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PubMed2004

Effect of cacao liquor extract on tumor marker enzymes during chemical hepatocarcinogenesis in rats.

This study investigated the effect of cacao liquor extract (CLE) on tumor marker enzymes--alkaline phosphatase (ALP), gamma-glutamyl transpeptidase (GGT), glutathione-S-transferase (GST), and glutathione reductase (GR) activities--in plasma and/or liver of hepatocarcinogenic rats, which were induced with diethylnitrosamine and 2-acetylaminofluorene. Twenty-nine male Sprague-Dawley rats (weighing 150-330 g) were divided into four groups (n = 6-8): normal control group (N), normal group + CLE (NE), cancer group (C), and cancer group + CLE (CE). Analysis of variance showed significant differences (P<.05) in the specific activities of ALP, GGT, and GST between the C and N groups. However, GR activity for the C group was not significantly different compared with the N group. In the CE group, the specific activities of ALP, GGT, GST, and GR were significantly lower (P<.05) compared with the C group. The findings showed that CLE could lower the activity of tumor marker enzymes of rats during hepatocarcinogenesis. Based on the results obtained, polyphenol compounds present in the cacao liquor, extracted by using ethanol, have the potential in decreasing the severity of hepatocarcinogenesis.

2-Acetylaminofluorene↗
PubMed1991

Characterization of the genome of cacao swollen shoot virus.

Cacao swollen shoot disease has been known to be caused by a small non-enveloped bacilliform virus for more than 25 years. Purification using a combination of celite filtration, polyethylene glycol concentration and sucrose density gradient centrifugation has yielded concentrated preparations of purified cacao swollen shoot virus (CSSV). Results of nuclease sensitivity tests indicated that the CSSV genome consists of dsDNA which has two single-stranded regions. The approximate size of CSSV DNA calculated from restriction enzyme digests is 7.4 kbp. It is very likely that CSSV is a member of the commelina yellow mottle virus group.

Blotting, Southern↗
PubMed2001

Identification of cariostatic substances in the cacao bean husk: their anti-glucosyltransferase and antibacterial activities.

The cacao bean husk has been shown to possess two types of cariostatic substances, one showing anti-glucosyltransferase (GTF) activity and the other antibacterial activity, and to inhibit experimental dental caries in rats infected with mutans streptococci. In the present study, chromatographic purification revealed high-molecular-weight polyphenolic compounds and unsaturated fatty acids as active components. The former, which showed strong anti-GTF activity, were polymeric epicatechins with C-4beta and C-8 intermolecular bonds estimated to be 4636 in molecular weight in an acetylated form. The latter, which showed bactericidal activity against Streptococcus mutans, were determined to be oleic and linoleic acids, and demonstrated a high level of activity at a concentration of 30 microgram/mL. The cariostatic activity of the cacao bean husk is likely caused by these biologically active constituents.

Anti-Infective Agents, Local↗
PubMed1998

The antioxidative substances in cacao liquor.

The antioxidative substances contained in cacao liquor, which is one of the major ingredients of chocolate, were separated by column chromatography and high-performance liquid chromatography. Three major compounds were purified and two of them were identified by 1H, 13C NMR and mass spectra as (-)-epicatechin (EC) and (+)-catechin (CA). Their antioxidative activity was measured by monitoring the peroxide value of linoleic acid and the thiobarbituric acid-reactive substance values of erythrocyte ghost membranes and microsomes. EC and CA had strong antioxidative effects in all three methods, but one unidentified peak was found to be less effective. Additionally, we analyzed the polyphenol concentration of cacao liquor extractions produced in several countries. The total polyphenol concentration was 7.0 to 13.0%, catechin concentration was 0.31 to 0.49%, and epicatechin concentration was 0.35 to 1.68% in the extractions. It is believed that chocolate is stable against oxidative deterioration on account of the presence of these polyphenolic compounds, and it is also expected to have a protective role against lipid peroxidation in living systems.

Alcoholic Beverages↗
PubMed1990

Inhibitory effects of aqueous extract of cacao bean husk on collagenase of Bacteroides gingivalis.

Natural materials containing tannin were examined to determine whether they possessed any inhibitory activity against collagenase obtained from the culture supernatant of Bacteroides gingivalis. The collagenolytic activity was determined with Collagenokit CLN-100 (Collagen Technological Co., Tokyo) using FITC-conjugated collagen type 1. Among the test samples, only an aqueous extract of cacao (Theobroma cacao) bean husk strongly inhibited the bacterial collagenase. This inhibitory potency was at the same level as that of tetracycline-HCl.

Cacao↗
PubMed2001

Auxin modification of the incompatibility response in Theobroma cacao.

The time course and control of floral abscission and fruit set in Theobroma cacao were studied after spray application of growth regulators. 1-Naphthaleneacetic acid (NAA) prevented flower abscission in a concentration dependent manner and induced the early stages of fruit development. The cytokinin benzylaminopurine (BAP) counteracted NAA but resulted in longer fruit retention. Measurements of endogenous levels of indole-3-acetic acid showed an inverse correlation between the number of flowers per plant and auxin content. The results suggest that the genetic control of self-incompatibility in T. cacao may be modulated by the hormonal content of the flower.

Journal Article↗
PubMed1991

Net CO(2) assimilation of cacao seedlings following dark chilling.

Overnight exposure of cacao (Theobroma cacao L.) seedlings to chilling temperatures between 4.7 and 15.8 degrees C reduced net CO(2) assimilation rate (A) and stomatal conductance to water vapor (g(s)), with temperatures below 10 degrees C causing severe inhibition. Net CO(2) assimilation rates of chilled seedlings recovered to those of nonchilled plants within 7 days. No differences in daytime intercellular CO(2) concentration (c(i)) with overnight temperature were observed on the first day after the chilling treatment, which indicates that the reduction in photosynthesis was not caused by the reduction in stomatal conductance. However, c(i) of chilled plants was much less than that of nonchilled plants on the second day after treatment, which suggests that chilling caused a change in stomatal response to CO(2) concentration. Even 7 days after treatment, when A had recovered to control values, g(s) of chilled leaves was only approximately 70% that of controls. Chilling did not inhibit A through an effect on leaf water potential, which was higher in chilled plants than in unchilled plants.

Journal Article↗
PubMed2005

Extraction and chromatographic separation of anticarcinogenic fractions from cacao bean husk.

The utilization of cacao bean husk (CBH), a by-product of chocolate manufacture, would be both environmentally and economically beneficial. For this purpose, a process for effectively separating and fractionating CBH fractions having cancer preventive potential was developed in this study. For screening the fractions with potent cancer preventive activity, we examined the effect of extracts and fractions of CBH on the inhibition of gap-junction intercellular communication (GJIC) and the DNA synthesis of cancer cells, both of which are characteristics of the promotion and progression stages in carcinogenesis. The extracts of CBH (especially, the 60% ethanol fraction after extraction with 50% acetone) containing 43 wt.% polyphenol exerted an excellent protective effect on H2O2-induced inhibition of GJIC in WB-F344 rat liver epithelial cells as determined by the scrape-loading/dye transfer assay. The enhancement of GJIC by the extracts of CBH was approximately 10-fold higher than that of a well-known dietary chemopreventive component, vitamin C. The extracts of CBH (especially, the 60% ethanol fraction) also suppressed DNA synthesis in all liver, stomach, and colon cancer cells as demonstrated by the ;3H-thymidine incorporation assay, by approximately four-fold higher than that of vitamin C. These results imply that the polyphenol extracts and fractions of CBH are effective functional materials to be used in either preventing or inhibiting cancer.

Animals↗
PubMed1996

Expression, purification and initial characterization of the recombinant storage protein precursor of Theobroma cacao.

The gene encoding the 67-kDa cocoa storage protein precursor has been cloned from Theobroma cacao and expressed in Escherichia coli using the pET expression system. The recombinant storage protein has been renatured from inclusion bodies at 30 degrees C using 20 mM glycine-NaOH buffer, pH 10.0, containing 1 mM oxidized glutathione and 0.1% Brij. The renatured protein was purified and demonstrated to adopt a stable native conformation by optical spectroscopy. Secondary structure analysis from circular dichroism indicated the protein to be 23% alpha-helix and 38% beta-sheet, in close agreement with values obtained using a secondary structure prediction program.

Cacao↗
PubMed1993

Nucleotide sequence and genomic organization of cacao swollen shoot virus.

Cacao swollen shoot virus is classified as a badnavirus based on its nonenveloped, bacilliform particle morphology and double-stranded DNA genome. A complete copy of the genome was cloned into a plasmid vector and the sequence was determined from 75 overlapping subclones covering both strands. The genome contains 7161 base pairs and possesses an intergenic region and five putative open reading frames (ORF) capable of coding for proteins > 10 kDa. All of the ORFs are present on the plus-strand. ORF 1 (17 kDa) and ORF 2 (14 kDa) encode proteins of unknown function. The large ORF 3 (211 kDa) encodes a polyprotein that can be divided into three regions. Based on distant homologies with viral movement proteins, region 1 may encode a protein involved in cell-to-cell spread, while region 2 encodes the viral capsid protein. Region 3 contains consensus sequences for viral aspartyl proteinase, reverse transcriptase, and ribonuclease H characteristic of pararetroviruses. The last two ORFs (13 and 14 kDa) overlap ORF 3 and are not present in the other badnaviruses described.

Amino Acid Sequence↗
PubMed1996

The open reading frame 2 product of cacao swollen shoot badnavirus is a nucleic acid-binding protein.

The function of the open reading frame 2 product (p2) of cacao swollen shoot virus (CSSV) and of other badnaviruses is not yet determined. Their carboxyl-termini are lysine and proline rich and also contain alanine residues, amino acids present at the C-termini of histone-like proteins. Full-length CSSV p2 (132 amino acids) or versions truncated at the C-terminus (128, 113, 103, or 101 amino acids) were expressed in Escherichia coli and partially purified. When assayed in nucleic acid-binding tests, p2 was able to interact with CSSV and other double-stranded DNAs and with CSSV and other single-stranded RNA transcripts in sequence-nonspecific manner. Moreover, this binding activity was progressively lost as the C-terminus was gradually deleted.

Amino Acid Sequence↗
PubMed2004

Creation of BAC genomic resources for cocoa ( Theobroma cacao L.) for physical mapping of RGA containing BAC clones.

We have constructed and validated the first cocoa ( Theobroma cacao L.) BAC library, with the aim of developing molecular resources to study the structure and evolution of the genome of this perennial crop. This library contains 36,864 clones with an average insert size of 120 kb, representing approximately ten haploid genome equivalents. It was constructed from the genotype Scavina-6 (Sca-6), a Forastero clone highly resistant to cocoa pathogens and a parent of existing mapping populations. Validation of the BAC library was carried out with a set of 13 genetically-anchored single copy and one duplicated markers. An average of nine BAC clones per probe was identified, giving an initial experimental estimation of the genome coverage represented in the library. Screening of the library with a set of resistance gene analogues (RGAs), previously mapped in cocoa and co-localizing with QTL for resistance to Phytophthora traits, confirmed at the physical level the tight clustering of RGAs in the cocoa genome and provided the first insights into the relationships between genetic and physical distances in the cocoa genome. This library represents an available BAC resource for structural genomic studies or map-based cloning of genes corresponding to important QTLs for agronomic traits such as resistance genes to major cocoa pathogens like Phytophthora spp ( palmivora and megakarya), Crinipellis perniciosa and Moniliophthora roreri.

Cacao↗
PubMed2006

In vitro production of biotrophic-like cultures of Crinipellis perniciosa, the causal agent of witches' broom disease of Theobroma cacao.

Witches' broom disease (WBD) of cacao, caused by the hemibiotrophic fungus, Crinipellis perniciosa, exhibits a succession of symptoms that are caused by the biotrophic phase of the fungus. However, the study of this biotrophic phase is limited by its exclusive growth inside the plant or in the presence of callus. Here we report for the first time a method for the growth and maintenance of the biotrophic-like phase of C. perniciosa on a defined medium with metabolites found in the diseased tissues. Our results suggest that glycerol is a key carbon source for this interaction. This is a crucial achievement toward understanding the biology of this fungus during the infectious phase of WBD.

Agaricales↗
PubMed2000

Isolation and characterisation of cell wall polysaccharides from cocoa (Theobroma cacao L.) beans.

Cell wall material (CWM) was prepared from sun-dried cocoa (Theobroma cacao L.) bean cotyledons before and after fermentation. The monosaccharide composition of the CWM was identical for unfermented and fermented beans. Polysaccharides of the CWM were solubilised by sequential extraction with 0.05 M trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA), 0.05 M Na2CO3, and 1 M, 4 M and 8 M KOH. The non-cellulosic sugar composition for each fraction was similar for unfermented and fermented samples, indicating that fermentation caused no significant modification of the structural features of individual cell wall polysaccharides. Pectic polysaccharides accounted for 60% of the cell wall polysaccharides but only small amounts could be solubilised in solutions of CDTA, Na2CO3, and 1 M and 4 M KOH. The bulk of the pectic polysaccharides were solubilised in 8 M KOH and were characterised by a rhamnogalacturonan backbone heavily substituted with side-chains of 5-linked arabinose and 4-linked galactose. Linkage analysis indicated the presence of additional acidic polysaccharides, including a xylogalacturonan and a glucuronoxylan. Cellulose, xyloglucan and a galactoglucomannan accounted for 28%, 8% and 3% of the cell wall polysaccharides, respectively. It is concluded that the types and structural features of cell wall polysaccharides in cocoa beans resemble those found in the parenchymatous tissue of many fruits and vegetables rather than those reported for many seed storage polysaccharides.

Cacao↗
PubMed2001

Cacao liquor polyphenols reduce oxidative stress without maintaining alpha-tocopherol levels in rats fed a vitamin E-deficient diet.

The effect of crude polyphenols (CLP) from cacao liquor on vitamin E-deficient rats was examined. The CLP fraction contained 49.8% antioxidative polyphenols such as catechins and their oligomers. Supplementation of the vitamin E-deficient diet with CLP for 7 wk did not prevent the decrease in alpha-tocopherol levels in the liver, kidney, heart, brain, and plasma. The lipid peroxide levels in these tissues increased in the group fed the vitamin E-deficient diet compared with the control group. However, these changes were inhibited in a dose-dependent manner as a result of supplementation of the vitamin E-deficient diet with 0.25, 0.5, or 1.0% CLP. The lipid peroxide levels in plasma increased in the group fed the vitamin E-deficient diet. This change tended to be suppressed as a result of supplementation of the diet with CLP, but the difference was not significant. There was no evidence of absorption and distribution of CLP to the tissues; however, CLP intake resulted in a decrease in oxidative stress without maintaining vitamin E levels in the plasma and the tissues.

Animals↗
PubMed2005

Suppressive effects of cacao liquor polyphenols (CLP) on LDL oxidation and the development of atherosclerosis in Kurosawa and Kusanagi-hypercholesterolemic rabbits.

We investigated the properties of cacao liquor polyphenols (CLP), which have an antioxidative effect on low-density lipoprotein (LDL) and an anti-atherosclerotic effect in the spontaneous familial hypercholesterolemic model, the Kurosawa and Kusanagi-hypercholesterolemic (KHC) rabbit. After 6 months of dietary administration of CLP at 1% (w/w) to the KHC rabbits, a higher total cholesterol concentration was observed in the treatment group compared to the control group. However, no other effects were noted in lipid profiles in plasma or lipoproteins. The plasma concentration of thiobarbituric acid reactive substances (TBARS), which is a lipid-peroxidation index, was significantly decreased 1 month after the start of CLP administration compared to that of the control group. The antioxidative effect of CLP on LDL was observed from 2 to 4 months of administration. The area of atherosclerotic lesions in the aorta in the CLP group (32.01+/-1.58%) was significantly smaller than that in the control group (47.05+/-3.29%), and the tissue cholesterol and TBARS concentrations were lower in the CLP group than in the control group. The anti-atherosclerotic effect of CLP was confirmed both rheologically and histopathologically. An in vitro study using KHC rabbit-derived LDL revealed that CLP significantly prolonged the lag time of LDL oxidation that was induced by a lipophilic azo-radical initiator, 2,2'-azobis(4-methoxy)-2,4-dimethylvaleronitrile (V-70), or Cu(2+) from a low concentration of 0.1 microg/mL. The antioxidative effect of CLP was superior to those of the well-known antioxidative substances, vitamin C, vitamin E and probucol. Therefore, CLP suppressed the generation of atherosclerosis, and its antioxidative effect appeared to have an important role in its anti-atherosclerotic activity.

Animals↗
PubMed2005

Preparation and characterization of solid lipid nanoparticles (SLN) made of cacao butter and curdlan.

Solid lipid nanoparticles (SLN) were prepared using cacao butter, as the lipid core, and curdlan, as the shell material. Tween 80 was used as a co-surfactant in order to prevent aggregation and gelling of the curdlan. Mannitol was used as a cryoprotectant in order to prevent aggregation during redispersion. No significant change in the size of the SLN was observed up to a lipid concentration of 1.0%, and the particle size ranged from 140 to 200 nm with a unimodal distribution. When an alternating pH between 7 and 11 was used to test the physical stability of an SLN solution, the change in the particle size remained within a narrow range up to a lipid concentration of 0.5%. Above 0.5%, the particles began to aggregate due to the insufficient amount of the coating material, curdlan and Tween 80. The critical aggregation concentration at pH 7.4 was found to be 6.95 x 10(-4) mg/ml. Pyrene was used as a fluorescence probe. As the temperature increased, pyrene was gradually released from the SLN. The loading efficiency was >75% when the verapamil to lipid ratios were 1:10 and 1:5 and decreased significantly as the ratio became 1:1. The release rate was significantly delayed when verapamil was loaded into the SLN.

Cacao↗
PubMed2000

Caries inhibitory activity of cacao bean husk extract in in-vitro and animal experiments.

Cacao bean husk extract (CBH) was examined for inhibitory effects on the caries-inducing properties of mutans streptococci in vitro and on caries development in specific pathogen-free Sprague-Dawley rats infected with mutans streptococci. CBH reduced the growth rate of almost all oral streptococci examined, which resulted in the reduction of acid production. Furthermore, insoluble glucan synthesis by the glucosyltransferases from Streptococcus mutans MT8148R and Streptococcus sobrinus 6715 was significantly inhibited by CBH. Hence, the sucrose-dependent cell adherence of mutans streptococci was also depressed by CBH. The administration of CBH in drinking water resulted in significant reductions of caries development and dental plaque accumulation in rats infected with either Strep. sobrinus 6715 or Strep. mutans MT8148R, and the minimum cariostatic concentration was 1.0 mg/ml. These results indicate that CBH possesses powerful anticariogenic potential.

Analysis of Variance↗