Christiaan J. Malherbe

Acute assessment of an aspalathin-enriched green rooibos (Aspalathus linearis) extract with hypoglycemic potential

Rooibos, an endemic South African plant, known for its use as herbal tea, has potential as an antidiabetic herbal product, following recent demonstration of the glucose lowering effect of its major flavonoid, the dihydrochalcone C-glucoside aspalathin. The purpose of this study was to confirm antidiabetic activity for rooibos extract high in aspalathin content. An extract (SB1) was selected after screening for high aspalathin content and α-glucosidase inhibition activity. On-line HPLC-biochemical detection confirmed α-glucosidase inhibitory activity for aspalathin. In vitro the extract induced a dose response increase in glucose uptake (5 × 10⁻⁵ to 5 μg/ml) on C2C12 myotubules. Aspalathin was effective at 1, 10 and 100 μM, while rutin was effective at 100 μM. In the Chang cells only the extract was effective. In vivo the extract sustained a glucose lowering effect comparable to metformin over a 6h period after administration (25mg/kg body weight (BW)) to STZ-induced diabetic rats. In an oral glucose tolerance test the extract (30 mg/kg BW) was more effective than vildagliptin (10mg/kg BW), a dipeptidyl peptidase-4 inhibitor. An aspalathin-rutin mixture (1:1; m/m) dosed at 1.4 mg/kg BW, but not the single compounds separately, reduced blood glucose concentrations of STZ-induced diabetic rats over a 6h monitoring period. The improved hypoglycemic activity of the aspalathin-rutin mixture and the extract illustrated synergistic interactions of polyphenols in complex mixtures.

Variation in phenolic content and antioxidant activity of fermented rooibos herbal tea infusions: role of production season and quality grade.

Data are required to calculate the dietary exposure to rooibos herbal tea flavonoids and phenolic acids. Representative content values for the principal phenolic compounds and total antioxidant capacity of fermented rooibos infusion, taking into account variation caused by production seasons (2009, 2010, and 2011) and quality grades (A, B, C, and D), were determined for samples (n = 114) from different geographical areas and producers. The major phenolic constituents were isoorientin and orientin (>10 mg/L), with quercetin-3-O-robinobioside, phenylpyruvic acid glucoside, and aspalathin present at >5 mg/L. Isovitexin, vitexin, and hyperoside were present at <3 mg/L. Rutin, ferulic acid, and isoquercitrin were present at <2 mg/L. Nothofagin was present at <1 mg/L. Only traces of luteolin-7-O-glucoside and the aglycones quercetin, luteolin, and chrysoeriol were present. Substantial variation was observed in the individual content values of the phenolic compounds and total antioxidant capacity within production seasons and quality grades.

Benzophenone C- and O-Glucosides from Cyclopia genistoides (Honeybush) Inhibit Mammalian α-Glucosidase

An enriched fraction of an aqueous extract prepared from the aerial parts of Cyclopia genistoides Vent. yielded a new benzophenone di-C,O-glucoside, 3-C-β-d-glucopyranosyl-4-O-β-d-glucopyranosyliriflophenone (1), together with small quantities of a known benzophenone C-glucoside, 3-C-β-d-glucopyranosylmaclurin (2). The isolated compounds showed α-glucosidase inhibitory activity against an enzyme mixture extracted from rat intestinal acetone powder. Compound 2 exhibited significantly (p < 0.05) higher inhibitory activity (54%) than 1 (43%) at 200 μM. In vitro tests in several cell models showed that 1 and its 3-C-monoglucosylated derivative (3-C-β-d-glucopyranosyliriflophenone) were marginally effective (p ≥ 0.05) in increasing glucose uptake.

Development of On-Line High Performance Liquid Chromatography (HPLC)-Biochemical Detection Methods as Tools in the Identification of Bioactives

Biochemical detection (BCD) methods are commonly used to screen plant extracts for specific biological activities in batch assays. Traditionally, bioactives in the most active extracts were identified through time-consuming bio-assay guided fractionation until single active compounds could be isolated. Not only are isolation procedures often tedious, but they could also lead to artifact formation. On-line coupling of BCD assays to high performance liquid chromatography (HPLC) is gaining ground as a high resolution screening technique to overcome problems associated with pre-isolation by measuring the effects of compounds post-column directly after separation. To date, several on-line HPLC-BCD assays, applied to whole plant extracts and mixtures, have been published. In this review the focus will fall on enzyme-based, receptor-based and antioxidant assays.

Occurrence and sensory perception of Z-2-(β-d-glucopyranosyloxy)-3-phenylpropenoic acid in rooibos (Aspalathus linearis).

Z-2-(β-d-glucopyranosyloxy)-3-phenylpropenoic acid (PPAG), a compound postulated to contribute to the taste and mouthfeel of fermented rooibos tea (Aspalathus linearis), was isolated from unfermented rooibos plant material. Its structure was unequivocally confirmed by LC-MS, -MS(2), FT-IR and NMR of the underivatised natural product, and optical rotation measurements of the hydrolysed sugar moiety. A similar compound, postulated to be E-2-(β-d-glucopyranosyloxy)-3-phenylpropenoic acid, was also detected. Analysis of the leaves of a large number of rooibos plants (n=54), sampled at commercial plantations, showed that PPAG is not ubiquitously present in detectable quantities in the leaves of different plants. This leads to large variation in the fermented plant material, infusions and food-grade extracts. PPAG was shown to have a slightly bitter to astringent taste and a detection threshold of 0.4 mg/l in water.

Mangiferin glucuronidation: Important hepatic modulation of antioxidant activity

Mangiferin displays an extensive spectrum of pharmacological properties, including antioxidant activity. Its phase II metabolism in the presence of Aroclor 1254-induced and un-induced microsomal and cytosolic fractions from rat liver and the antioxidant potency of the glucuronidated conjugates were investigated. Mangiferin was not a substrate for the cytosolic sulphotransferases. Glucuronidation led to the formation of two monoglucuronidated metabolites of mangiferin and a monoglucuronidated metabolite of homomangiferin (a minor constituent of the mangiferin standard). Deconjugation utilising glucuronidase resulted in the disappearance of the metabolites, with the concomitant formation of the two parent compounds. Considering steric hinderance caused by the C-2 glucosyl moiety and the relative acidity of the xanthone OH groups, the 6-OH of mangiferin and, to a lesser degree the 7-OH, are likely to be the primary glucuronidation targets. The ferric iron reducing ability of the glucuronidated reaction mixture was reduced, while the free radical scavenging abilities of mangiferin, utilising on-line post-column HPLC-DAD-DPPH· and HPLC-DAD-ABTS·+ assays, were eliminated, providing further evidence that the catechol arrangement at C-6 and C-7 was the preferred site of conjugation. This paper provides the first evidence that the glucuronidated metabolites of mangiferin resulted in a loss in free radical scavenging and ferric iron reducing ability.

Isolation of aspalathin and nothofagin from rooibos (Aspalathus linearis) using high-performance countercurrent chromatography: Sample loading and compound stability considerations

Aspalathin and nothofagin, the major dihydrochalcones in rooibos (Aspalathus linearis), are valuable bioactive compounds, but their bioactivity has not been fully elucidated. Isolation of these compounds using high-performance countercurrent chromatography (HPCCC), a gentle, support-free, up-scalable technique, offers an alternative to synthesis for obtaining sufficient amounts. An HPLC-DAD method was adapted to allow rapid (16 min from injection to injection) quantification of the four major compounds (aspalathin, nothofagin, isoorientin, orientin) during development of the isolation protocol. The traditional shake-flask method, used to determine distribution constants (K(D)) for target compounds, was also adapted to obtain higher repeatability. Green rooibos leaves with a high aspalathin and nothofagin content were selected as source material. Sample loading of the polyphenol-enriched extract was limited due to constituents with emulsifying properties, but could be increased by removing ethanol-insoluble matter. Furthermore, problems with degradation of aspalathin during HPCCC separation and further processing could be limited by acidifying the HPCCC solvent system. Aspalathin was shown to be fairly stable at pH 3 (91% remaining after 29 h) compared to pH 7 (45% remaining after 29 h). Aspalathin and nothofagin with high purities (99% and 100%, respectively) were obtained from HPCCC fractions after semi-preparative HPLC.

Potential of Rooibos, its Major C-Glucosyl Flavonoids and Z-2-(β-D-Glucopyranoloxy)-3-phenylpropenoic acid in Prevention of Metabolic Syndrome

ABSTRACT Risk factors of type 2 diabetes mellitus (T2D) and cardiovascular disease (CVD) cluster together and are termed the metabolic syndrome. Key factors driving the metabolic syndrome are inflammation, oxidative stress, insulin resistance (IR), and obesity. IR is defined as the impairment of insulin to achieve its physiological effects, resulting in glucose and lipid metabolic dysfunction in tissues such as muscle, fat, kidney, liver, and pancreatic β-cells. The potential of rooibos extract and its major C-glucosyl flavonoids, in particular aspalathin, a C-glucoside dihydrochalcone, as well as the phenolic precursor, Z-2-(β-D-glucopyranosyloxy)-3-phenylpropenoic acid, to prevent the metabolic syndrome, will be highlighted. The mechanisms whereby these phenolic compounds elicit positive effects on inflammation, cellular oxidative stress and transcription factors that regulate the expression of genes involved in glucose and lipid metabolism will be discussed in terms of their potential in ameliorating features of the metabolic syndrome and the development of serious metabolic disease. An overview of the phenolic composition of rooibos and the changes during processing will provide relevant background on this herbal tea, while a discussion of the bioavailability of the major rooibos C-glucosyl flavonoids will give insight into a key aspect of the bioefficacy of rooibos.

Modeling of the total antioxidant capacity of rooibos (Aspalathus linearis) tea infusions from chromatographic fingerprints and identification of potential antioxidant markers.

Models to predict the total antioxidant capacity (TAC) of rooibos tea infusions from their chromatographic fingerprints and peak table data (content of individual phenolic compounds), obtained using HPLC with diode array detection, were developed in order to identify potential antioxidant markers. Peak table data included the content of 12 compounds, namely phenylpyruvic acid-2-O-glucoside, aspalathin, nothofagin, isoorientin, orientin, ferulic acid, quercetin-3-O-robinobioside, vitexin, hyperoside, rutin, isovitexin and isoquercitrin. The TAC values, measured using the oxygen radical absorbance capacity (ORAC) and DPPH radical scavenging assays, could be predicted from the peak table data or the chromatographic fingerprints (prediction errors 9-12%) using partial least squares (PLS) regression. Prediction models created from samples of only two production years could additionally be used to predict the TAC of samples from another production year (prediction errors<13%) indicating the robustness of the models in a quality control environment. Furthermore, the uninformative variable elimination (UVE)-PLS method was used to identify potential antioxidant markers for rooibos infusions. All individual phenolic compounds that were quantified were selected as informative variables, except vitexin, while UVE-PLS models developed from chromatographic fingerprints indicated additional antioxidant markers, namely (S)-eriodictyol-6-C-glucoside, (R)-eriodictyol-6-C-glucoside, aspalalinin and two unidentified compounds. The potential antioxidant markers should be validated prior to use in quality control of rooibos tea.

Polyphenol-Enriched Fractions of Cyclopia intermedia Selectively Affect Lipogenesis and Lipolysis in 3T3-L1 Adipocytes

Cyclopia species are increasingly investigated as sources of phenolic compounds with potential as therapeutic agents. Recently, we demonstrated that a crude polyphenol-enriched organic fraction (CPEF) of Cyclopia intermedia, currently forming the bulk of commercial production, decreased lipid content in 3T3-L1 adipocytes and inhibited body weight gain in obese db/db mice. The aim of the present study was to determine whether a more effective product and/or one with higher specificity could be obtained by fractionation of the CPEF by purposely increasing xanthone and benzophenone levels. Fractionation of the CPEF using high performance counter-current chromatography (HPCCC) resulted in four fractions (F1-F4), predominantly containing iriflophenone-3-C-β-D-glucoside-4-O-β-D-glucoside (benzophenone: F1), hesperidin (flavanone: F2), mangiferin (xanthone: F3), and neoponcirin (flavone: F4), as quantified by high-performance liquid chromatography with diode array detection (HPLC-DAD), and confirmed by LC-DAD with mass spectrometric (MS) and tandem MS (MSE) detection. All fractions inhibited lipid accumulation in 3T3-L1 pre-adipocytes and decreased lipid content in mature 3T3-L1 adipocytes, although their effects were concentration-dependent. F1-F3 stimulated lipolysis in mature adipocytes. Treatment of mature adipocytes with F1 and F2 increased the messenger RNA expression of hormone sensitive lipase, while treatment with F1 and F4 increased uncoupling protein 3 expression. In conclusion, HPCCC resulted in fractions with different phenolic compounds and varying anti-obesity effects. The activities of fractions were lower than the CPEF; thus, fractionation did not enhance activity within a single fraction worthwhile for exploitation as a nutraceutical product, which illustrates the importance of considering synergistic effects in plant extracts.