Comprehensive evaluation of phenolic profile in dark chocolate and dark chocolate enriched with Sakura green tea leaves or turmeric powder

Recently, a huge number of studies have confirmed the important role of chocolate polyphenols in human health, underlining its beneficial effects especially in the treatment of cardiovascular diseases. However, a thorough evaluation of chocolate phenolic profile is still lacking. This study aimed at a comprehensive characterization of dark chocolate phenolic profile, using non-targeted mass spectrometry identification. This approach allowed a tentative identification of 158 individual phenolic compounds: 67 were newly detected in dark chocolate, among these 38 were observed for the first time in chocolate as well as in cocoa beans or products. Ellagitannins, which have never been reported in cocoa or chocolate, represented about the 10% of the phenolic profile of dark chocolate. The enrichment of dark chocolate with Sakura green tea leaves or turmeric powder influenced and modified the phenolic profile, resulting in a phenolic concentration increase. In this way, this functional chocolate might maximize the beneficial effect of chocolate consumption, combining the positive health effects of chocolate, turmeric and green tea and, at the same time, reducing the amount of sugars and calories introduced with chocolate.


Introduction 18
Western lifestyle built-around a highly refined diet rich in saturated fat and sugars but low in 19 complex plant carbohydrates, phytochemicals and vitamins is a hot research topic in the field of 20 nutrition. It is widely known that diet is the cause of many pathogenic age-related conditions. The 21 intake of certain dietary components is plays an essential role in the prevention or management of 22 these diseases (Del Rio et al., 2013). Increasing interest has pointed to naturally occurring 23 compounds, which have been considered non-nutritive for a long time. Polyphenols are a 24 representative class of these compounds and can be summarised into several groups, i.e. 25 hydroxybenzoic acids, hydroxycinnamic acids, flavan-3-ols, flavonols, flavones, flavanones, 26 isoflavones, anthocyanins, ellagitannins, stilbenes, and lignan. They occur in all fruits, vegetables, 27 nuts, seeds, flowers, bark, beverages and processed food. As reviewed by Wollgast, & Anklam 28 (2000a;2000b) polyphenols are characterised by several beneficial effects including anti-29 carcinogenic, anti-atherogenic, anti-inflammatory, immunomodulating and vasodilatory activities. 30 They can exert their protective effects through several mechanisms such as plasma cholesterol 31 reduction, modulation of lipid and lipoprotein metabolism, modulation of enzymes (phase I and 32 phase II) and apoptosis as well as their activity against reactive oxygen species (Del Rio et al., 33 2013). 34 Cocoa (Theobroma cacao) is known as a rich source of dietary phenolic compounds. Cocoa-derived 35 products such as dark chocolate are widely studied for their beneficial effects ascribed to 36 polyphenols. There is good evidence to suggest that cocoa derived polyphenols may have beneficial 37 effects on cardiovascular disease risk factors (Del Rio et al., 2013). Short-term dark chocolate 38 intake has been shown to reduce blood pressure in hypertensive subjects, to improve endothelial 39 function and insulin resistance as well as to inhibit platelet activation (Del Rio et al., 2013). As 40 reported by Rusconi, & Conti (2010), cocoa beans are characterised by phenolic compounds of the 41 flavan-3-ol group (catechin, epicatechin, gallocatechin and epigallocatechin) comprising oligomeric 42  (Hvattum, & Ekeberg, 2003;Waridel et al., 2001). The Therefore, compound 19.2 was speculated to be eriodictiol-C-hexoside-7-O-hexoside isomer (De 320 Beer at al. 2012;Hvattum, & Ekeberg, 2003). The negative ionization mode of compound 32.3 321 exhibited a [M-H]precursor ion at m/z 435.1376, with MS 2 product ions at m/z 345 and 315, losing 322 90 and 120 amu, respectively. This fragmentation pattern has been previously described for 323 phloretin-C-hexoside isomer (Kazuno, Yanagida, Shindo, & Murayama, 2005 tentatively identified as ellagic acid-galloyl-hexoside (Teixeira,Bertoldi,Lajolo,Mariko,& 337 Hassimotto, 2015) 338 339

Phenolic compounds in chocolate 360
Table 2 provides information about the amount of the 158 tentatively identified phenolic 361 compounds in the different types of chocolate. In order to quantify the amount of total phenolic 362 compounds in chocolates, seven calibration curves were prepared with the available authentic 363 standards: epicatechin, coumaric and ferulic acids, quercetin-3-O-rutinoside, ellagic acid, 364 protocatechuic acid and curcumin. In all cases, the linearity was better than 0.99. The other 365 compounds, for which no commercial standards were available, were tentatively quantified using 366 the standards with similar structural characteristics and considering the functional groups that may 367 affect the ionisation properties. As shown in Figure 3, even if flavan-3-ols were the most 368 representative class in each type of chocolate, the phenolic profile is thoroughly influenced by the 369 addition of Sakura green tea or turmeric powder. 370 371

Dark chocolate (DC) phenolic profile 372
As determined by LC-MS/MS experiments, the total phenolic concentration in DC was 787.63 ± 373 10.90 mg/100 g of chocolate, representing about 30.0% of total phenolic compounds determined 374 with the Folin-Ciocalteau assay (2624.15 ± 112.36 mg/100 g of chocolate). The ABTS radical 375 scavenging and Fe 3+ -reducing ability of DC (Figure 4) were tested (11.00 ± 0.26 and 6.29 ± 0.13 376 mmol trolox equivalents/100 g of chocolate, respectively) resulting in line with the findings 377 proposed by Batista et al. (2016). As reported by Wollgast & Anklam (2000a), catechins and 378 procyanidins represent more than 90% of phenolic profile of cocoa beans and cocoa-products. We 379 found out that total flavan-3-ols amount in DC was 503.76 ± 8.98 mg/100 g of chocolate 380 representing the 64.0% of total polyphenols identified by MS experiments. Considering monomeric 381 structures, epicatechin and catechin were the major represented flavan-3-ols, whose estimated 382 concentrations were higher than those reported so far (Gu et al., 2006). Epicatechin alone 383 represented the 40.4 % of total flavan-3-ols and the 25.8% of total phenolic identified by MS 384 experiments, resulting the most present compounds in DC. Large amounts of oligomeric structures 385 were also found, displaying a total concentration value of 166.28± 4.13 mg/100 g of chocolate and 386 reaching approximately 33.0% of flavan-3-ols class. Epicatechin has been causally linked to the 387 reported cardiovascular effects observed after the consumption of cocoa (Schroeter et al., 2006). 388 The ingestion of flavanol-rich cocoa in healthy adult males was associated with acute elevations in 389 levels of circulating nitric oxide, an enhanced flow-mediated dilation response of conduit arteries, 390 and an augmented microcirculation in humans and the results were repeatable with pure epicatechin 391 intake (70 mg/day; equivalent to 35-40 g of DC). Indeed, elderly men with a median epicatechin 392 intake of 22 mg/day (equivalent to 10-15 g of DC) had a 38% lower risk of cardiovascular disease 393 mortality than that of subjects with a median intake of 8 mg/day (Dower, Geleijnse, 394 Hollman, Soedamah-Muthu, & Kromhout, 2016). 395 The hydroxycinnamic acids made up about 20.6% of DC phenolic profile, among these ferulic acid, 396 di-hydroxycinnamic aspartate and coumaroyl aspartate were the main hydroxycinnamic acids 397 detected in DC. The largest contribution was given by ferulic acid, with a concentration of 61.23 ± 398 3.74 mg/100 g of chocolate. Among the N-phenylpropenoyl-L-amino acids, clovamide or caffeoyl-399 tyrosine, described for the first time in cocoa by Sanbongi et al. (1998), was the main representative 400 with total concentration of its two isomers of 9.54 ± 0.54 mg/100 g of chocolate. Previous studies 401 19 found that clovamide exhibited antiradical properties (Locatelli et al., 2013;Sanbongi et al., 1998), 402 neuroprotective effects (Fallarini et al., 2009) and anti-inflammatory properties (Zeng et al., 2011). 403 Ellagitannins, which were identified for the first time in dark chocolate and cocoa in this study, 404 made up about 10% of DC phenolic profile. The higher amount was ascribed to ellagic acid (56.16 405 ± 3.58 mg/100 g of chocolate), followed by HHDP-galloyl-hexoside (15.79 ± 1.20 mg/100 g of 406 chocolate). Ellagic acid and ellagitannins can be metabolized by human microbiota in urolithins, 407 which are responsible for the health effects attributed to the consumption of ellagic acid and 408 ellagitannins-rich food (Tomás-Barberán et al., 2017). 409 410

Sakura green tea dark chocolate (GTDC) phenolic profile 411
The content of total polyphenolic compounds in GTDC displayed a significant increase (P value 412 <0.001) in respect to that of DC, recording a total concentration value of 1035.45 ± 14.81 mg/100 g 413 of chocolate (Figure 3). This value represented the 30.3% of total phenolic compounds determined 414 with the Folin-Ciocalteau assay (3417.81 ± 229.45 mg/100 g of chocolate). The increased phenolic 415 concentration resulted in increased antioxidant properties in comparison with DC, which gave rise 416 to 40% and 144% enhancements of GTDC ABTS radical scavenging and ferric-reducing power, 417 respectively ( Figure 4). The major phenolics in GTDC were still flavan-3-ols accounting for about 418 70.1% of total phenolic compounds, displaying a concentration value of 726.03 ± 14.53 mg/100 g 419 of chocolate, significantly different from DC flavan-3-ols content (503.76 ± 8.98 mg/100 g of 420 chocolate, P value <0.001). This flavan-3-ols increase was related to the Sakura green tea leaves 421 enrichment of dark chocolate formulation and was clearly reflected in the significant increase in 422 epicatechin (303.69 ± 11.65 mg/100 g of chocolate, P value <0.001, detailing about 30% of GTDC 423 phenolic profile), epigallocatechin (29.76 ± 1.74 mg/100 g of chocolate, P value <0.001) and total 424 procyanidins (230.76 ± 15.73 mg/100 g of chocolate, P value <0.001). The Sakura green tea 425 contribution was also confirmed by the presence of typical green tea gallate flavan-3-ols, especially 426 20 epigallocatechin gallate, showing a remarkable concentration value of 33.54 ± 2.16 mg/100 g of 427 chocolate. The hydroxycinnamic acids were still the second most representative class of phenolic 428 profile in GTDC, explaining about 15.3% of GTDC phenolic profile (Figure 2). Ellagitannins 429 showed a significant content increasing in GTDC respect to DC (89.12 ± 1.50 mg/100 g of 430 chocolate, P value <0.001) with an incidence rate of 8.6%. These results may confirm a possible 431 polyphenols enrichment of dark chocolate profile which can lead to a potential combination of the 432 positive health effects and properties derived from both chocolate and green tea. LC-MS 433 experiments showed that GTDC contained 49% more epicatechin and 43% more flavan-3-ols than 434 DC. This can result in a lower intake to achieve the same biological effects. This seems a promising 435 way to maximise the potential beneficial effect of epicatechin consumption, contemporaneously 436 reducing the amount of sugars and calories introduced with chocolate. 437 438

Turmeric dark chocolate (TDC) phenolic profile 439
The TDC phenolic amount showed a significant increase (P value <0.001) respect to that of DC 440 which recorded a total concentration value of 1094.03 ± 10.15 mg/100 g of chocolate (Figure 3), 441 representing about 36% of total phenolic compounds assayed with the Folin-Ciocalteau method 442 (3043.81 ± 294.64 mg/100 g of chocolate). Despite that, single phenolic classes did not show a 443 significant and remarkable increase respect to those of DC. This higher concentration can be 444 ascribed to turmeric powder contribution as well as the related curcuminoids, which accounted for 445 about 25% of TDC total phenolic profile, displaying a concentration value of 272.73 ± 2.58 mg/100 446 g of chocolate (Figure 3). ABTS radical scavenging ability and ferric-reducing power were tested, 447 resulting in 12.30 ± 0.27 and 10.57 ± 0.2 mmol trolox equivalents/100 g of chocolate, respectively 448 ( Figure 4). Bisdemethoxycurcumin was the most concentrated curcuminoid (115.55 ± 2.16 mg/100 449 g of chocolate), followed by demethoxycurcumin (82.64 ± 1.33 mg/100 g of chocolate) which are 450 considered to be curcumin natural analogues and were reported to have a similar biological activity 451 21 to curcumin itself (Kocaadam, & Şanlier, 2017). Curcumin was found at the lowest concentration of 452 74.55 ± 0.47 mg/100 g of chocolate. Normally, curcumin is present at a concentration higher or 453 similar to the demethoxylated analogue (Jayaprakasha, Rhao, & Sakariah, 2002). Since the phenolic 454 composition of spices (and of vegetable food in general) is greatly variable depending on the 455 cultivar and agro-climatic factors (such as growing, harvesting time, seasonal variability) as well as 456 technological processes, it is plausible that different turmeric powder preparation had different 457 phenolic composition. Moreover, in the case of dark chocolate enriched with turmeric powder a 458 possible food matrix effect should be considered since some macromolecules such as proteins and 459 polysaccharide may interact with curcuminoids reducing their extractability. It is important also to 460 note that only free and extractable phenolic compounds were considered and analysed in this study.