David Stuart

Inhibition of Key Digestive Enzymes by Cocoa Extracts and Procyanidins

This study determined the in vitro inhibitory effects of cocoa extracts and procyanidins against pancreatic α-amylase (PA), pancreatic lipase (PL), and secreted phospholipase A(2) (PLA(2)) and characterized the kinetics of such inhibition. Lavado, regular, and Dutch-processed cocoa extracts as well as cocoa procyanidins (degree of polymerization (DP) = 2-10) were examined. Cocoa extracts and procyanidins dose-dependently inhibited PA, PL, and PLA(2). Lavado cocoa extract was the most potent inhibitor (IC(50) = 8.5-47 μg/mL). An inverse correlation between log IC(50) and DP (R(2) > 0.93) was observed. Kinetic analysis suggested that regular cocoa extract, the pentamer, and decamer inhibited PL activity in a mixed mode. The pentamer and decamer noncompetitively inhibited PLA(2) activity, whereas regular cocoa extract inhibited PLA(2) competitively. This study demonstrates that cocoa polyphenols can inhibit digestive enzymes in vitro and may, in conjunction with a low-calorie diet, play a role in body weight management.

Impact of Alkalization on the Antioxidant and Flavanol Content of Commercial Cocoa Powders

Cocoa is a food ingredient that is important for the contribution of flavor to foods but is also associated with potential health benefits. The chemistry thought to be responsible for its cardiovascular health benefits is the flavanol (flavan-3-ol) antioxidants. Evidence from the literature indicates that natural cocoas are high in flavanols, but when the cocoa is processed with alkali, also known as Dutch processing or Dutching, the flavanols are substantially reduced. This paper provides a survey of the physical and chemical composition of representative natural cocoas and lightly, medium, and heavily alkalized cocoas. As part of the survey, both brown/black and red/brown alkali-processed cocoas were measured. Natural cocoa powders have an extractable pH of 5.3-5.8. Alkalized cocoa powders were grouped into lightly treated (pH 6.50-7.20), medium-treated (pH 7.21-7.60), and heavily treated (pH 7.61 and higher). The natural, nonalkalized powders had the highest ORAC and total polyphenols and flavanols (including procyanidins). These chemical measurements showed a linear decrease as the extractable pH of the cocoa powder increased. Likewise, the flavanol monomers, oligomers, and polymers all showed a linear decrease with increasing pH of the final cocoa powder. When brown/black cocoa powders were compared to red cocoa powders, similar decreases in flavanols were observed with increased alkalization. The average total flavanol contents were 34.6 +/- 6.8 mg/g for the natural cocoas, 13.8 +/- 7.3 mg/g for the lightly processed cocoas, 7.8 +/- 4.0 mg/g for the medium processed cocoas, and 3.9 +/- 1.8 mg/g for the heavily processed cocoa powders. The observed linear and predictable impact of alkalization on flavanol content is discussed with respect to other reports in the literature as well as what implications it may have on diet and food manufacturing.

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.

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 Residues in Ancient Maya Vessels from Rio Azul, Guatemala

Results of chemical analyses on residues collected from ceramic vessels found in an Early Classic period Maya tomb revealed that certain of the residues contained theobromine and caffeine, a clear indication that the corresponding vessels once contained cacao in some form. One of the vessels yielding cacao residues is decorated with hieroglyphs, two of which we believe have the phonetic values for the word “cacao” in the Mayan language. These findings are significant for three reasons: (1) a new method for recognizing ancient cacao use is demonstrated, (2) a novel way of verifying glyph interpretations is presented, and (3) data are generated that indicate what contents certain Maya vessels actually held, thus permitting useful functional interpretations.

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.

Characterization of primary standards for use in the HPLC analysis of the procyanidin content of cocoa and chocolate containing products.

This report describes the characterization of a series of commercially available procyanidin standards ranging from dimers DP = 2 to decamers DP = 10 for the determination of procyanidins from cocoa and chocolate. Using a combination of HPLC with fluorescence detection and MALDI-TOF mass spectrometry, the purity of each standard was determined and these data were used to determine relative response factors. These response factors were compared with other response factors obtained from published methods. Data comparing the procyanidin analysis of a commercially available US dark chocolate calculated using each of the calibration methods indicates divergent results and demonstrate that previous methods may significantly underreport the procyanidins in cocoa-containing products. These results have far reaching implications because the previous calibration methods have been used to develop data for a variety of scientific reports, including food databases and clinical studies.

Hieroglyphs and Archaeology at Copan

The study of Copans extensive hieroglyphic texts has grown hand in hand with long-term architectural excavations. Recent progress in Maya decipherment, much of it derived from work at Copan, now allows for new and potentially rewarding opportunities to integrate epigraphy and “dirt” archaeology. Toward this end, this paper provides an overview of the current knowledge of Copans elite history, summarizing current knowledge of the ruler sequence and attributions of monuments and architecture to individual reigns. Thematic and stylistic changes are evident in the inscriptions between the Early and Late Classic periods, including a proliferation of subordinate elite texts in the decades leading up to political collapse. Also, new information is provided on the changing relations between Copan and Quirigua throughout the Classic period.

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.

Astronomical Implications of Maya Hieroglyphic Notations at Xultun

IntroductionRecently, we reported on the existence of a group of numerically related texts painted on the walls of a room in an early ninth-century A.D. residential house (structure 10K-2) in the Classic Maya city of Xultun, Guatemala. The texts, carefully executed in fine black line over colourful underlying murals pertaining to royal rituals, with which they appear to have no apparent connection, consist of a 162-lunar synodic month semester table and a group of four relatively large day tallies, ranging in length between 935 and 6704 tropical years, used to calculate commensurations among important calendrical and astronomical cycles. Though executed in a different medium, the numbers resemble those found in the codices, or bark paper books, dated to the late fifteenth and early sixteenth centuries; therefore the major significance of the discovery is that it pushes back by several centuries the archaeological evidentiary baseline for sophisticated Maya astronomical computations.Here we analyse the aforementioned inscriptions in detail not permitted, for want of space, in our original report. We also describe and interpret additional texts discovered since the submission of that report. We refer to three text areas: Area A, near the middle of the East Wall of Structure 10K-2; Area B, immediately to the left of Area A; and Area C, on the northeast corner of the north wall2 (Figure 1).Area A: A Lunar Semester TableA neatly composed, but badly eroded, 5 cm wide by 48 cm long text located in the middle of the east wall, about 40 cm above the level of the raised floor, consists of 27 columns of black dot and bar numerals. Each column is topped by one of three alternating forms of a glyph (Glyph C) identifiable with that used on monumental inscriptions to record moon positions in a semester cycle.3 It has long been known from studies of the monumental inscriptions that the Maya grouped lunar synodic months in semesters, or lunar half-years, consisting of six lunar synodic months made up of combinations of 29 and 30 days, totaling 177 (or 178) days.As first recognized by one of the authors (Stuart), the readable portion of the text below each of the glyphs leaves little doubt of its lunar function. The last two (the 26th and 27th) columns read 4606 (12.14.6) and 4784 (13.5.4), respectively, the difference between the two being 178 days. Moreover, subtracting 177 (178) from the 26th entry, one arrives at 4429 (4428) or 12.5.9 (12.5.8), which is consistent with the readable portion, 12.5.(7), of entry 25. Fragments of the remainder of the text are consistent with its being a table of 27 lunar semesters, 22 consisting of 177 days and five (including the 26th) of 177, totaling 4784 days,4 or 162 lunar synodic months. The implied average value of the lunar synodic month is 4784 days, or 29d.530864. This is a remarkable 0d.000278 short of the value ofthat period for epoch 800 A.D. (29d.530586).5 Absent the five days added in the 178-day entry columns, the time averaged synodic period would have produced a much less accurate, though nevertheless still impressive, lunar synodic period of 29?500000, 0d.030586 short of the epoch 800 A.D. value.The restored version of the Xultun Lunar Semester Table (hereinafter LST) is given in Figure 2. This text, the only one of its kind, surely functioned as an instrument to calculate, either backward or forward, the position of a particular moon in a Lunar Series statement. Such statements are found on numerous monumental inscriptions. They consist (usually) of the name of the semester in a trimester system, Jaguar Lord Semester (columns A, D, G, ... of Figure 1), Death Skull Semester (columns B, E, H, . . .), and Female Moon semester (columns C, F, I, . . .), followed by the numbered moon (1-6) in that particular semester, along with the age (phase) ofthat particular moon, usually measured from new moon = 0d; e.g. in Column B: Skull semester, fourth moon, day 2 (a thin waxing crescent, visible low in the west after sunset). …