Superior reducing carbon dots from proanthocyanidin for free-radical scavenging and for cell imaging.

The presence of excessive ROS can cause much harm to the human body and can even cause diseases. Therefore, it is important to detect and remove ROS, but there is no ideal method available for this at present. In this research, using procyanidins, a type of plant extract with strong reducibility, as raw materials, fluorescent carbon dots (CDs) were prepared by a hydrothermal method. The proanthocyanidin-based carbon dots (PCDs) emit a light-green colored light under UV irradiation. The PCDs retain the strong reducibility of procyanidins and are highly water-soluble compared with procyanidins. The PCDs, in addition to having good biocompatibility, also have the superior properties of radical scavenging activity and cell imaging. In in vitro experiments, 1,1-diphenyl-2-picrylhydrazyl (DPPH; 100 µM) was reduced by 30% when PCDs were added up to a concentration of 87.5 µg mL-1. At the same time, the fluorescence quenching correlates with the concentration of hypochlorite and hydrogen peroxide and has a good linearity in the range of 250-2250 nM and 60-180 µM with a detection limit of 3.676 nM and 0.602 µM, respectively. Based on the previously described advantages, PCDs have potential as a biomedicine.

Proanthocyanidins: Components, Pharmacokinetics and Biomedical Properties.

Proanthocyanidins (PAs) are a group of polyphenols enriched in plant and human food. In recent decades, epidemiological studies have upheld the direct relationship between PA consumption and health benefits; therefore, studies on PAs have become a research hotspot. Although the oral bioavailability of PAs is quite low, pharmacokinetics data revealed that some small molecules and colonic microbial metabolites of PAs could be absorbed and exert their health beneficial effects. The pharmacological effects of PAs mainly include anti-oxidant, anticancer, anti-inflammation, antimicrobial, cardiovascular protection, neuroprotection, and metabolism-regulation behaviors. Moreover, current toxicological studies show that PAs have no observable toxicity to humans. This review summarizes the resources, extraction, structures, pharmacokinetics, pharmacology, and toxicology of PAs and discusses the limitations of current studies. Areas for further research are also proposed.

Acute and Subacute Toxicity Studies of the Ethyl Acetate Soluble Proanthocyanidins of the Immature Inflorescence of Cocos nucifera L. in Female Wistar Rats.

Ayurvedic and traditional medical practitioners of Sri Lanka use the decoction of the immature inflorescence of Cocos nucifera L. (IC) variety aurantiaca for the treatment of menorrhagia. The progestogenic effect of the ethyl acetate soluble proanthocyanidins (EASPA) of the IC in female rats at a dose of 3.5 mg/kg body weight has been reported. Acute and subacute toxicity studies of EASPA of the IC carried out using female Wistar rats according to Organization for Economic Co-operation and Development (OECD) guidelines 423 and 407, respectively, are reported herein. In the acute toxicity study, a single dose of EASPA (2000 mg/kg body weight) was orally administered to rats, which were monitored for 14 days. In the subacute toxicity study, rats were orally administered with EASPA daily for 28 days at doses of 1.75, 3.5, 7, and 14 mg/kg body weight. No rat in either the acute or subacute toxicity study exhibited mortality or clinical signs of toxicity. Further, these rats did not show any significant change in their mean body weight, food, and water intake, haematological and biochemical parameters as well as in the results of their histopathological examinations compared to those of control group rats. According to results of the acute toxicity, the LD50 of EASPA is estimated to be greater than 2000 mg/kg body weight. Considering the results of the subacute toxicity study, the oral administration of EASPA daily for 28 days was well tolerated up to the dose, 14 mg/kg by rats. These results will be useful in the development of a novel therapeutic agent from EASPA of the IC for the treatment of menorrhagia, which incapacitates a considerable proportion of women worldwide.

Bioactive Compounds from Theobroma cacao: Effect of Isolation and Safety Evaluation.

Plants, including most food and feed plants, produce a broad range of bioactive chemical compounds. Among these compounds, polyphenols are reported to provide beneficial effects as anti-carcinogenic, anti-atherogenic, anti-inflammatory, immune modulating, anti-microbial, vasodilatory and analgesic. Cocoa (Theobroma cacao), a major, economically important, international crop, has been related to several nutritional benefits, which have been associated with the phenolic fraction. The main subclass of flavonoids found in cocoa is flavanols, particularly (epi)catechins monomers, and their oligomers, also known as procyanidins. In this study, these compounds were isolated by different methodologies as solid phase extraction (SPE), semi-preparative high-performance liquid chromatography (HPLC) and membrane technologies to obtain different polyphenolic profiles by HPLC coupled to electrospray time-of-flight mass spectrometry (ESI-TOF-MS) and to test their cytotoxicity. Finally, different polyphenolic profiles were collected, where the combination of both semi-preparative HPLC and SPE technologies provided the most purified fractions. Filtration with membranes and SPE provide extracts with different composition depending on the pore size of membranes and on the solvent, respectively. In addition, the results of toxicity assay indicated low levels in all fractions.

In silico approach to study the metabolism and biological activities of oligomeric proanthocyanidin complexes.

OBJECTIVES: Over the past three decades, numerous studies have focused on the biological activities of oligomeric proanthocyanidins (OPCs) in the prevention of many diseases such as neurodegeneration, atherosclerosis, tumorigenesis, and microbial infections. OPC has redox-active metabolites which could modulate the intracellular redox equilibrium to maintain the antioxidant homeostasis. This redox-modulating efficiency of OPC could provide new insights into therapeutic approaches that could reduce the burden of cardiovascular diseases. The main objective of this study was to explore the biological and metabolic activities of OPC using in silico approaches. METHODS: To validate the above objective, chemoinformatic tools were used to predict the metabolism of OPC after ingestion, based on both the ligand and structure of the constituent compounds. RESULTS: OPC showed possible sites for Phase I metabolism by cytochrome P450, and the metabolites obtained thereafter may be responsible for its biological activities. Absorption, distribution, metabolism, elimination, and toxicity properties showed efficient absorption, distribution, and metabolism of OPC, without toxicity. CONCLUSION: Thus, from the results obtained, OPC could be strongly recommended as a cardioprotective drug.

Preparation of decellularized vascular matrix by co-crosslinking of procyanidins and glutaraldehyde.

BACKGROUND: Vascular extracellular matrices (vECMs) have shown potential for small-diameter blood vessel tissue engineering applications. However, problems such as chemical instability and easy calcification are still remained. Chemical crosslinking using crosslinkers such as glutaraldehyde (GA) can improve mechanical properties and proteolysis resistance of vECMs, but leads to calcification and cytotoxicity. Procyanidins (PC) can crosslink ECMs with anti-calcification property and cytocompatibility, but the mechanical properties and chemical stability are unsatisfactory. OBJECTIVE: A novel co-crosslinking technique using PC and GA was developed, which combines the advantages of both PC and GA for enhancing mechanical properties and stability of vECMs with reduced calcification and cytotoxicity. METHODS: Fresh carotid were decellularized and then crosslinked by PC and subsequent GA for 6 h respectively. The mechanical properties, dynamic release of PC, enzymatic degradation, calcification and cytotoxicity of crosslinked samples were evaluated. RESULTS: The co-crosslinked vECMs showed enhanced tensile strength, chemical and biological stability, comparable anti-calcification property as compared to pure PC-crosslinked samples. Cytotoxicity assay showed that the co-crosslinked vECMs were cytocompatible for supporting the adhesion and proliferation of HUVECs. CONCLUSIONS: Co-crosslinking with PC and GA might be a useful method for preparation of vECM scaffolds with potential applications in small-diameter blood vessel tissue engineering.

Impact of Procyanidins from Different Berries on Caspase 8 Activation in Colon Cancer.

SCOPE: The aim of this work is to identify which proapoptotic pathway is induced in human colon cancer cell lines, in contact with proanthocyanidins extracted from various berries. METHODS AND RESULTS: Proanthocyanidins (Pcys) extracted from 11 berry species are monitored for proapoptotic activities on two related human colon cancer cell lines: SW480-TRAIL-sensitive and SW620-TRAIL-resistant. Apoptosis induction is monitored by cell surface phosphatidylserine (PS) detection. Lowbush blueberry extract triggers the strongest activity. When tested on the human monocytic cell line THP-1, blueberry Pcys are less effective for PS externalisation and DNA fragmentation is absent, highlighting a specificity of apoptosis induction in gut cells. In Pcys-treated gut cell lines, caspase 8 (apoptosis extrinsic pathway) but not caspase 9 (apoptosis intrinsic pathway) is activated after 3 hours through P38 phosphorylation (90 min), emphasizing the potency of lowbush blueberry Pcys to eradicate gut TRAIL-resistant cancer cells. CONCLUSION: We highlight here that berries Pcys, especially lowbush blueberry Pcys, are of putative interest for nutritional chemoprevention of colorectal cancer in view of their apoptosis induction in a human colorectal cancer cell lines.

The cranberry flavonoids PAC DP-9 and quercetin aglycone induce cytotoxicity and cell cycle arrest and increase cisplatin sensitivity in ovarian cancer cells.

Cranberry flavonoids (flavonols and flavan-3-ols), in addition to their antioxidant properties, have been shown to possess potential in vitro activity against several cancers. However, the difficulty of isolating cranberry compounds has largely limited anticancer research to crude fractions without well-defined compound composition. In this study, individual cranberry flavonoids were isolated to the highest purity achieved so far using gravity and high performance column chromatography and LC-MS characterization. MTS assay indicated differential cell viability reduction of SKOV-3 and OVCAR-8 ovarian cancer cells treated with individual cranberry flavonoids. Treatment with quercetin aglycone and PAC DP-9, which exhibited the strongest activity, induced apoptosis, led to caspase-3 activation and PARP deactivation, and increased sensitivity to cisplatin. Furthermore, immunofluorescence microscopy and western blot study revealed reduced expression and activation of epidermal growth factor receptor (EGFR) in PAC DP-9 treated SKOV-3 cells. In addition, quercetin aglycone and PAC DP-9 deactivated MAPK-ERK pathway, induced downregulation of cyclin D1, DNA-PK, phospho-histone H3 and upregulation of p21, and arrested cell cycle progression. Overall, this study demonstrates promising in vitro cytotoxic and anti-proliferative properties of two newly characterized cranberry flavonoids, quercetin aglycone and PAC DP-9, against ovarian cancer cells.

Pine bark extracts: nutraceutical, pharmacological, and toxicological evaluation.

Proanthocyanidins are among the most abundant constituents in pine bark extracts (PBEs). This review summarizes medical research on PBEs from Pinus pinaster, Pinus radiata, Pinus massoniana, and other less well characterized species. The precise mechanisms of the important physiologic functions of PBE components remain to be elucidated, but there is evidently great potential for the identification and development of novel antioxidant, anti-inflammatory, cardiovascular, neuroprotective, and anticancer medicines. Although toxicological data for PBEs are limited, no serious adverse effects have been reported. PBEs, therefore, may have potential as nutraceuticals and pharmaceuticals and should be safe for use as food ingredients.

Preliminary in vitro and ex vivo evaluation of afzelin, kaempferitrin and pterogynoside action over free radicals and reactive oxygen species.

Biological activities of flavonoids have been extensively reviewed in literature. The biochemical profile of afzelin, kaempferitrin, and pterogynoside acting on reactive oxygen species was investigated in this paper. The flavonoids were able to act as scavengers of the superoxide anion, hypochlorous acid and taurine chloramine. Although flavonoids are naturally occurring substances in plants which antioxidant activities have been widely advertised as beneficial, afzelin, kaempferitrin, and pterogynoside were able to promote cytotoxic effect. In red blood cells this toxicity was enhanced, depending on flavonoids concentration, in the presence of hypochlorous acid, but reduced in the presence of 2,2'-azo-bis(2-amidinopropane) free radical. These flavonoids had also promoted the death of neutrophils, which was exacerbated when the oxidative burst was initiated by phorbol miristate acetate. Therefore, despite their well-known scavenging action toward free radicals and oxidants, these compounds could be very harmful to living organisms through their action over erythrocytes and neutrophils.

Oligomeric proanthocyanidins alleviate hexabromocyclododecane-induced cytotoxicity in HepG2 cells through regulation on ROS formation and mitochondrial pathway.

Hexabromocyclododecane (HBCD), a type of brominated flame retardants (BFR), has become ubiquitous organic contaminants in recent years. However, studies on HBCD toxicity and the related molecular mechanisms are so far limited. The objective of the present study was to investigate the effects of oligomeric proanthocyanidins (OPCs) on cytotoxicity induced by HBCD and the underlying molecular mechanisms. HepG2 cells were treated with HBCD and/or OPCs at different concentrations, and cell viability, cell apoptosis, reactive oxygen species (ROS) production, cellular Ca(2+) level, mitochondrial membrane potential (ΔΨ), cytochrome C (Cyt-c) release, and nuclear factor-erythroid 2-related factor 2 (Nrf2) proteins expression were evaluated. Results showed that HBCD induced toxic effects in HepG2 cells in a concentration-dependent manner. HBCD at high concentrations (40 and 60µM) caused a significant decrease of cell viability and led to elevated cell apoptosis ratio, intracellular Ca(2+) level, cytoplasmic Cyt-c level, and ROS production, together with a loss of ΔΨ and mobilization of Nrf2. Pretreatment with OPCs effectively attenuated the cytotoxic effects and ROS production, as well as mitochondrial responses induced by HBCD. Thus, OPCs could alleviate cytotoxicity in HepG2 cells induced by HBCD through regulation on intracellular Ca(2+) level and ROS formation in a mitochondrial pathway.

Production, composition and toxicology studies of Enzogenol® Pinus radiata bark extract.

Enzogenol® pine bark extract is a dietary supplement and food ingredient produced by water extraction of Pinus radiata. We present production method, composition, and safety data from rat and dog toxicological and human clinical studies. The dry powder contains proanthocyanidins (>80%), taxifolin (1-2%), other flavonoids and phenolic acids (up to 8%), and carbohydrates (5-10%). Reverse mutation assays showed lack of mutagenic activity. Single and 14-day repeat dosing in rats and dogs had no influence on body weight, feed consumption, blood chemistry, and haematology at any dose level. There were no treatment related findings on gross and detailed necroscopy, organ weights, organ weight ratios and histology. The only adverse events were emesis and diarrhoea in dogs occurring mainly in un-fed condition and at the highest dose level in a total of 18% of applications. The MTD and NOAEL in the present rat and dog studies were 2500 and 750 mg/kg/day, respectively. Consumption of 480 mg/day for 6 months and 960 mg/day for 5 weeks in two human studies showed Enzogenol® had no adverse influence on liver and kidney function, haematology, and did not cause any adverse events. Our studies indicate lack of toxicity of Enzogenol® and support safe use as a food ingredient.

Antiproliferative and apoptotic effects triggered by Grape Seed Extract (GSE) versus epigallocatechin and procyanidins on colon cancer cell lines.

Grape seed extract has been proven to exert anticancer effects on different tumors. These effects are mainly ascribed to catechin and procyanidin content. Analytical studies demonstrated that grape seed extract composition is complex and it is likely other components could exert biological activities. Using cell count and flow cytometry assays, we evaluated the cytostatic and apoptotic effects produced by three different grape seed extracts from Italia, Palieri and Red Globe cultivars, on Caco2 and HCT-8 colon cancer cells. These effects were compared to those induced by epigallocatechin and procyanidins, alone or in association, on the same cell lines. All the extracts induced growth inhibition and apoptosis in Caco2 and HCT-8 cells, along the intrinsic apoptotic pathway. On both cell lines, growth inhibition induced by Italia and Palieri grape seed extracts was significantly higher than that it has been recorded with epigallocatechin, procyanidins and their association. In Caco2 cells, the extract from Red Globe cultivar was less effective in inducing growth inhibition than procyanidins alone and in association with epigallocatechin, whereas, in HCT-8 cells, only the association of epigallocatechin and procyanidins triggers a significant proliferation decrease. On both cell lines, apoptosis induced by Italia, Palieri and Red Globe grape seed extracts was considerably higher than has been recorded with epigallocatechin, procyanidins and their association. These data support the hypothesis by which other compounds, present in the grape seed extracts, are likely to enhance the anticancer effects.

The mutagenic, DNA-damaging and antioxidative properties of bark and leaf extracts from Coutarea hexandra (Jacq.) K. Schum.

Coutarea hexandra is a species commonly known in Brazil as quina, and its bark is used in folk medicine. In this study, we assess the mutagenic and DNA-damaging effects of ethanol extracts from C. hexandra stem bark (SCH) and leaves (LCH) by employing the Ames test on the TA98 and TA100 strains of Salmonella typhimurium in addition to a plasmid treatment test. Furthermore, we performed a phytochemical analysis by TLC and HPLC, a quantification of the phenolic constituents and an assessment of the antioxidative activity. SCH and LCH showed mutagenic action in the Ames test for TA98 strains after metabolic activation. LCH also showed mutagenicity for the TA100 strain after metabolic activation. The findings from the plasmid treatment test did not indicate any DNA-damaging activity for either of the extracts with the tested dosages. SCH showed greater flavonoid content and greater antioxidative potential in relation to LCH. This study suggests that caution is advisable in the use of this plant. However, in vivo studies should be conducted to confirm these data.

High-throughput screening for anti-influenza A virus drugs and study of the mechanism of procyanidin on influenza A virus-induced autophagy.

In this research, we have established a high-throughput screening (HTS) platform based on the influenza A virus (IAV) vRNA promoter. Using this HTS platform, we selected 35 medicinal plants out of 83 examples of traditional Chinese medicine and found that 7 examples had not been reported. After examining many previous reports, we found that Vaccinium angustifolium Ait., Vitis vinifera L, and Cinnamomum cassia Presl had a common active compound, procyanidin, and then determined the anti-IAV effect of procyanidin and explored its mechanism of action. With a plaque inhibition assay and a time-of-addition experiment, we found that procyanidin could inhibit the IAV replication at several stages of the life cycle. In the Western blot and EGFP-LC3 localization assays, we found that procyanidin could inhibit the accumulation of LC3II and the dot-like aggregation of EGFP-LC3. In the RT-PCR and Western blot assays, we found procyanidin could inhibit the expression of Atg7, Atg5, and Atg12. Finally, by the bimolecular fluorescence complementation-fluorescence resonance energy transfer and co-immunoprecipitation assays, we found that procyanidin could inhibit the formation of the Atg5-Atg12/Atg16 heterotrimer and the dissociation of the beclin1/bcl2 heterodimer. In conclusion, we have established an HTS platform and identified procyanidin as a novel and promising anti-IAV agent.

Toxicology evaluation of a procyanidin-rich extract from grape skins and seeds.

The procyanidin-rich extract from grape seeds and skins (GSSE) has antioxidant properties which may have cardioprotective effects. Since it might be interesting to incorporate this extract into a functional food, toxicological tests need to be made to determine how safe it is. In this study we carried out a limit test to determine the acute oral toxicity and the lethal dose 50 (LD50) and some genotoxicity tests of the extract in rats. The LD50 was higher than 5000 mg/kg. Doses of up to 2000 mg/kg showed no increase in micronucleated erythrocytes 72 h after treatment. The bacterial reverse mutation test showed that the extract was weakly mutagenic to the dose of 5 mg/plate and 19.5 and 9.7 µg/ml of GSSE did not show significant differences in the frequency of aberrant metaphases in relation to negative controls. Our results indicated slight mutagenicity under the study conditions, so further studies should be conducted at lower doses to demonstrate that this extract is not toxic.

Safety evaluation of proanthocyanidin polymer-rich fraction obtained from stem bark of Stryphnodendron adstringens (BARBATIMAO) for use as a pharmacological agent.

The widespread use of medicinal plants among the Brazilian population warrants an assessment of the potential risks associated with their intake. Stryphnodendron adstringens (barbatimão) is one of the most frequently used medicinal plants in Brazil, and the risks associated with its use have yet to be investigated. This study evaluated the genotoxic safety of the use of the proanthocyanidin polymer-rich fraction (F2) of stem bark of S. adstringens. The micronucleus test with 750, 1500, and 2250 mg kg(-1) of F2 administered in Mus musculus (Swiss) outbred mice, showed respectively, 5.0±0.8 (Mean±S.D.), 9.1±1.7, and 10.6±1.9 micronucleated polychromatic erythrocytes (MNPCE). A positive control with cyclophosphamide resulted in 21.0±3.8 MNPCE. Antimutagenicity was also evaluated, by adding 750 mg kg(-1) to cyclophosphamide; the result of 8.7±1.4 showed a protective cytotoxic effect. For the Artemia salina test, 10, 100, and 1000 mg L(-1) of F2 showed, respectively, 8.7±0.6, 7.7±0.6, and 5.7±1.2 survival, i.e., F2 did not inhibit 50% of the population when compared to the control (9.7±0.6). These results indicated that F2 obtained from stem bark of S. adstringens has no genotoxic activity.

Development of a grape seed polyphenolic extract with anti-oligomeric activity as a novel treatment in progressive supranuclear palsy and other tauopathies.

A diverse group of neurodegenerative diseases - including progressive supranuclear palsy (PSP), corticobasal degeneration and Alzheimer's disease among others, collectively referred to as tauopathies - are characterized by progressive, age-dependent intracellular formations of misfolded protein aggregates that play key roles in the initiation and progression of neuropathogenesis. Recent studies from our laboratory reveal that grape seed-derived polyphenolic extracts (GSPE) potently prevent tau fibrillization into neurotoxic aggregates and therapeutically promote the dissociation of preformed tau aggregates [J. Alzheimer's Dis. (2009) vol. 16, pp. 433]. Based on our extensive bioavailability, bioactivity and functional preclinical studies, combined with the safety of GSPE in laboratory animals and in humans, we initiated a series of studies exploring the role of GSPE (Meganatural-Az(®) GSPE) as a potential novel botanical drug for the treatment of certain forms of tauopathies including PSP, a neurodegenerative disorder involving the accumulation and deposition of misfolded tau proteins in the brain characterized, in part, by abnormal intracellular tau inclusions in specific anatomical areas involving astrocytes, oligodendrocytes and neurons [J. Neuropathol. Exp. Neurol. (2002) vol. 61, pp. 33]. In this mini-review article, we discuss the biochemical characterization of GSPE in our laboratory and its potential preventative and therapeutic role in model systems of abnormal tau processing pertinent to PSP and related tauopathies.

Condensed tannin intake in spiny-forest-dwelling Lemur catta at Berenty reserve, Madagascar, during reproductive periods.

Primates vary in their choice or avoidance of plant foods high in condensed tannins (CT). Our study focused on CT intake in two groups of Lemur catta residing in spiny forest habitat during two reproductive periods. We examined whether L. catta in this habitat avoided plant foods high in CT, and whether reproductive females ingested lower concentrations of CT compared with males, since CT consumption compromises protein absorption. Feeding data and plant food samples were collected during reproductive periods in 2006 (early/mid-lactation) and 2007 (early gestation). Food samples were assayed for CT content, and average CT intake was determined for all focal animals. No significant difference was found in CT content of the most commonly consumed foods compared with other foods in each season or between seasons. No sex differences occurred in CT consumption in either reproductive period. These L. catta did not avoid plant foods high in CT, and three strategies - ingesting small amounts of CT regularly, high amounts only over short periods, and geophagy - may assist these lemurs in coping with CT content in their highly seasonal diet. L. catta may also be somewhat physiologically adapted to cope with CT concentrations in their plant foods.

Highly oligomeric procyanidins from areca nut induce lymphocyte apoptosis via the depletion of intracellular thiols.

Procyanidins are plant-derived polyphenolic compounds possessing a variety of biological activities, such as immunomodulation, and induction of tumor cell apoptosis. We previously reported that total extract of areca nut exhibited a suppressive effect on the metabolic activity and cytokine expression in normal splenic lymphocytes. As areca nut contains a rich amount of polyphenols, the objective of the present study was to investigate the pro-apoptotic effect of polyphenol-enriched areca nut extract (PANE) and its fractionated oligomeric procyanidins in splenic lymphocytes. Our data showed that PANE markedly induced lymphocyte apoptosis in a concentration- and time-dependent manner. Notably, the fractionated oligomeric procyanidins from pentamers to decamers were active in inducing the apoptosis, whereas monomers to tetramers were inactive. In addition, a marked diminishment in the level of intracellular thiols was revealed in lymphocytes treated with pentamers to decamers. Pretreatment with N-acetyl-L-cysteine, a precursor of glutathione, resulted in significant attenuation of both apoptosis and thiol diminishment induced by areca procyanidins. Taken together, our results indicated that highly oligomeric procyanidins derived from areca nut exhibited a chain length-dependent pro-apoptotic effect in primary lymphocytes, which is mediated, at least in part, by the diminishment of intracellular thiols.