The beverages with the highest values of overall liking—the E2 formulation in both capsules and tea bags—and therefore the greatest potential to be commercialized as final products underwent In Vitro GID in order to assess the bioaccessibility of their bioactive compounds, comparing the results obtained before digestion with those obtained after the digestion process. In particular, phenolic content through TPC and HPLC analyses (the latter for methylxanthines as well), RSA, and α-glucosidase inhibition capacity were evaluated.
3.3.1. Determination of Polyphenol and Methylxanthine Composition of Undigested and Digested Beverages through RP-HPLC-PDA.
shows the compounds detected in the beverages through liquid chromatography before and after GID (two methylxanthines and nine polyphenols). As far as polyphenols are concerned, phenolic acids (protocatechuic acid and caffeic acid), flavan-3-ols (catechin-3-O
-glucoside, catechin and epicatechin), B-type procyanidins (PCB isomers), and flavonols (quercetin-3-O
-glucoside and quercetin-3-O
-rhamnoside) were identified.
In non-digested beverages (PRE), the most abundant compound was theobromine, a methylxanthine alkaloid that characterizes cocoa (72.34% and 74.89% of the total identified compounds for capsules and tea bags, respectively) followed by caffeine (17.18% and 14.24% of the total compounds identified for capsules and tea bags, respectively), another biologically active alkaloid. The total methylxanthine content was equal to 212.99 mg/L and 171.93 mg/L for capsules and tea bags, respectively. The ratio between these two alkaloids was approximately 5:1, which is in line with the data published by Rojo-Poveda et al. [16
]. Concerning polyphenol composition, capsule and tea bag beverages showed the same profile with PCB2, PCB, and epicatechin as the most abundant compounds. In fact, for the total quantified polyphenols in capsule drinks, PCB2, PCB, and epicatechin were 25.47%, 19.75%, and 18.60% with respect to the total polyphenols quantified, respectively. Likewise, in tea bag drinks, PCB2, PCB, and epicatechin were 24.59%, 21.05%, and 18.45%, respectively.
After GID, the content of methylxanthines remained unchanged in both beverages, as they are stable under gastric and intestinal conditions and not degraded by pH and enzymes. Hence, the 5:1 ratio between theobromine and caffeine was maintained after GID. On the contrary, the concentration of polyphenols changed during GID with capsule and tea bag beverages showing the same behaviour. The total content of polyphenols quantified before GID was 24.95 and 20.99 mg/L for capsule and tea bag beverages, respectively. After GID, these values decreased, reaching a total of 12.45 and 11.56 mg/L with bioaccessibility of 50.00% and 55.46% for capsules and tea bags, respectively. These values represent the amount of soluble and accessible polyphenols not only to be absorbed but also to potentially exert their functions at the intestinal level, such as the ability to inhibit the α-glucosidase enzyme and the anti-inflammatory effects demonstrated by Rossin et al. [28
]. The phenolic acids in both drinks degraded considerably with bioaccessibility that ranged from 37.43–38.21% for protocatechuic acid and 22.17–22.82% for caffeic acid. With regard to flavan-3-ols, in both capsule and tea bag beverages, catechin-3-O
-glucoside degraded partially during GID (bioaccessibility of 47.41% and 55.75%, respectively) while epicatechin degraded almost completely (bioaccessibility of 10.55% and 12.50%, respectively). These results agree with the literature, which reports a poor availability of flavan-3-ols due to their instability in the gastrointestinal environment [13
]. In fact, pH plays a key role in the stability of catechins and catechin glucosides, making them very unstable and rapidly subjected to degradation in neutral or alkaline solutions, whereas they are relatively stable in acidic solution [32
]. Moreover, the high binding capacity of these compounds to digestive enzymes, which entail the polymerization and thus formation of insoluble aggregates, has been broadly reported [13
]. The considerable loss of epicatechin during GID could therefore be explained by its high affinity for such enzymes and the mild alkaline milieu that typifies the intestinal phase. On the other hand, after GID, catechin content in capsule and tea bag beverages increased significantly (p
< 0.01) with a bioaccessibility exceeding 100%. This boost could be derived from three different pathways. First, catechin-3-O
-glucoside may have been hydrolysed in the intestinal phase, releasing aglycone. In fact, as reported by Raab et al. [32
-glucoside shows a consistent degradation starting at pH 7 while it remains stable at low pH values. Hence, since glucoside shows a better resistance to degradation compared to aglycone [32
], glycosylation could increase the bioaccessibility of catechin, which is delivered intact to the small intestine in an absorbable form. The second hypothesis is the epimerization of epicatechin into catechin under acidic conditions, as has been widely reported by many authors [34
]. Lastly, a depolymerization of type B procyanidin with a consequent liberation of free catechin may have occurred, and this result could be confirmed by several studies on polyphenol bioaccessibility after GID [13
]. Finally, regarding flavonols, both quercetin-3-O
-glucoside and quercetin-3-O
-rhamnoside degraded completely after In Vitro GID.
Comparing the beverages obtained with the two different extraction techniques, significant differences (p
< 0.05) were found for protocatechuic acid after GID, catechin before GID, and caffeine and catechin-3-O
-glucoside both before and after GID, which were found to be higher in capsules drink (Table 4
). However, it should be highlighted that, once again, the solid/liquid ratio was different between capsules and tea bags (7 g of preparation in 120 mL of water in capsule beverages and 3 g in 100 mL in tea bag beverages). Thus, the results obtained from HPLC were normalized based on the grams of preparation contained in the capsules and tea bags (Table 5
Considering the results, the tea bag method had a greater efficiency in extracting bioactive compounds (the reasons for which have already been discussed above), including 56.96% more methylxanthines, 63.64% more polyphenols, and 57.66% more total bioactive compounds. In fact, all compounds with the exceptions of caffeine and epicatechin were significantly higher in tea bags than capsules (p < 0.05).
Considering the quantity that may be consumed for one cup of each beverage (120 mL for capsules and 200 mL for tea bags), the dose intake of theobromine and caffeine would be 20.65 mg and 4.91 mg for capsules and 28.87 mg and 5.51 mg for tea bags, respectively, which also represents the potential amount available to be absorbed into the bloodstream. On the other hand, the dose intake of total polyphenols detected in this study would be 2.99 mg for capsules and 4.20 mg for tea bags with a potential post-GID availability of 1.49 mg and 2.31 mg, respectively. The U.S. Food and Drug Administration (FDA) has established 400 mg of caffeine per day in adults as a dose not generally related to dangerous and negative effects; however, this is contingent on individual sensitivity to the alkaloid and how fast it is metabolized [37
]. Likewise, the European Food Safety Authority (EFSA) states that caffeine intake from all sources up to 400 mg per day (about 5.7 mg/kg body weight (bw) per day for a 70 kg adult) does not give rise to safety concerns for healthy adults with the exceptions of pregnant and lactating women, adolescents, and children, for whom the EFSA sets a limit of 200 mg per day (approximately 3 mg/kg bw per day) [38
]. As for theobromine, firm conclusions have not been drawn yet. In fact, while in clinical studies of with a three- to four-week duration, dose levels of 150 mg theobromine/day (1.5–2.1 mg/kg bw) were well tolerated and adverse effects (such as nausea, vomiting, headache and diarrhoea) were only observed from doses higher than 500 mg theobromine/day, an actual level of no safety concern in humans has not yet been identified; however, the EFSA suggests that it is probably higher than 150 mg/day [39
]. Based on these findings, the EFSA decided to derive its reference dose from the caffeine data since the results of pharmacokinetics studies of caffeine and its metabolites suggest that about 11% of caffeine oral intake is converted into theobromine and the two substances show a similar pharmacological profile [39
]. Moreover, although the pharmacological effects of caffeine and theobromine can overlap, the latter shows a much lower potency than caffeine with respect to effects on the central nervous system, kidneys, or heart [39
]. Therefore, the EFSA predicts a level of 0.6 mg/kg bw per day for healthy adults and 0.3 mg/kg bw per day for pregnant and lactating women, adolescents, and children to be of no safety concern [39
] but also suggests that exceeding these doses would not necessarily result in a health risk. In view of this, considering that their values of caffeine and theobromine are lower than the recommended doses, both capsule and tea bag beverages could be considered safe for human health and are not expected to cause negative effects generally associated with overconsumption of these two methylxanthines, such as insomnia, anxiousness, tachycardia and nausea [37