Biocontrol of Fusarium Species Utilizing Indigenous Rooibos and Honeybush Extracts.

Mycotoxins produced by several Fusarium species have a significant effect on reducing maize yield and grain quality and have led to food safety concerns. The antifungal activities of rooibos (Aspalathus linearis) and honeybush (Cyclopia species) tea extracts reduced the growth of plant pathogen Botrytis cinerea, but their efficacy against Fusarium spp. is unknown. In this study, we examined the effects of fermented and unfermented rooibos (A. linearis) and honeybush (Cyclopia subternata) aqueous extracts as well as green tea (Camellia sinensis) against 10 Fusarium species. Conidial viability was assessed by fluorescence microscopy dyes, ATP production was determined using the BacTiter-Glo assay, the mode of action was analyzed by scanning electron microscopy (SEM), and quantification of polyphenols was done using high-performance liquid chromatography with diode array detection (HPLC-DAD). Fermented rooibos extract demonstrated the highest antifungal activity (P < 0.0001) against Fusarium verticillioides MRC 826-E, Fusarium subglutinans MRC 8553, Fusarium proliferatum MRC 8549, and Fusarium globosum MRC 6647, with only 9.53%, 9.26%, 11.0%, and 12.7% ATP production, respectively, followed by antifungal activity of the fermented C. subternata extract against F. subglutinans MRC 8553, F. subglutinans MRC 8554, F. proliferatum MRC 8550, and F. verticillioides MRC 826-E with 3.79%, 6.04%, 6.04%, and 8.40% ATP production, respectively. Extract-treated conidia examined by SEM exhibited disruption of conidial hyphae and collapsed spores. Overall, the fermented rooibos and C. subternata extracts showed higher antifungal activity against the Fusarium species than the unfermented extracts. IMPORTANCE In maize subsistence farming areas in South Africa, daily consumption of maize contaminated by high level of mycotoxins contributes to long-term health effects such as immune deficiency and cancer. Biocontrol methods that are safe and cost-effective are critical to addressing this public health problem. Plant extracts known as biocides or green pesticides are alternatives to chemical pesticides due to their safety and eco-friendly properties. In South Africa, rooibos (Aspalathus linearis) and honeybush (Cyclopia species) contain polyphenols with significant antioxidant and antimicrobial properties. These indigenous herbal teas are widely available and consumed in South Africa and have potential as an innovative approach to reduce mycotoxin levels and, subsequently, human and animal exposure to these toxins. This study evaluates the efficacy of the antifungal activities of several aqueous extracts prepared from fermented and unfermented rooibos (A. linearis), honeybush (Cyclopia subternata), and green tea (Camellia sinensis) on 10 Fusarium strains.

Differential Cytotoxicity of Rooibos and Green Tea Extracts against Primary Rat Hepatocytes and Human Liver and Colon Cancer Cells - Causal Role of Major Flavonoids.

Differential anti-proliferative and pro-apoptotic effects of aqueous extracts of green rooibos (Rg; Aspalathus linearis) and green tea (GT; Camellia sinensis) and an aspalathin-enriched extract of green rooibos (GRE), were investigated in primary rat hepatocytes (PH) and human liver (HepG2) and colon (HT-29) cancer cells. Rooibos flavonoids, aspalathin and luteolin, and the green tea flavanol, epigallocatechin gallate (EGCG), were included to assess their contribution relative to their extract concentrations. GRE was the most effective in reducing cell growth parameters which was associated with a high total polyphenol content and high ferric reducing potential. Differential cell responses were noticed with HepG2 cells more sensitive than PH toward the induction of apoptosis by GRE. Luteolin induced apoptosis in PH and HepG2 cells while aspalathin lacked any effect. EGCG induced apoptosis in HepG2 cells while PH were resistant. HT-29 cells were resistant to apoptosis induction by the tea and pure flavonoids. Differences existed in the individual effects of the major rooibos and GT flavonoids against cell growth parameters compared to their equivalent concentrations in the extract mixtures. Diversity of the flavonoid constituents, physicochemical properties and cellular redox status governing cell survival are likely to explain the differential cell responses.

Differential Modulation of Gene Expression Encoding Hepatic and Renal Xenobiotic Metabolizing Enzymes by an Aspalathin-Enriched Rooibos Extract and Aspalathin.

Modulation of the expression of hepatic and renal genes encoding xenobiotic metabolizing enzymes by an aspalathin-enriched green rooibos (Aspalathus linearis) extract (GRE) was investigated in the liver and kidneys of F344 rats following dietary exposure of 28 d, as well as selected xenobiotic metabolizing genes in rat primary hepatocytes. In the liver, GRE upregulated genes (p < 0.05) encoding aldehyde dehydrogenase, glucose phosphate isomerase, and cytochrome P450 while 17ß-hydroxysteroid dehydrogenase 2 (Hsd17ß2) was downregulated. In primary hepatocytes, GRE lacked any effect, while aspalathin downregulated Hsd17ß2, mimicking the effect of GRE in vivo, and upregulated catechol-O-methyl transferase and marginally (p < 0.1) cytochrome P450 2e1. In the kidneys, GRE upregulated (p < 0.05) genes encoding the phase II xenobiotic metabolism enzymes, glutathione-S-transferase mµ and microsomal glutathione-S-transferase, while downregulating genes encoding the ATP binding cassette transporter, cytochrome P450, gamma glutamyltransferase 1, and N-acetyltransferase 1. Differential modulation of the expression of xenobiotic metabolizing genes in vivo and in vitro by GRE is dose-related, duration of exposure, the tissue type, and interactions between specific polyphenol and/or combinations thereof. Aspalathin is likely to be responsible for the downregulation of estradiol and testosterone catabolism by GRE in the liver. The differential gene expression by GRE in the liver and kidneys could, depending on the duration exposure and dose utilized, determine the safe use of such an extract in humans for specific health and/or disease outcomes.

The mutagenic and antimutagenic activity of Sutherlandia frutescens extracts and marker compounds.

BACKGROUND: Sutherlandia frutescens (L.) R. Br is endemic to Southern Africa where it has been traditionally used for cancer and diabetes. In recent times it has been marketed for its reputed (but not proven) anticancer, antidiabetic and anti-HIV properties. Little is known about the mutagenic and antimutagenic potential of extracts and common marker compounds of Sutherlandia frutescens. Therefore this study aimed to investigate the putative efficacy and possible long-term adverse effects of using this herb. METHODS: Ethylacetate (EA) and 50% Methanol (MeOH) extracts were screened for mutagenic and antimutagenic activity using the Ames assay utilising TA97a, TA98, TA100 and TA102 in the presence and absence of metabolic activation. Four compounds, L-arginine, L-canavanine, GABA and D-pinitol known to occur in sutherlandia were also included. The total polyphenolic content of the both extracts was determined using the Folin-Ciocalteau method and FRAP and ABTS were used to determine the anti-oxidant potential of the extracts. RESULTS: The extracts and the standards did not show any cytotoxicity except in TA97a. The EA extract exhibited antimutagenicity against all the bacterial strains at all concentrations tested. The MeOH extract showed both pro-mutagenic and antimutagenic activities with 2-acetamidofluorene and aflatoxin B1 in the presence of metabolic activation of TA98 and TA100, respectively. All compounds, except L-canavanine exhibited antimutagenic activity against all strains. L-canavanine, on the other hand showed co-mutagenicity with 9-aminoacridine on TA97a, at all test concentrations. The extracts and pure compounds exhibited their antimutagenic activity in a dose response manner. L-arginine and GABA showed an some antimutagenic response. EA extract had three times the total phenolic content (12.56 µg GE / mg) observed in the MeOH extract. There was correlation between total phenolic content, antioxidant potential and antimutagenicity. CONCLUSION: Both extracts exhibited a protective effect, with the EA extract exhibiting greater potency. L-canavanine acted as a co-mutagen in a dose response manner without metabolic activation. It is suggested that the EA extract be priotized for future development work as it showed a better risk profile and activity.

In Vitro Chemopreventive Properties of Green Tea, Rooibos and Honeybush Extracts in Skin Cells.

The chemopreventive properties of the herbal teas rooibos (Aspalathus linearis) and honeybush (Cyclopia spp.) have been demonstrated on mouse skin in vivo but the underlying mechanisms are not clear. The aim of the current study was to determine the anti-proliferative and pro-apoptotic activity of methanol and aqueous extracts of rooibos and two Cyclopia species in different skin cells, using green tea (Camellia sinensis) as a benchmark. Extracts were also characterised for their major individual polyphenols by high performance liquid chromatography and spectroscopically for the total polyphenol (TP) groups. The methanol extract of rooibos, containing higher levels of polyphenols than its aqueous extract, displayed similar activity to green tea as it selectively targeted premalignant cells by inhibiting cell proliferation at lower concentrations whilst inducing apoptosis via membrane depolarisation at higher concentrations. Specific roles of the major rooibos dihydrochalcones and flavanol/proanthocyanidin-type (FLAVA) compounds are likely to be involved. The aqueous extracts of the Cyclopia species were more active against cell proliferation and at inducing apoptosis which was associated with a higher FLAVA content and a reduced TP/FLAVA ratio. In contrast, their methanol extracts exhibited a cytoprotective effect against apoptosis which was related to their monomeric xanthone and flavanone content. The underlying chemopreventive properties of green tea and the herbal teas appear to be associated with diverse and complex monomeric/polymeric polyphenolic cell interactions.

Anti-Inflammatory Effects of Aspalathus linearis and Cyclopia spp. Extracts in a UVB/Keratinocyte (HaCaT) Model Utilising Interleukin-1α Accumulation as Biomarker.

Ultraviolet B (UVB) radiation is one of the major predisposing risk factors of skin cancer. The anticancer and photoprotective effects of unoxidized rooibos (Aspalathus linearis) and honeybush (Cyclopia) herbal teas, containing high levels of dihydrochalones and xanthones, respectively, have been demonstrated in skin cancer models in vivo. In the current study, the anti-inflammatory effects of methanol and aqueous extracts of these herbal teas were investigated in a UVB/HaCaT keratinocyte model with intracellular interleukin-1α (icIL-1α) accumulation as a biomarker. Extracts of green tea (Camellia sinensis) served as benchmark. Both extracts of green tea and rooibos, as well as the aqueous extract of C. intermedia, enhanced UVB-induced inhibition of cell viability, proliferation and induction of apoptosis, facilitating the removal of icIL-1α. The underlying mechanisms may involve mitochondrial dysfunction exhibiting pro-oxidant responses via polyphenol-iron interactions. The methanol extracts of honeybush, however, protected against UVB-induced reduction of cell growth parameters, presumably via antioxidant mechanisms that prevented the removal of highly inflamed icIL-1α-containing keratinocytes via apoptosis. The dual antioxidant and/or pro-oxidant role of the polyphenolic herbal tea constituents should be considered in developing preventive strategies against UVB-induced skin carcinogenesis. The indirect removal of UVB damaged keratinocytes by herbal tea extracts via apoptosis may find application in the prevention of photo-induced inflammation.

The potential role of polyphenols in the modulation of skin cell viability by Aspalathus linearis and Cyclopia spp. herbal tea extracts in vitro.

OBJECTIVES: The relationship between polyphenol constituents, antioxidant properties of aqueous and methanol extracts of green tea (Camellia sinensis), the herbal teas, rooibos (Aspalathus linearis) and honeybush (Cyclopia spp.), against skin cell viability was investigated in vitro. METHODS: The effect of extracts, characterised in terms of polyphenol content and antioxidant properties, on cell viability of premalignant, normal and malignant skin cells was determined. KEY FINDINGS: Phenolic composition, particularly high levels of potent antioxidants, of rooibos and green tea methanol extracts was associated with a strong reduction in cell viability specifically targeting premalignant cells. In contrast, the aqueous extracts of Cyclopia spp. were more effective in reducing cell viability. This correlated with a relatively high flavanol/proanthocyanidin content and ABTS radical cation scavenging capacity. The major green tea flavanol (epigallocatechin gallate) and rooibos dihydrochalcone (aspalathin) exhibited differential effects against cell viability, while the major honeybush xanthone (mangiferin) and flavanone (hesperidin) lacked any effect presumably due to a cytoprotective effect. The underlying mechanisms against skin cell viability are likely to involve mitochondrial dysfunction resulting from polyphenol-iron interactions. CONCLUSIONS: The polyphenol constituents and antioxidant parameters of herbal tea extracts are useful tools to predict their activity against skin cell survival in vitro and potential chemopreventive effects in vivo.

Biologically Based Methods for Control of Fumonisin-Producing Fusarium Species and Reduction of the Fumonisins.

Infection by the fumonisin-producing Fusarium spp. and subsequent fumonisin contamination of maize adversely affect international trade and economy with deleterious effects on human and animal health. In developed countries high standards of the major food suppliers and retailers are upheld and regulatory controls deter the importation and local marketing of fumonisin-contaminated food products. In developing countries regulatory measures are either lacking or poorly enforced, due to food insecurity, resulting in an increased mycotoxin exposure. The lack and poor accessibility of effective and environmentally safe control methods have led to an increased interest in practical and biological alternatives to reduce fumonisin intake. These include the application of natural resources, including plants, microbial cultures, genetic material thereof, or clay minerals pre- and post-harvest. Pre-harvest approaches include breeding for resistant maize cultivars, introduction of biocontrol microorganisms, application of phenolic plant extracts, and expression of antifungal proteins and fumonisin degrading enzymes in transgenic maize cultivars. Post-harvest approaches include the removal of fumonisins by natural clay adsorbents and enzymatic degradation of fumonisins through decarboxylation and deamination by recombinant carboxylesterase and aminotransferase enzymes. Although, the knowledge base on biological control methods has expanded, only a limited number of authorized decontamination products and methods are commercially available. As many studies detailed the use of natural compounds in vitro, concepts in reducing fumonisin contamination should be developed further for application in planta and in the field pre-harvest, post-harvest, and during storage and food-processing. In developed countries an integrated approach, involving good agricultural management practices, hazard analysis and critical control point (HACCP) production, and storage management, together with selected biologically based treatments, mild chemical and physical treatments could reduce fumonisin contamination effectively. In rural subsistence farming communities, simple, practical, and culturally acceptable hand-sorting, maize kernel washing, and dehulling intervention methods proved to be effective as a last line of defense for reducing fumonisin exposure. Biologically based methods for control of fumonisin-producing Fusarium spp. and decontamination of the fumonisins could have potential commercial application, while simple and practical intervention strategies could also impact positively on food safety and security, especially in rural populations reliant on maize as a dietary staple.

Short-Term and Sub-Chronic Dietary Exposure to Aspalathin-Enriched Green Rooibos (Aspalathus linearis) Extract Affects Rat Liver Function and Antioxidant Status.

An aspalathin-enriched green rooibos (Aspalathus linearis) extract (GRE) was fed to male Fischer rats in two independent studies for 28 and 90 days. The average dietary total polyphenol (TP) intake was 756 and 627 mg Gallic acid equivalents (GAE)/kg body weight (bw)/day over 28 and 90 days, respectively, equaling human equivalent doses (HEDs) of 123 and 102 GAE mg/kg bw/day. Aspalathin intake of 295 mg/kg bw/day represents a HED of 48 mg/kg bw/day (90 day study). Consumption of GRE increased feed intake significantly (p < 0.05) compared to the control after 90 days, but no effect on body and organ weight parameters was observed. GRE significantly (p < 0.05) reduced serum total cholesterol and iron levels, whilst significantly (p < 0.05) increasing alkaline phosphatase enzyme activity after 90 days. Endogenous antioxidant enzyme activity in the liver, i.e., catalase and superoxide dismutase activity, was not adversely affected. Glutathione reductase activity significantly (p < 0.05) increased after 28 days, while glutathione (GSH) content was decreased after 90 days, suggesting an altered glutathione redox cycle. Quantitative Real Time polymerase chain reaction (PCR) analysis showed altered expression of certain antioxidant defense and oxidative stress related genes, indicative, among others, of an underlying oxidative stress related to changes in the GSH redox pathway and possible biliary dysfunction.

The influence of Aspalathus linearis (Rooibos) and dihydrochalcones on adrenal steroidogenesis: quantification of steroid intermediates and end products in H295R cells.

The steroid hormone output of the adrenal gland is crucial in the maintenance of hormonal homeostasis, with hormonal imbalances being associated with numerous clinical conditions which include, amongst others, hypertension, metabolic syndrome, cardiovascular disease, insulin resistance and type 2 diabetes. Aspalathus linearis (Rooibos), which has been reported to aid stress-related symptoms linked to metabolic diseases, contains a wide spectrum of bioactive phenolic compounds of which aspalathin is unique. In this study the inhibitory effects of Rooibos and the dihydrochalcones, aspalathin and nothofagin, were investigated on adrenal steroidogenesis. The activities of both cytochrome P450 17α-hydroxylase/17,20 lyase and cytochrome P450 21-hydroxylase were significantly inhibited in COS-1 cells. In order to study the effect of these compounds in H295R cells, a human adrenal carcinoma cell line, a novel UPLC-MS/MS method was developed for the detection and quantification of twenty-one steroid metabolites using a single chromatographic separation. Under both basal and forskolin-stimulated conditions, the total amount of steroids produced in H295R cells significantly decreased in the presence of Rooibos, aspalathin and nothofagin. Under stimulated conditions, Rooibos decreased the total steroid output 4-fold and resulted in a significant reduction of aldosterone and cortisol precursors. Dehydroepiandrosterone-sulfate levels were unchanged, while the levels of androstenedione (A4) and 11ß-hydroxyandrostenedione (11ßOH-A4) were inhibited 5.5 and 2.3-fold, respectively. Quantification of 11ßOH-A4 showed this metabolite to be a major product of steroidogenesis in H295R cells and we confirm, for the first time, that this steroid metabolite is the product of the hydroxylation of A4 by human cytochrome P450 11ß-hydroxylase. Taken together our results demonstrate that Rooibos, aspalathin and nothofagin influence steroid hormone biosynthesis and the flux through the mineralocorticoid, glucocorticoid and androgen pathways, thus possibly contributing to the alleviation of negative effects arising from elevated glucocorticoid levels.

Modulating effects of rooibos and honeybush herbal teas on the development of esophageal papillomas in rats.

Widespread consumption of herbal teas has stimulated interest in their role as cancer preventive agents. The present investigation monitored the modulation of methylbenzylnitrosamine (MBN)-induced esophageal squamous cell carcinogenesis by rooibos (Aspalathus linearis) and honeybush (Cyclopia intermedia) herbal and Camellia sinensis teas in male F344 rats. The tumor multiplicity was significantly (P < 0.05) inhibited by unfermented honeybush (45.5%), green (50%), and black (36%) teas, while the other teas exhibited weaker effects (<30% inhibition). The mean total papilloma size was reduced by unfermented rooibos (87%), unfermented honeybush (94%), and fermented honeybush (74%) due to the absence of large papillomas (>10 mm(3)). Reduction of the mean total papilloma number correlated with the total polyphenol (TPP) (r = 0.79; P < 0.02) and flavanol/proanthocyanidin (FLAVA) (r = 0.89; P < 0.008) intake (mg/100 g body weight) of the teas and the FLAVA (r = 0.89; P < 0.04) and flavonol/flavones/xanthones (r = 0.99; P < 0.002) intake when considering only the herbal teas. A daily TPP intake threshold of 7 mg/100 g body weight existed below where no inhibition of papilloma development was observed. Fermentation of herbal teas reduced the inhibitory effects on papilloma development associated with a reduction in the polyphenolic constituents. The inhibitory effect of herbal teas on papilloma development is associated with different flavonoid subgroups and/or combination thereof.

In vitro hepatic biotransformation of aspalathin and nothofagin, dihydrochalcones of rooibos (Aspalathus linearis), and assessment of metabolite antioxidant activity.

Aspalathin (2',3,4,4',6'-pentahydroxy-3'-C-beta-d-glucopyranosyldihydrochalcone) is the major flavonoid present in the South African herbal tea rooibos. In vitro metabolism of aspalathin and a structural analogue nothofagin, lacking the A ring catechol group, was investigated by monitoring the formation of glucuronyl and sulfate conjugates using Aroclor 1254 induced and uninduced rat liver microsomal and cytosolic subcellular fractions. Following glucuronidation of both aspalathin and nothofagin, HPLC-DAD, LC-MS, and LC-MS/MS analyses indicated the presence of two metabolites: one major and one minor. Only one aspalathin metabolite was obtained after sulfation, while no metabolites were observed for nothofagin. Two likely sites of conjugation for aspalathin are 4-OH or 3-OH on the A-ring. For nothofagin, the 4-OH (A-ring) and 6'-OH (B-ring) seem to be involved. The glucuronyl conjugates of aspalathin lack any radical scavenging properties in online postcolumn DPPH radical and ABTS radical cation assays. Deconjugation assays utilizing glucuronidase and sulfatase resulted in the disappearance of the metabolites, with the concomitant formation of the unconjugated form in the case of the glucuronidated product. The balance between conjugated and unconjugated forms of aspalathin could have important implications regarding its role in affecting oxidative status in intra- and extracellular environments in vivo.

Phenolic contribution of South African herbal teas to a healthy diet.

South African herbal teas, rooibos and honeybush, are increasingly enjoyed as healthy alternatives to Camellia sinensis teas. They contribute to the diet with bioactive phytochemicals not commonly found in foods. Major compounds of rooibos are the unique dihydrochalcone, aspalathin, and its flavone isomers, orientin and isoorientin. Honeybush contributes the xanthones, mangiferin and isomangiferin and the flavanones, eriocitrin, narirutin and hesperidin. All these compounds are either C-glucosides or O-rhamnoglucosides, which are poorly absorbed. Phase II metabolism and degradation by intestinal bacteria are important factors in their absorption. Modulation of drug metabolising enzymes is indicated which not only could affect the therapeutic window of drugs, but also the bioavailability of other dietary flavonoids.

Chemoprotective properties of rooibos (Aspalathus linearis), honeybush (Cyclopia intermedia) herbal and green and black (Camellia sinensis) teas against cancer promotion induced by fumonisin B1 in rat liver.

The chemoprotective properties of unfermented and fermented rooibos (Aspalathus linearis) and honeybush (Cyclopia intermedia) herbal teas, and green and black teas (Camellia sinensis) were investigated against fumonisin B1 (FB1) promotion in rat liver utilizing diethylnitrosamine (DEN) as cancer initiator. The various teas differently affected the clinical chemical parameters associated with liver and kidney damage associated with FB1 suggesting specific FB1/iron/polyphenolic interactions. Green tea enhanced (P<0.05) the FB1-induced reduction of the oxygen radical absorbance capacity, while fermented herbal teas and unfermented honeybush significantly (P<0.05) decreased FB1-induced lipid peroxidation in the liver. The teas exhibited varying effects on FB1-induced changes in the activities of catalase, glutathione peroxidase (GPx) glutathione reductase (GR) as well as the glutathione (GSH) status. Unfermented rooibos and honeybush significantly (P<0.05) to marginally (P<0.1) reduced the total number of foci (>10microm), respectively, while all the teas reduced the relative amount of the larger foci. Fermentation seems to reduce the protective effect of the herbal teas. Differences in the major polyphenolic components and certain FB1/polyphenolic/tissue interactions may explain the varying effects of the different teas on the oxidative parameters, hepatotoxic effects and cancer promotion in rat liver.

Effect of species variation and processing on phenolic composition and in vitro antioxidant activity of aqueous extracts of Cyclopia spp. (Honeybush Tea).

The in vitro antioxidant activity of aqueous extracts prepared from four Cyclopia spp. (unfermented and fermented) was assessed using radical (ABTS *+) scavenging, ferric ion reduction, and inhibition of Fe2+-induced microsomal lipid peroxidation as criteria. Aqueous extracts of unfermented and fermented Aspalathus linearis (rooibos) and Camellia sinensis teas (green, oolong, and black) were included as reference samples. Qualitative and quantitative differences in phenolic composition were demonstrated for the Cyclopia spp. The xanthone glycoside, a.k.a. mangiferin, was the major monomeric polyphenol present in the Cyclopia extracts, with both unfermented and fermented C. genistoides extracts containing the highest quantities. Fermentation resulted in a significant reduction in extract yields and their total polyphenolic and individual polyphenol contents. Unfermented plant material should preferentially be used for preparation of extracts, as fermentation significantly ( P < 0.05) lowered antioxidant activity of all species, except in the case of C. genistoides, where the ability to inhibit lipid peroxidation was not affected. Unfermented plant material also retained the highest concentration of mangiferin. Overall, extracts of unfermented Cyclopia were either of similar or lower antioxidant activity as compared to the other teas. However, the presence of high levels of mangiferin merits the use of Cyclopia spp. and, in particular, C. genistoides, as an alternative herbal tea and potential dietary supplement.

Antioxidant and pro-oxidant activities of aqueous extracts and crude polyphenolic fractions of rooibos (Aspalathus linearis).

Unfermented rooibos tea is known to contain higher levels of total polyphenols and flavonoids than its fermented counterpart, making it the obvious choice for the preparation of flavonoid-enriched fractions. Evaluation of aqueous extracts and crude polyphenolic fractions of unfermented and fermented rooibos showed anti- and/or pro-oxidant activities, using a linoleic acid-Tween-buffer emulsion for lipid peroxidation and the deoxyribose degradation assay, based on a Fenton reaction model system containing FeCl3-EDTA and H2O2 for the generation of hydroxyl radicals. Except for the ethyl acetate fraction, with the highest total polyphenol (TP) content and offering the least protection presumably due to pro-oxidant activity, the inhibition of lipid peroxidation by the samples correlated moderately with their TP content in a linear relationship (r = 0.896, P < 0.01). Using the deoxyribose degradation assay, the pro-oxidant activity of the aqueous extracts and their crude polymeric fractions (0.1 mg/mL in the reaction mixture) was linear with respect to their dihydrochalcone (aspalathin and nothofagin) (r = 0.977, P = 0.023) and flavonoid (r = 0.971, P = 0.029) content. Pro-oxidant activity was demonstrated for pure aspalathin. Using the same assay, but with ascorbate added to regenerate Fe3+ to Fe2+, the aqueous extract and crude polymeric fraction of fermented rooibos displayed hydroxyl radical scavenging activity. Fermentation (i.e., oxidation) of rooibos decreased the pro-oxidant activity of aqueous extracts, which was contributed to a decrease in their dihydrochalcone content. The in vitro pro-oxidant activity displayed by flavonoid-enriched fractions of rooibos demonstrates that one must be aware of the potential adverse biological properties of potent antioxidant extracts utilized as dietary supplements.

Inhibition of tumour promotion in mouse skin by extracts of rooibos (Aspalathus linearis) and honeybush (Cyclopia intermedia), unique South African herbal teas.

The modulating effect of ethanol/acetone (E/A) soluble fractions, prepared from methanolic extracts of processed and unprocessed rooibos (Aspalathus linearis) and honeybush (Cyclopia intermedia) as well as green (Camellia sinensis) teas was established in a two-stage mouse skin carcinogenesis assay. Topical application of the tea fractions prior to the tumour promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), on ICR mouse skin initiated with 7,12-dimethylbenz[a]anthracene (DMBA) suppressed skin tumorigenesis significantly (P<0.001) with the green tea E/A fraction exhibiting a 100% inhibition, unprocessed honeybush 90%, processed honeybush 84.2%, processed rooibos 75% and unprocessed rooibos 60%. The green tea fraction, with the highest flavanol/proanthocyanidin content, also exhibited the highest protective activity (99%) against hepatic microsomal lipid peroxidation, and completely inhibited skin tumour formation. Differences in the flavanol/proanthocyanidin and flavonol/flavone composition and/or non polyphenolic constituents are likely to be important determinants in the inhibition of tumour promotion by the herbal tea E/A fractions in mouse skin.

Fumonisins disrupt sphingolipid metabolism, folate transport, and neural tube development in embryo culture and in vivo: a potential risk factor for human neural tube defects among populations consuming fumonisin-contaminated maize.

Fumonisins are a family of toxic and carcinogenic mycotoxins produced by Fusarium verticillioides (formerly Fusarium moniliforme), a common fungal contaminant of maize. Fumonisins inhibit ceramide synthase, causing accumulation of bioactive intermediates of sphingolipid metabolism (sphinganine and other sphingoid bases and derivatives) as well as depletion of complex sphingolipids, which interferes with the function of some membrane proteins, including the folate-binding protein (human folate receptor alpha). Fumonisin causes neural tube and craniofacial defects in mouse embryos in culture. Many of these effects are prevented by supplemental folic acid. Recent studies in LMBc mice found that fumonisin exposure in utero increases the frequency of developmental defects and administration of folate or a complex sphingolipid is preventive. High incidences of neural tube defects (NTD) occur in some regions of the world where substantial consumption of fumonisins has been documented or plausibly suggested (Guatemala, South Africa, and China); furthermore, a recent study of NTD in border counties of Texas found a significant association between NTD and consumption of tortillas during the first trimester. Hence, we propose that fumonisins are potential risk factors for NTD, craniofacial anomalies, and other birth defects arising from neural crest cells because of their apparent interference with folate utilization.

Modulation of hepatic drug metabolizing enzymes and oxidative status by rooibos (Aspalathus linearis) and Honeybush (Cyclopia intermedia), green and black (Camellia sinensis) teas in rats.

Rooibos and honeybush teas significantly (P < 0.05) enhanced the activity of cytosolic glutathione S-transferase alpha. A significant (P < 0.05) to marginal (P < 0.1) increase in the activity of the microsomal UDP-glucuronosyl transferase was obtained with unprocessed rooibos and honeybush teas, respectively. Oxidized glutathione (GSSG) levels were significantly (P < 0.05) reduced in the liver of all tea treated rats while reduced glutathione (GSH) was markedly increased in the liver of the herbal tea treated rats. These changes resulted in a significant (P < 0.05) increase in the GSH/GSSG ratio by the unprocessed, processed rooibos and unprocessed honeybush teas. Green and black teas markedly to significantly decreased the oxygen radical absorbance capacity in liver homogenates, respectively. Modulation of phase II drug metabolizing enzymes and oxidative status in the liver may be important events in the protection against adverse effects related to mutagenesis and oxidative damage.