Bioactive compounds and antioxidant potential for polyphenol-rich cocoa extract obtained by agroindustrial residue

Abstract Processing of cocoa (Theobroma cacao L.) beans responsible for agricultural exports leads to large amounts of solid waste that were discarded, however, this one presents high contents of metabolites with biological activities. The major objective of this study was to valorise cocoa agroindustrial residue obtained by hydraulic pressing for extract rich in antioxidants. For it, the centesimal composition of residue was investigated, the green extraction was carried out from the residue after, the bioactive compounds, sugar contents and screaming by HPTLC were quantified for extract. The extract has a total polyphenol content of 229.64 mg/g and high antioxidant activity according to ABTS 225.0 μM/g. HTPLC analysis confirmed the presence in the extract, residue of terpenes, sesquiterpenes, flavonoids and antioxidant activity. These results, as a whole, suggest that the extract from the cocoa residue has interesting characteristics to alternative crops with potential industrial uses.


Introduction
Brazilian economy is largely sustained by the agri-food sector, which accounts for 6% of gross domestic product and for about 38% of all exports, and in this context, coffee and cocoa (Theobroma cacao L.) are the major agricultural commodities of the country (Seyffarth 2015). Cocoa secondary metabolites include polyphenols which exhibit beneficial effects against cancer, oxidative stress, neurodegenerative diseases (Hu et al. 2016) and antioxidant activities (Belmokhtar and Harche 2014). Residual co-products after obtaining cocoa dry bean are composed mainly of three fractions namely, pod husk, bean shells and mucilage, which are often underexploited and considered an undesirable waste of cocoa/chocolate industry (Martínez et al. 2012). One way to lower costs of these activities is to recycle materials, reduce losses and possible environmental damage and obtain matrices with excellent properties (Gabbay Alves et al. 2017). Therefore, the aim of this study was to valorise extract cocoa residue for its potential industrial use in order to add value to the production chain and cost savings.

Results and discussion
The HPTLC illustrated in Figure 1 shows that the 60:30:10 (v/v/v) dichloromethane : acetone: formic acid mobile phase was suitable for carrying out the chromatographic fingerprinting (Table S1), being able to separate the compounds of interest. In particular, the panel A of this figure highlights the presence, in all the extract samples, of compounds with retention factor (RF) values of 0.2 and 0.7-0.8, which are suggestive of terpenes and sesquiterpenes, respectively, while green fluorescent bands evident in panel B, with RF values of 0.4 and 0.5, may be ascribed to flavonoids (Wagner and Bladt 2001;Caprioli et al. 2016) other than rutin. Neither blue fluorescent bands with RF (0.5-0.65) after staining with NP/PEG ( Figure 1C) or orange pigment bands ( Figure 1D) were observed in the extract, indicating the absence of tannins (Riffault et al. 2014) and alkaloids (Wagner and Bladt 2001), respectively. On the other hand, Figure 1E shows the presence of yellow bands with positive RF (0.4), which are similar to those revealed with NP/PEG (flavonoids). According to Hosu et al. (2015), the intensity of these yellow bands would be directly proportional to the antioxidant potential.
The chemical composition of residue listed in Table S2 shows amounts of nutrients, in particular lipids and carbohydrates, which suggest its promising reuse as raw material. These values are not so far from those reported for unroasted cocoa beans from Ghana and Ecuador (Torres-Moreno et al. 2015), and for cocoa beans from Cameroon (Caprioli et al. 2016) pointing out that bean pressing did not exert any significant influence on waste composition. The polyphenol content was much higher than those of cocoa powder (45.3-60.2 mg/g), dark chocolate (11.7-14.9 mg/g), (Othman et al. 2007), hexane (44.94-70.9 mg/g) (Carrillo et al. 2014) and methanolic (14.41-27.74 mg/g) extracts (Hu et al. 2016) of cocoa beans. This result suggests that this extraction is efficient and the beans pressing was crucial for the production of an extract with high content of polyphenols. The flavonoid content obtained in this study was lower than those of methanolic extracts of cocoa bean (7.19-18.97 mg/g) (Hu et al. 2016). The content of condensed tannins obtained in the present study is similar to those reported for cocoa liquor (4.75 mg/g) and higher than those obtained for milk chocolate (up to 1.00 mg/g) (Belščak et al. 2009). The antioxidant activity of the extract measured by the ABTS method was similar to that observed for cocoa powder extracts (241.1-252.3 μM/g) (Todorovic et al. 2015), higher than those of extracts of cocoa bean shell, cocoa mucilage and cocoa pod husks obtained using either ethanol (2.56-2.89, 2.48-2.56 and 23.03-24.13 μM/g, respectively) or methanol : acetone (4. 45-4.56, 4.10-4.17 and 37.97-42.93 μM/g, respectively) (Martínez et al. 2012). The antioxidant activity measured by the DPPH method was higher than those reported for ethanol extracts of cocoa beans (1.57-1.71 μM/g) (Martínez et al. 2012), but well lower than those of cocoa co-products such as milk chocolate (13-14.9 μM/g), dark chocolate (54.7-90.9 μM/g), cocoa powder (236.5-242.3 μM/g) (Todorovic et al. 2015), unroasted (298 mM/g) and roasted (173 mM/g) cocoa beans, natural cocoa liquor (268 mM/g), alkalised cocoa liquor (112 mM/g) and cocoa powder (110 mM/g) (Gültekin-Özgüven et al. 2016). Such variability of results not only may have been related to the solvent type and extraction conditions, but also suggests that bean pressing could have concentrated the main compounds responsible for the antioxidant activity it in the residue.

Experimental
See supplementary material.

Conclusion
The cocoa residue contained large amounts of dietary components, especially lipids and carbohydrates. The extract obtained from this waste showed high contents of total polyphenols (229.64 mg/g). Its antioxidant activity, according to both the ABTS (225 μMTrolox/g) and DPPH (6.74 μMTrolox/g), was quite high. The HPTLC confirmed the presence of terpenes, sesquiterpenes, flavonoids and antioxidant activity, which suggests that the green extraction and the beans pressing were crucial for the production of an extract rich in antioxidants that could be industrially exploited for active pharmaceutical use.

Disclosure statement
No potential conflict of interest was reported by the authors.