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Introduction
Consumers’ affluence and increasing prevalence of lifestyle-related diseases, such as obesity and diabetes, encourage the consumers to selectively consume beverage products that support healthy lifestyles and potentially reduce the risk of chronic diseases. This group of modern consumers begins to pay attention to the nutraceutical properties of their beverages in addition to the nutritional ones. Chocolate, as a food or confectionery product, has been widely enjoyed by consumers, and therefore was extensively studied in the food science and nutrition domains. However, cocoa or chocolate-flavored beverages were comparatively less studied. Cocoa or chocolate-flavored beverage is a popular beverage preparation of the ground, roasted cocoa beans 1. It is commonly enjoyed in different formats by consumers in various geographical regions and cultures. The results from several meta-analyses suggested health benefits from the consumption of cocoa 2-4. However, little is known about the possible health benefits of cocoa beverage consumption. Numerous studies had associated antioxidant capacity of plant- based food, like fruits and vegetables to their flavonoid and polyphenol contents 5-7. Similar to cocoa and other plant-based food products, the nutraceutical properties of cocoa beverages are likely contributed by their innate phytochemicals, such as flavonoids and polyphenols, via antioxidant and anti-inflammatory activities 7,8. The flavonoid, polyphenol contents, antioxidant capacity, and anti-inflammatory activity of commercial cocoa beverage products are relatively unknown.
The food additives are a group of substances added to the food and beverage products to preserve flavor or enhance their tastes, appearances, stability, and other qualities, in order to meet the consumers’ requirements. Recent studies had linked food additives, like preservatives and colorants, to detrimental physiological events 9,10. Limited data are available on the uses of food additives in the manufacture of commercial cocoa beverage products. The effects of food additives on flavonoid, polyphenol contents, antioxidant capacity, and anti-inflammatory activity of commercial cocoa beverage products are also relatively unknown.
Cocoa beverage products are predominantly sold in the form of powder premix in Singapore. The study examined the presence of food additives in these commercial cocoa beverage premix products. The same study also evaluated if the presence of food additives affects the total flavonoid, polyphenol contents, antioxidant capacity, cellular antioxidant and anti-inflammatory activity of these beverage products.
Materials and Methods
Chemicals and Materials
F2-isoprostanes-d4, F2-isoprostanes, leukotriene B4 (LTB4), leukotriene B4-d4, and arachidonic acid (AA) were purchased from Cayman Chemical (Ann Arbor, MI, USA). Glucose, dextran 500, sodium carbonate, Folin–Ciocalteu’s reagent, gallic acid, aluminium chloride, quercetin, 1,1-diphenyl-2-picrylhydrazyl radical (DPPH), vitamin C, phorbol 12-myristate 13-acetate (PMA), calcium ionophore, trypan blue, phosphate-buffered saline (PBS), pyridine, toluene, isooctane, hydrogens peroxide (50% by volume), guaiacol, xylenol orange, ammonium ferrous sulfate, 2,2’-Azobis(2-methylpropionamidine) dihydrochloride (AAPH), 2,3,4,5,6-pentafluorophenylbromide, and bis(trimethylsilyl)trifluoroacetamide were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile, ethyl acetate, methanol, ethanol, and sulfuric acid were purchased from Tedia (Fairfield, OH, USA). Ficoll-paque was purchased from GE Healthcare (Uppsala, Sweden).
Food Additive Profiling
All the cocoa beverage powder premix products found on the shelves of ten randomly selected local supermarkets were included in this study. The supermarkets were selected through cluster sampling based on the geographical locations (two from The Central, North, South, East, and West of Singapore). The name of each beverage item and the food additives used in its production declared on the product label were recorded by trained research personnel.
Total Polyphenol, Total Flavonoid, and Free Radical Scavenging Capacity Analysis
The nutraceutical contents of the commercial cocoa beverages were determined by measuring their total flavonoid, polyphenol contents and DPPH radical scavenging capacity. The cocoa beverage premix powder (10 g) was mixed with deionized water (10 mL) before extracting with 100% ethanol (2 x 5 mL). The ethanolic extract was topped up to 10 mL to give a final concentration of 1 g/ mL.
The total polyphenol content of the ethanolic extract was determined by a modified Folin-Ciocalteu assay 11. The total polyphenol content was expressed as mg gallic acid equivalents (GAE)/ 100g beverage. The total flavonoid contents were measured by a modified Dowd colorimetric method 12. The total flavonoid content was expressed as the quercetin-equivalence in 100 g beverage.
The radical scavenging capacity of the ethanolic extract was determined using DPPH radical scavenging assay 13. The radical scavenging results were expressed in mg vitamin C/ 100 g.
Cellular Antioxidant and Anti-Inflammatory Studies
Human neutrophils were isolated from the neutrophil/ erythrocyte pellet of fresh human whole blood after Ficoll-Paque gradient centrifugation and dextran sedimentation of red blood cells 14. The whole human blood was obtained in kind from the study researchers, as such human ethics approval is not required. The freshly isolated neutrophils were resuspended in phosphate-buffered saline at a concentration of 5×106 cells mL-1. Cell viability was assessed using trypan blue exclusion and was typically >98%.
The antioxidant and anti-inflammatory properties of the cocoa beverages were evaluated by measuring their abilities to inhibit the productions of F2-isoprostanes (marker of oxidative stress), LPO (lipid hydroperoxides, marker of lipid oxidation), LTB4, and MPO (myeloperoxidase) activity (markers of cellular inflammation) from freshly-isolated human neutrophils. Briefly, the freshly-isolated human neutrophils (5×106 cells/ mL in PBS, 1mL) were incubated with cocoa beverage ethanolic extract (10mL, final concentrations, 10 mg/ mL) and AA (final concentration, 10 mmol/ L) at 37 °C for 5 minutes before stimulation. The neutrophils were incubated with PMA (final concentration, 200 nmol/ L) at 37 °C for 15 minutes to stimulate the F2-isoprostanes production. For LPO productions, the neutrophils were stimulated with AAPH (final concentration, 5 mmol/ L) at 37 °C for 15 minutes. The neutrophils were incubated with calcium ionophore (final concentration, 200 nmol/ L) at 37 °C for 15 minutes to stimulate the cellular production of LTB415. Positive control experiments were performed by incubating neutrophils with AA before activating with either PMA, AAPH, or calcium ionophore. Negative control experiments were carried out by incubating neutrophils with AA only. The supernatant from the cell suspension was collected and stored at -80 °C before F2-isoprostanes, LPO, and LTB4 analysis. F2-isoprostanes and LTB4 were quantified using stable isotope-labeled Gas Chromatography-Mass Spectrometry 11, 15. The formation of LPO was quantified using the Ferrous Oxidation-Xylenol Orange assay 16.
To examine the effects of the cocoa beverages on MPO activity, freshly isolated neutrophils (1 x 106 cells/ mL in PBS) were incubated with their ethanolic extracts (final concentrations, 10 mg/ mL) for 5 minutes at 37 °C before the incubate was removed. The neutrophils were resuspended in fresh PBS and lyzed by sonication. Untreated neutrophils were used as positive controls. Functional MPO activity was determined by measuring its catalytic action on the oxidation of guaiacol in the presence of hydrogen peroxide 17.
Statistical Analysis
Statistical analyses were performed using IBM SPSS Statistics version 26.0 (USA). Data were presented as mean ± standard deviation (SD). Differences between two groups were compared using two-sample independent t-tests. A significant difference was observed when p<0.05.
Results
Food Additive Profiling
Twenty five cocoa beverage powder premix products were included in this study. 28% of the studied cocoa beverages were free from food additives. Flavorings are the most prevalent additives present in 60% of these beverages. They were followed closely behind by antioxidants (56%), pH regulators (40%), and emulsifiers (36%). Colorings (4%) were less commonly added, and preservatives were totally absent. The specific antioxidants, colorings, emulsifiers, flavorings, and pH regulators were not stated on the product labels.
Total Polyphenol, Total Flavonoid, and Free Radical Scavenging Capacity Analysis
Significant concentrations of flavonoids (13.27±1.08 mg quercetin/ 100 mL) and polyphenols (137.8±14.0 mg GAE/ 100 mL) were present in the studied cocoa beverage products. The additive-free cocoa beverage products contained significantly lower concentrations of flavonoids and polyphenols than those with food additives (Figure 1a,b). After stratifying to different food additive groups, the presence of flavorings, pH regulators, and emulsifiers did not influence the concentrations of flavonoids and polyphenols in the cocoa beverages. Cocoa beverages with added antioxidants contained significantly higher concentrations of flavonoids and polyphenols than those without the designated additives (Figure 1a,b). Correspondingly, the presence of food additives and antioxidants in cocoa beverages significantly increased DPPH radical scavenging capacity (Figure 1c). The other food additives did not influence DPPH radical scavenging activity.
Cellular Experiments
The F2-isoprostanes, LPO, LTB4, and MPO activity inhibitory actions of cocoa beverage ethanolic extracts in human neutrophils are presented in Figure 2. The inhibitory activity was expressed as the percentage reduction in F2-isoprostanes, LPO, LTB4, and MPO activity compared to the untreated positive controls. None of the negative controls showed measurable F2-isoprostanes, LPO, and LTB4 productions.
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Figure 1: Total (a) flavonoid (mg quercetin/ 100mL), (b) polyphenol (mg gallic acid equivalent/ 100mL) contents, and (c) diphenyl-2-picrylhydrazyl free radical scavenging activity. |
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Figure 2: Inhibition (%, mean±SD) of (a) F2-isoprostanes, (b) lipid hydroperoxides, (c) leukotriene B4 formations, and (d) myeloperoxidase activity. |
The cocoa beverage ethanolic extracts decreased the cellular formations of F2-isoprostanes, LPO, LTB4, and MPO activity of the freshly isolated human neutrophils (Figure 2a-d). The presence of food additives in the cocoa beverages inhibited LTB4 formation by activated human neutrophils to significantly greater extents when compared to the cocoa beverages without food additives (Figure 2c). The formation of F2-isoprostanes, LPO, and MPO activity were unaffected by the presence or absence of food additives. After stratifying to the different food additive types, the amounts of inhibition of F2-isoprostanes, LPO, and LTB4 formation in the freshly isolated human neutrophils were significantly increased by the presence of antioxidants (Figure 2a(ii), 2b(ii), and 2c(ii)) Added flavorings, pH regulators, and emulsifiers did not exhibited significant influence on the measured cellular activities.
Discussion
Nearly three-quarters of the commercial cocoa beverage powder premix products contained food additives. Consumers are avoiding products with food additives, especially food colorants and preservatives, as recent studies reported that these food additives may pose undesired health effects 9,10. It becomes important for food manufacturers to justify, try to avoid or reduce the uses of food additives. Flavorings and antioxidants were the more prevalent food additives added into more than half of the cocoa beverages. Cocoa beverages offers unique flavor and aroma profiles that enable the easy identification. Flavorings are presumably used in cocoa beverage products to restore the flavors lost during the manufacturing processes and/or to enhance the original flavors. They may also be used to mask the undesirable roasted cocoa flavor. Antioxidants were likely added to prevent oxidation and photooxidation, to achieve shelf-life stability, and to maintain or enhance flavor, aroma, and color 18. Stabilization of the beverage rheological property is an important process during the manufacture of the cocoa beverages, as evidenced by the presence of emulsifiers in the cocoa beverages. The pH regulators were likely added into the cocoa beverages to maintain a mildly acidic pH of 5-6 19,20 to improve shelf-life stability and to reduce the alkalinity or bitter taste profiles. Color preservation was more likely achieved through the uses of antioxidants, in place of colorants. This was evidenced as less than 5% of the cocoa beverages had added colorings. The cocoa beverages have their unique natural colors, which have been universally accepted by their consumers. Adding food colorants into these beverages offers limited advantages to the sensory attributes of the final products. The absence or low prevalence of added colorings and preservatives are considered as welcome news to the consumers as recent studies had shown deleterious physiological effects of commonly used food colorants and preservatives 9,10.
The ethanolic extracts of the cocoa beverages inhibited the formations of the markers of oxidative stress (F2-isoprostanes and LPO) and inflammation (LTB4 and MPO activity) by freshly isolated human neutrophils. The results of the DPPH radical scavenging activity, cellular inhibition of F2-isoprostanes, and LPO productions affirmed that these commercial cocoa beverages are likely to exert antioxidant property. Those from the MPO activity assay and LTB4, cellular productions suggested that these beverages also exert anti-inflammatory activity. Cocoa possesses unique phytochemical compositions, especially the flavonoids and polyphenols 21, which might contribute to the demonstrated antioxidant and anti-inflammatory activities 7. The study results showed that the commercial cocoa beverage premix products contained significant concentrations of flavonoids and polyphenols. The innate flavonoids and polyphenols in cocoa were previously shown to be retained in their beverages 21, albeit of losses due to handling and processing. Therefore, the cocoa beverages may offer flavonoid- and polyphenol-associated nutraceutical benefits beyond the conventional macro- and micronutrients 7. The study demonstrated that the food additives altered the antioxidant and anti-inflammatory properties of the cocoa beverages, dependent of the flavonoid and polyphenol contents. After stratification based on the types of food additives, similar significant difference in DDPH and cellular results were demonstrated only between cocoa beverages with and without added antioxidants. The added antioxidants may have prevented against the degradation of the innate cocoa flavonoids and polyphenols during the manufacturing processes 22 and thereby indirectly preserve the potential antioxidant and anti-inflammatory capacities 22. Technically, the added antioxidants might directly contribute to the antioxidant and anti-inflammatory properties. The flavonoid and polyphenol molecules are required to be easily oxidized to act as effective antioxidant 23 or possibly anti-inflammatory agents 24. Unlike the antioxidants which are likely to stabilise the flavonoid and polyphenol molecules via oxidative-reductive mechanisms, emulsifiers and pH regulators may instead form relatively stable complexes with the flavonoid and polyphenol molecules 25,26, and make the latter less susceptible to oxidation and consequently decrease their antioxidant and anti-inflammatory efficacies.
The results and its accompanying discussion, although limited by the observational nature of the study, provided crucial information on the antioxidant and anti-inflammatory activities of real commercial cocoa beverage products, and the effects of food additives on these activities. More cellular, animal or human studies are required to ascertain the effects of food additives on the antioxidant and anti-inflammatory activities offered by these flavonoid- and polyphenol-rich beverages. Mechanistic studies are also required to elucidate the effects of specific food additives on their nutraceutical properties.
Conclusion
The commercial cocoa beverage powder premix products contained significant concentrations of flavonoids and polyphenols, and may offer antioxidant and anti-inflammatory benefits. The uses of food additives may exert profound and differential effects on the antioxidant and anti-inflammatory activities of cocoa beverages.
Acknowledgements
Nil.
Funding Sources
The authors received no financial support for the research, authorship, and/ or publication of this article.
Conflict of Interest
The authors declare no conflict of interest.
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