Ritual drinks in the pre-Hispanic US Southwest and Mexican Northwest.

Chemical analyses of organic residues in fragments of pottery from 18 sites in the US Southwest and Mexican Northwest reveal combinations of methylxanthines (caffeine, theobromine, and theophylline) indicative of stimulant drinks, probably concocted using either cacao or holly leaves and twigs. The results cover a time period from around A.D. 750-1400, and a spatial distribution from southern Colorado to northern Chihuahua. As with populations located throughout much of North and South America, groups in the US Southwest and Mexican Northwest likely consumed stimulant drinks in communal, ritual gatherings. The results have implications for economic and social relations among North American populations.

Ritual Black Drink consumption at Cahokia.

Chemical analyses of organic residues in fragments of pottery from the large site of Cahokia and surrounding smaller sites in Illinois reveal theobromine, caffeine, and ursolic acid, biomarkers for species of Ilex (holly) used to prepare the ritually important Black Drink. As recorded during the historic period, men consumed Black Drink in portions of the American Southeast for ritual purification. This first demonstrated discovery of biomarkers for Ilex occurs in beaker vessels dating between A.D. 1050 and 1250 from Cahokia, located far north of the known range of the holly species used to prepare Black Drink during historic times. The association of Ilex and beaker vessels indicates a sustained ritual consumption of a caffeine-laced drink made from the leaves of plants grown in the southern United States.

Evidence of cacao use in the Prehispanic American Southwest.

Chemical analyses of organic residues in fragments of ceramic vessels from Pueblo Bonito in Chaco Canyon, New Mexico, reveal theobromine, a biomarker for cacao. With an estimated 800 rooms, Pueblo Bonito is the largest archaeological site in Chaco Canyon and was the center of a large number of interconnected towns and villages spread over northwestern New Mexico. The cacao residues come from pieces of vessels that are likely cylinder jars, special containers occurring almost solely at Pueblo Bonito and deposited in caches at the site. This first known use of cacao drinks north of the Mexican border indicates exchange with cacao cultivators in Mesoamerica in a time frame of about A.D. 1000-1125. The association of cylinder jars and cacao beverages suggests that the Chacoan ritual involving the drinking of cacao was tied to Mesoamerican rituals incorporating cylindrical vases and cacao. The importance of Pueblo Bonito within the Chacoan world likely lies in part with the integration of Mesoamerican ritual, including critical culinary ingredients.

Chemical and archaeological evidence for the earliest cacao beverages.

Chemical analyses of residues extracted from pottery vessels from Puerto Escondido in what is now Honduras show that cacao beverages were being made there before 1000 B.C., extending the confirmed use of cacao back at least 500 years. The famous chocolate beverage served on special occasions in later times in Mesoamerica, especially by elites, was made from cacao seeds. The earliest cacao beverages consumed at Puerto Escondido were likely produced by fermenting the sweet pulp surrounding the seeds.

Antioxidant activity and polyphenol and procyanidin contents of selected commercially available cocoa-containing and chocolate products in the United States.

In the United States, commercially available foods, including cocoa and chocolate, are being marketed with statements referring to the level of antioxidant activity and polyphenols. For cocoa-containing foods, there has been no comprehensive survey of the content of these and other chemistries. A survey of cocoa and chocolate-containing products marketed in the United States was conducted to determine antioxidant activity and polyphenol and procyanidin contents. Commercially available samples consisted of the top market share products in each of the following six categories: natural cocoa, unsweetened baking chocolate, dark chocolate, semisweet baking chips, milk chocolate, and chocolate syrup. Composite samples were characterized using four different methods: oxygen radical absorbance capacity (ORAC), vitamin C equivalence antioxidant capacity (VCEAC), total polyphenols, and procyanidins. All composite lots were further characterized for percent nonfat cocoa solids (NFCS) and percent fat. Natural cocoas had the highest levels of antioxidant activities, total polyphenols, and procyanidins followed by baking chocolates, dark chocolates and baking chips, and finally milk chocolate and syrups. The results showed a strong linear correlation between NFCS and ORAC (R (2) = 0.9849), total polyphenols (R (2) = 0.9793), and procyanidins (R (2) = 0.946), respectively. On the basis of principal component analysis, 81.4% of the sample set was associated with NFCS, antioxidant activity, total polyphenols, and procyanidins. The results indicated that, regardless of the product category, NFCS were the primary factor contributing to the level of cocoa antioxidants in the products tested. Results further suggested that differences in cocoa bean blends and processing, with the possible exception of Dutching, are minor factors in determining the level of antioxidants in commercially available cocoa-containing products in the United States.

Quantitative analysis of phytosterols in edible oils using APCI liquid chromatography-tandem mass spectrometry.

Previous methods for the quantitative analysis of phytosterols have usually used GC-MS and require elaborate sample preparation including chemical derivatization. Other common methods such as HPLC with absorbance detection do not provide information regarding the identity of the analytes. To address the need for an assay that utilizes mass selectivity while avoiding derivatization, a quantitative method based on LC-tandem mass spectrometry (LC-MS-MS) was developed and validated for the measurement of six abundant dietary phytosterols and structurally related triterpene alcohols including brassicasterol, campesterol, cycloartenol, ß-sitosterol, stigmasterol, and lupeol in edible oils. Samples were saponified, extracted with hexane and then analyzed using reversed phase HPLC with positive ion atmospheric pressure chemical ionization tandem mass spectrometry and selected reaction monitoring. The utility of the LC-MS-MS method was demonstrated by analyzing 14 edible oils. All six compounds were present in at least some of the edible oils. The most abundant phytosterol in all samples was ß-sitosterol, which was highest in corn oil at 4.35 ± 0.03 mg/g, followed by campesterol in canola oil at 1.84 ± 0.01 mg/g. The new LC-MS-MS method for the quantitative analysis of phytosterols provides a combination of speed, selectivity and sensitivity that exceed those of previous assays.

Impact of fermentation, drying, roasting and Dutch processing on flavan-3-ol stereochemistry in cacao beans and cocoa ingredients.

This paper reports a systematic study of the level of flavan-3-ol monomers during typical processing steps as cacao beans are dried, fermented and roasted and the results of Dutch-processing. Methods have been used that resolve the stereoisomers of epicatechin and catechin. In beans harvested from unripe and ripe cacao pods, we find only (-)-epicatechin and (+)-catechin with (-)-epicatechin being by far the predominant isomer. When beans are fermented there is a large loss of both (-)-epicatechin and (+)-catechin, but also the formation of (-)-catechin. We hypothesize that the heat of fermentation may, in part, be responsible for the formation of this enantiomer. When beans are progressively roasted at conditions described as low, medium and high roast conditions, there is a progressive loss of (-)-epicatechin and (+)-catechin and an increase in (-)-catechin with the higher roast levels. When natural and Dutch-processed cacao powders are analyzed, there is progressive loss of both (-)-epicatechin and (+)-catechin with lesser losses of (-)-catechin. We thus observe that in even lightly Dutch-processed powder, the level of (-)-catechin exceeds the level of (-)-epicatechin. The results indicate that much of the increase in the level of (-)-catechin observed during various processing steps may be the result of heat-related epimerization from (-)-epicatechin. These results are discussed with reference to the reported preferred order of absorption of (-)-epicatechin > (+)-catechin > (-)-catechin. These results are also discussed with respect to the balance that must be struck between the beneficial impact of fermentation and roasting on chocolate flavor and the healthful benefits of chocolate and cocoa powder that result in part from the flavan-3-ol monomers.

Impact of fermentation, drying, roasting, and Dutch processing on epicatechin and catechin content of cacao beans and cocoa ingredients.

Low molecular weight flavan-3-ols are thought to be responsible, in part, for the cardiovascular benefits associated with cocoa powder and dark chocolate. The levels of epicatechin and catechin were determined in raw and conventionally fermented cacao beans and during conventional processing, which included drying, roasting, and Dutch (alkali) processing. Unripe cacao beans had 29% higher levels of epicatechin and the same level of catechin compared to fully ripe beans. Drying had minimal effect on the epicatechin and catechin levels. Substantial decreases (>80%) in catechin and epicatechin levels were observed in fermented versus unfermented beans. When both Ivory Coast and Papua New Guinea beans were subjected to roasting under controlled conditions, there was a distinct loss of epicatechin when bean temperatures exceeded 70 °C. When cacao beans were roasted to 120 °C, the catechin level in beans increased by 696% in unfermented beans, by 650% in Ivory Coast beans, and by 640% in Papua New Guinea fermented beans compared to the same unroasted beans. These results suggest that roasting in excess of 70 °C generates significant amounts of (-)-catechin, probably due to epimerization of (-)-epicatechin. Compared to natural cocoa powders, Dutch processing caused a loss in both epicatechin (up to 98%) and catechin (up to 80%). The epicatechin/catechin ratio is proposed as a useful and sensitive indicator for the processing history of cacao beans.

Stability of cocoa antioxidants and flavan-3-ols over time.

Several recent reports have been published indicating that the antioxidant activity of olive oil and tea leaves is not stable over product shelf lives of about one year. We have measured the antioxidant activity, total polyphenols, flavan-3-ols monomers, and procyanidin levels in milk and dark chocolate, in cocoa powder, and in cocoa beans. Results show that for the cocoa products studied, antioxidant activity, and flavan-3-ol levels are stable over typical shelf lives of one year under controlled storage and over 2 years in ambient storage in the laboratory. We also show that 80 year old cocoa powder and 116 year old cocoa beans still show very high levels of antioxidant activity and flavan-3-ol content.

Screening antioxidants using LC-MS: case study with cocoa.

Oxidative stress enhances pathological processes contributing to cancer, cardiovascular disease, and neurodegenerative diseases, and dietary antioxidants may counteract these deleterious processes. Because rapid methods to evaluate and compare food products for antioxidant benefits are needed, a new assay based on liquid chromatography-mass spectrometry (LC-MS) was developed for the identification and quantitative analysis of antioxidants in complex natural product samples such as food extracts. This assay is based on the comparison of electrospray LC-MS profiles of sample extracts before and after treatment with reactive oxygen species such as hydrogen peroxide or 2,2-diphenyl-1-picrylhydrazyl radical (DPPH). Using this assay, methanolic extracts of cocoa powder were analyzed, and procyanidins were found to be the most potent antioxidant species. These species were identified using LC-MS, LC-MS/MS, accurate mass measurement, and comparison with reference standards. Furthermore, LC-MS was used to determine the levels of these species in cocoa samples. Catechin and epicatechin were the most abundant antioxidants followed by their dimers and trimers. The most potent antioxidants in cocoa were trimers and dimers of catechin and epicatechin, such as procyanidin B2, followed by catechin and epicatechin. This new LC-MS assay facilitates the rapid identification and then the determination of the relative antioxidant activities of individual antioxidant species in complex natural product samples and food products such as cocoa.

Survey of commercially available chocolate- and cocoa-containing products in the United States. 2. Comparison of flavan-3-ol content with nonfat cocoa solids, total polyphenols, and percent cacao.

A survey of a broad range of chocolate- and cocoa-containing products marketed in the United States was conducted to provide a more detailed analysis of flavan-3-ol monomers, oligomers, and polymers, which can be grouped into a class of compounds called procyanidins. Samples consisted of the three or four top-selling products within the following six categories: natural cocoa powder, unsweetened baking chocolate, dark chocolate, semisweet baking chips, milk chocolate, and chocolate syrup. Composite samples were characterized for percent fat (% fat), percent nonfat cocoa solids (% NFCS), antioxidant level by ORAC, total polyphenols, epicatechin, catechin, total monomers, and flavan-3-ol oligomers and polymers (procyanidins). On a gram weight basis epicatechin and catechin content of the products follow in decreasing order: cocoa powder > baking chocolate > dark chocolate = baking chips > milk chocolate > chocolate syrup. Analysis of the monomer and oligomer profiles within product categories shows there are two types of profiles: (1) products that have high monomers with decreasing levels of oligomers and (2) products in which the level of dimers is equal to or greater than the monomers. Results show a strong correlation (R(2) = 0.834) of epicatechin to the level of % NFCS and also very good correlations for N = 2-5 oligomers to % NFCS. A weaker correlation was observed for catechin to % NFCS (R(2) = 0.680). Other analyses show a similar high degree of correlation with epicatechin and N = 2-5 oligomers to total polyphenols, with catechin being less well correlated to total polyphenols. A lesser but still good correlation exists between the calculated percent cacao (calcd % cacao) content, a proxy for percent cacao, and these same flavanol measures, with catechin again showing a lesser degree of correlation to calcd % cacao. Principal component analysis (PCA) shows that the products group discretely into five classes: (1) cocoa powder, (2) baking chocolate, (3) dark chocolate and semisweet chips, (4) milk chocolates, and (5) syrup. PCA also shows that most factors group closely together including the antioxidant activity, total polyphenols, and the flavan-3-ol measures with the exception of catechin and % fat in the product, which group separately. Because catechin distribution appears to be different from the other flavan-3-ol measures, an analysis of the epicatechin to catechin ratio was done, indicating there is a >5-fold variation in this measure across the products studied. The cocoa-containing products tested range from cocoa powder with 227.34 +/- 17.23 mg of procyanidins per serving to 25.75 +/- 9.91 mg of procyanidins per serving for chocolate syrup. These results are discussed with respect to other studies on commercial products, the bioavailability of the flavanols, and the possible role of processing on the amount of catechin in products.

Survey of the trans-resveratrol and trans-piceid content of cocoa-containing and chocolate products.

Dietary resveratrol (3,4',5-trihydroxystilbene) has been implicated in the health benefits associated with grapes and red wine, more specifically with potential benefits for metabolic syndrome, energy use, and increased endurance. Levels of trans-resveratrol and its glucoside, trans-piceid, were determined in 19 top selling commercially available cocoa-containing and chocolate products from the U.S. market. Amounts of trans-resveratrol and trans-piceid were closely correlated with the amount of nonfat cocoa solids (NFCS) in the cocoa-containing products. Among these products, trans-resveratrol levels were highest in cocoa powders (1.85 +/- 0.43 microg/g), followed by unsweetened baking chocolates (1.24 +/- 0.22), semisweet chocolate baking chips (0.52 +/- 0.14), dark chocolates (0.35 +/- 0.08), milk chocolates (0.10 +/- 0.05), and chocolate syrups (0.09 +/- 0.02). These cocoa-containing and chocolate products have about 3-5 times more trans-piceid than trans-resveratrol. Levels of trans-piceid were highest in the cocoa powders (7.14 +/- 0.80 microg/g), followed by unsweetened baking chocolates (4.04 +/- 0.14), semisweet chocolate baking chips (2.01 +/- 0.18), dark chocolates (1.82 +/- 0.36), milk chocolates (0.44 +/- 0.06), and chocolate syrups (0.35 +/- 0.06). On an equal weight basis, cocoa powder had about half as much trans-resveratrol as the average California red wine. On a per serving basis, cocoa-containing and chocolate products had less trans-resveratrol than red wine and grape juice but more than roasted peanuts. Overall, these cocoa-containing and chocolate products rank second after red wines and grape juice in foods with the highest levels of total trans-resveratrol in the diet.

Cacao usage by the earliest Maya civilization.

The Maya archaeological site at Colha in northern Belize, Central America, has yielded several spouted ceramic vessels that contain residues from the preparation of food and beverages. Here we analyse dry residue samples by using high-performance liquid chromatography coupled to atmospheric-pressure chemical-ionization mass spectrometry, and show that chocolate (Theobroma cacao) was consumed by the Preclassic Maya as early as 600 bc, pushing back the earliest chemical evidence of cacao use by some 1,000 years. Our application of this new and highly sensitive analytical technique could be extended to the identification of other ancient foods and beverages.