for developing and optimizing a cocoa nutraceutical supplement in tablet form by leveraging QbD principles and advanced pharmaceutical science (21).
The most advanced form of thin-layer chromatography (TLC) is known as high-performance thin-layer chromatography (HP-TLC) (22). Every stage of the procedure, including exact sample implementation, universally reliable chromatogram development, and software-managed evaluation, uses chromatographic layers with the highest separation efficiency. HP-TLC employs a widely recognized methodology based on scientific principles as well as the utilization of approved quantitative and qualitative analytical methods (23). HP-TLC covers all the quantitative measurement requirements of modern analytical laboratories because of its high resolution and precision (24).
2 Methods
2.1 Materials
EGCG (99%) and CTN (96%) analytical standards were obtained from Otto Chemicals, Mumbai. Ethanol was sourced from Merck. Petroleum ether (85%) LR grade, methanol, ethyl acetate, toluene, and formic acid HPLC grade solvents were procured from Thermofischer scientific – Navi Mumbai, India. Lactose monohydrate, croscarmellose sodium, and magnesium stearate were supplied by Hi media- Nashik, India Laboratories. Avicel PH − 101 was provided by Molychem- Badalapur, Maharashtra.
2.2 Collection and authentication of cocoa pods
Cocoa pods were procured from Kadamba Marketing Souharda Sahakari Niyamita (KMSSN) in Sirsi, Uttara Kannada District of Karnataka, India, and authenticated by the taxonomist at ICMR-NITM Belagavi. The herbarium (voucher number: RMRC−1392) was archived at ICMR-NITM, Belagavi, for future reference.
2.3 Extraction and spray drying
Cocoa beans were deshelled, pulverized, and defatted using petroleum ether in a Soxhlet apparatus at 40–60°C. The cycles were repeated until all the fat wax was deposited in the round bottom flask. Subsequently, it was dried at an ambient and dark conditions with 25°C room temperature and pulverized into a coarse powder (25). This powder was sieved through a number 40 sieve to achieve a finer particle size, enhancing extraction efficiency. For extraction, the cold maceration method was obtained using ethanol and water (hydroalcoholic) in a ratio of 80:20 v/v in a conical flask. The mixture was stirred at 100 rpm in an incubator shaker at 37°C for 24 h (26, 27). The liquid extract was filtered, and the filtrate was concentrated using a rota-evaporator (Hei VAP ultimate, Heidolph) at 45°C and 100 rpm. Then, the extract was spray-dried into powder using Spray Mate (Jay Instruments and Systems). The process parameters are shown in Table 1 (28, 29). Physicochemical analysis of extracts, such as loss on drying, total ash, water-soluble ash, acid-insoluble ash, and extractive value, was evaluated, and powder characteristics of the spray-dried CE, such as the angle of repose, Carr’s index, process yield, and hygroscopicity, were also assessed.
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Muppayyanamath, Patil, Patil, Mastiholimath, Hegde, DARASAGUPPE RAMACHANDRA and Roy. Quality by design-based optimization and HP-TLC densitometric standardization of Theobroma cacao L. extract as a nutraceutical supplement. Front. Nutr., Sec. Clinical Nutrition, Volume 12 – 2025
doi: 10.3389/fnut.2025.1537963
