Development of sensory tools for green rooibos (Aspalathus linearis (Burm.f.) R.Dahlgren) and changes in quality attributes during shelf-life storage.

BACKGROUND: Green rooibos (Aspalathus linearis (Burm.f.) R.Dahlgren) herbal tea is popular due to its health-promoting properties. Information on its characteristic sensory profile is scarce and sensory tools to define product variation are needed. The storage conditions and time during its shelf-life are hypothesized to affect the product quality. RESULTS: Production batches from two producers spanning 5 years (n = 57) were analyzed using descriptive sensory analysis. Primary attributes (>30 median intensity; 100% occurrence frequency) included 'hay/dried grass', 'cooked oats', 'tobacco', 'honey' and 'caramel' aromas, and astringent mouthfeel. 'Cooked vegetables', 'green grass', 'stewed fruit', 'rooibos-woody', 'marmalade' and 'cardboard' aromas, sweet taste and bitter taste were secondary attributes (10-20 median intensity; 100% occurrence frequency). The same flavor attributes were present, except for sweet-associated and fruity notes. A sensory lexicon and sensory wheels for aroma and palate attributes were constructed from the data. The shelf-life stability of green rooibos was evaluated in moisture-impermeable (pouches) and moisture-permeable (sachets) packaging at 25 and 40 °C at 60% relative humidity over 24 weeks. Green rooibos samples stored in pouches at 4 °C were also evaluated. Storage in sachets led to moisture uptake (~10 g (100 g)-1 dry basis) and an increase in water activity (>0.6), causing degradation of chlorophyll and dihydrochalcones. Changes in color and sensory profile (decreased vegetal, cereal and cardboard aromas and increased sweet-associated and fruity aromas) were evident and more pronounced at the higher storage temperature. CONCLUSIONS: Storage at ≤25 °C in moisture-impermeable packaging material is recommended for green rooibos herbal tea. © 2024 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Food-grade phytosome vesicles for nanoencapsulation of labile C-glucosylated xanthones and dihydrochalcones present in a plant extract matrix-Effect of process conditions and stability assessment.

Phytosomes consist of a phytochemical bound to the hydrophilic choline head of a phospholipid. Their use in food products is gaining interest. However, literature on the use of food-grade solvents, crude plant extracts as opposed to pure compounds, and unrefined phospholipids to prepare phytosomes is limited. Furthermore, studies on compound stability are lacking. This study aimed to develop nano-phytosome vesicles prepared from inexpensive food-grade ingredients to improve the stability of polyphenolic compounds. Cyclopia subternata extract (CSE) was selected as a source of phenolic compounds. It contains substantial quantities of C-glucosyl xanthones, benzophenones, and dihydrochalcones, compounds largely neglected to date. The effect of process conditions on the complexation of CSE polyphenols with minimally refined food-grade fat-free soybean lecithin (PC) was studied. The PC:CSE ratio, sonication time, and reaction temperature were varied. This resulted in phytosomes ranging in vesicle size (113.7-312.7 nm), polydispersity index (0.31-0.48), and zeta potential (-55.0 to -38.9 mV). Variation was also observed in the yield (93.5%-96.0%), encapsulation efficiency (3.7%-79.0%), and loading capacity (LC, 1.3%-14.7%). Vesicle size and LC could be tailored by adjusting the sonication time and PC:CSE ratio, respectively. Chemical interaction between the lipid and the phenolic compounds was confirmed with nuclear magnetic resonance. Phytosomal formulation protected the compounds against degradation when freeze-dried samples were stored at 25 and 40°C for 6 months at low relative humidity. The study provided valuable information on the importance of specific process parameters in producing food-grade phytosomes with improved phenolic stability.

Urinary Peptidomics and Pulse Wave Velocity: The African-PREDICT Study.

Increased arterial stiffness is related to early vascular aging and is an independent predictor for cardiovascular disease and mortality. Molecular mechanisms underlying increased arterial stiffness are largely unexplored, especially at the proteome level. We aimed to explore the relationship between pulse wave velocity and urinary proteomics. We included 919 apparently healthy (no chronic illnesses) Black and White men and women (equally distributed) between 20 and 30 years from the African-PREDICT study. Capillary electrophoresis time-of-flight mass spectrometry was used to analyze the urinary proteome. We measured the carotid-femoral pulse wave velocity to estimate arterial stiffness. In the total group, pulse wave velocity correlated positively with collagen-derived peptides including collagen types I, II, III, IV, V, and IX and inversely with collagen type XI (adjusted for mean arterial pressure). Regarding noncollagen-derived peptides, pulse wave velocity positively correlated with polymeric immunoglobulin receptor peptides (n = 2) (all q-value ≤0.05). In multivariable adjusted analyses, pulse wave velocity associated positively and independently with seven urinary peptides (collagen type I, n = 5) (all p-value ≤0.05). We found significant positive and independent associations between pulse wave velocity and the collagen type I-derived peptides, suggesting that dysregulation of collagen type I in the extracellular matrix scaffold could lead to early onset of increased arterial stiffness.

Reaction kinetics of aspalathin degradation and flavanone isomer formation in aqueous model solutions: Effect of temperature, pH and metal chelators.

The poor stability of aspalathin in aqueous solutions is a major challenge in delivering a shelf-stable ready-to-drink (RTD) green rooibos iced tea. The kinetics of aspalathin degradation and the formation of eriodictyol glucoside isomers [(S/R)-6-ß-D-glucopyranosyleriodictyol and (S/R)-8-ß-D-glucopyranosyleriodictyol] in aqueous buffers were modeled to understand and predict aspalathin losses during heat processing. The effects of temperature and pH on the rate constants of aspalathin degradation and eriodictyol glucoside isomer formation were determined in a 0.1 M phosphate buffer with 5.7 mM citric acid. The zero-order model best described the reaction kinetics of aspalathin degradation and eriodictyol glucoside isomer formation. Increasing the temperature and pH increased the reaction rate constants. The activation energies of the reactions were much lower at pH 6 than at pH 4, indicating that pH affected the temperature dependence of the reactions. The 8-C-glucosyl eriodictyol derivatives (RE8G and SE8G) formed at much lower rates than the 6-C-glucosyl eriodictyol derivatives (RE6G and SE6G). The metal chelators, citric acid, citrate and EDTA, drastically reduced the reaction rate constants, indicating the catalytic role of metal ions in aspalathin autoxidation. The results of the study could assist manufacturers to improve the shelf life of rooibos RTD beverages by changing the formulation and adjusting heat processing conditions.

Stability of labile xanthones and dihydrochalcones in a ready-to-drink honeybush beverage during storage.

BACKGROUND: The shelf-life of a functional herbal tea-based beverage is important not only for consumer acceptability, but also for the retention of bioactive compounds. The present study aimed to clarify the role of common iced tea beverage ingredients (citric and ascorbic acids) on the shelf-life stability of an herbal tea-based beverage. A hot water extract of green Cyclopia subternata, also used as honeybush tea, was selected as the main ingredient because it provides different types of phenolic compounds associated with bioactive properties (i.e. xanthones, benzophenones, flavanones, flavones and dihydrochalcones). RESULTS: The model solutions were stored for 180 and 90 days at 25 and 40 °C, respectively. Changes in their volatile profiles and color were also quantified as they contribute to product quality. 3',5'-Di-ß-d-glucopyranosyl-3-hydroxyphloretin (HPDG; dihydrochalcone) and, to a lesser extent, mangiferin (xanthone), were the most labile compounds. Both compounds were thus identified as critical quality indicators to determine shelf-life. The stability-enhancing activity of the acids depended on the compound; ascorbic acid and citric acid enhanced the stability of HPDG and mangiferin, respectively. However, when considering all the major phenolic compounds, the base solution without acids was the most stable. This was also observed for the color and major volatile aroma-active compounds [α-terpineol, (E)-ß-damascenone, 1-p-menthen-9-al and trans-ocimenol]. CONCLUSION: The addition of acids, added for stability and taste in ready-to-drink iced tea beverages, could thus have unwanted consequences in that they could accelerate compositional changes and shorten the shelf-life of polyphenol-rich herbal tea beverages. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Identifying a urinary peptidomics profile for hypertension in young adults: The African-PREDICT study: Urinary peptidomics and hypertension: Urinary peptidomics and hypertension.

Hypertension is one of the most important and complex risk factors for cardiovascular diseases (CVDs). By using urinary peptidomics analyses, we aimed to identify peptides associated with hypertension, building a framework for future research towards improved prediction and prevention of premature development of CVD. We included 78 hypertensive and 79 normotensive participants from the African-PREDICT study (aged 20-30 years), matched for sex (51% male) and ethnicity (49% black and 51% white). Urinary peptidomics data were acquired using capillary-electrophoresis-time-of-flight-mass-spectrometry. Hypertension-associated peptides were identified and combined into a support vector machine-based multidimensional classifier. When comparing the peptide data between the normotensive and hypertensive groups, 129 peptides were nominally differentially abundant (Wilcoxon p < 0.05). Nonetheless, only three peptides, all derived from collagen alpha-1(III), remained significantly different after rigorous adjustments for multiple comparisons. The 37 most significant peptides (all p ≤ 0.001) served as basis for the development of a classifier, with 20 peptides being combined into a unifying score, resulting in an AUC of 0.85 in the ROC analysis (p < 0.001), with 83% sensitivity at 80% specificity. Our study suggests potential value of urinary peptides in the classification of hypertension, which could enable earlier diagnosis and better understanding of the pathophysiology of hypertension and premature cardiovascular disease development.

A urinary peptidomics approach for early stages of cardiovascular disease risk: The African-PREDICT study.

Cardiovascular disease (CVD) affects individuals across the lifespan, with multiple cardiovascular (CV) risk factors increasingly present in young populations. The underlying mechanisms in early cardiovascular disease development are complex and still poorly understood. We therefore employed urinary proteomics as a novel approach to gain better insight into early CVD-related molecular pathways based on a CVD risk stratification approach. This study included 964 apparently healthy (no self-reported chronic illnesses, free from clinical symptoms of CVD) black and white men and women (aged 20-30 years old) from the African Prospective study on the Early Detection and Identification of Cardiovascular disease and Hypertension (African-PREDICT) study. Cardiovascular risk factors used for stratification included obesity, physical inactivity, tobacco use, high alcohol intake, hyperglycemia, dyslipidemia and hypertension. Participants were divided into low (0 risk factors), medium (1-2 risk factors) and high (≥3 risk factors) CV risk groups. We analyzed urinary peptidomics by capillary electrophoresis time-of-flight mass spectrometry. After adjusting for ethnicity, sex and age, 65 sequenced urinary peptides were differentially expressed between the CV risk groups (all q-values ≤ 0.01). These peptides included a lower abundance of collagen type I- and III-derived peptides in the high compared to the low CV risk group. With regard to noncollagen peptides, we found a lower abundance of alpha-1-antitrypsin fragments in the high compared to the low CV risk group (all q-values ≤ 0.01). Our findings indicate lower abundances of collagen types I and III in the high compared to the low CV risk group, suggesting potential early alterations in the CV extracellular matrix.

Assessment of the stability of compounds belonging to neglected phenolic classes and flavonoid sub-classes using reaction kinetic modeling.

Phenolic compounds are known to degrade and/or undergo changes during food production and storage. Reaction kinetic modeling is generally used to define kinetic parameters of a food system and predict changes during thermal processing and storage. Data for phenolic acids and flavonoids, such as anthocyanins and flavan-3-ols, have been reviewed in detail, but the flavonoid sub-classes, dihydrochalcones and flavanones, have been mostly neglected. Other neglected phenolic classes are xanthones and benzophenones. The stability of these types of compounds is important as they are present in fruits and exposed to heat when processed into juice and jam. Other sources of the compounds are herbal teas, which are also subjected to thermal processing, either during the primary processing of the plant material, or the production of extracts for use as food ingredients. The theoretical background is given to understand the review of literature on these classes/sub-classes. Results of research on kinetic modeling are discussed in detail, while research on compound stability without the application of reaction kinetic modeling is briefly mentioned to provide context. The studies discussed included those focusing on heating during the processing and storage of model solutions, liquid foods, plant material, dried extracts, and extracts formulated with other food ingredients.

Class-modelling of overlapping classes. A two-step authentication approach.

Honeybush is an indigenous herbal tea highly valued for its aroma, flavour and medicinal properties. It is protected as Geographical Indication (GI) since it is produced from a number of Cyclopia species that are endemic to South Africa. Most commonly used for honeybush tea production are C. intermedia, C. subternata and C. genistoides, differing slightly, but distinctly in flavour. Demand for species-specific honeybush tea instead of mixtures have increased, meriting a strategy for authentication of C. intermedia, C. subternata and C. genistoides. Samples of these three species were analysed, using hyperspectral imaging (HSI) in the near-infrared spectral range. The data were pre-processed and used for class-modelling, a general approach well suited for authentication purposes. Unfortunately, since the HSI data of Cyclopia species studied are very similar, the classification results obtained with individual class-models are unsatisfactory, e.g., class-models constructed for C. genistoides and C. subternata yielded correct classification rate (CCR) values of 76.4 and 83.1%, respectively. On the other hand, discriminant modelling, which is another type of classification technique, led to good classification outcomes (CCR 98.9%). However, the classical discriminant model cannot be applied for authentication purposes since it always assigns a new sample to one of the classes studied, even if in reality, it belongs to none of them. Counterfeits or non-representative samples would be incorrectly assigned by the discriminant model to one of the authentic classes. Therefore, in this study, a two-step authentication of overlapping classes is proposed, which combines the advantages of class-modelling and discriminant methods. When applied to the authentication of Cyclopia species studied, the two-step approach yielded a CCR of 97.4%, which is a significant improvement compared to results obtained with the individual class-models. The proposed approach is general and can be applied when classes studied are very similar, and individual class-models lead to unsatisfactory results.

Heat treatment improves the sensory properties of the ultrafiltration by-product of honeybush (Cyclopia genistoides) extract.

BACKGROUND: Ultrafiltration of green honeybush (Cyclopia genistoides) extract results in a by-product (retentate). Application of further separation processes for recovery of polyphenols would entail creation of additional waste. Repurposing the retentate as a food flavour ingredient provides an alternative valorization approach. RESULTS: The retentate, suspended in water (270 g L-1 ), was heat-treated at 80 °C for 2, 4, 8 and 16 h, and at 90 °C for 2, 4, 6 and 8 h to change its sensory profile. The heat-treated retentate, diluted to beverage strength (2.15 g L-1 ), had prominent 'grape/Muscat-like' and 'marmalade/citrus' aroma and flavour notes. Overall, heating for ≤ 4 h increased the intensities of positive flavour and aroma notes, while reducing those of 'green/grass', 'hay' and bitterness, whereafter further heating only had a slight effect on the aroma profile at 80 °C (P < 0.05), but not at 90 °C (P ≥ 0.05). The heat treatments, 80 °C/4 h and 90 °C/4 h, were subsequently applied to different batches of retentate (n = 10) to accommodate the effect of natural product variation. Heating at 90 °C produced higher intensities of positive aroma attributes (P < 0.05), but was more detrimental to the phenolic stability, compared to 80 °C. CONCLUSION: After heat treatment, the phenolic content of C. genistoides retentate, reconstituted to beverage strength, still fell within the range of a typical 'fermented' (oxidized) honeybush leaf tea infusion. The change in phenolic composition will not diminish the benefit of an improved sensory profile for the retentate by-product through heating. © 2021 Society of Chemical Industry.

Shelf-Life Stability of Ready-to-Use Green Rooibos Iced Tea Powder-Assessment of Physical, Chemical, and Sensory Properties.

Green rooibos extract (GRE), shown to improve hyperglycemia and HDL/LDL blood cholesterol, has potential as a nutraceutical beverage ingredient. The main bioactive compound of the extract is aspalathin, a C-glucosyl dihydrochalcone. The study aimed to determine the effect of common iced tea ingredients (citric acid, ascorbic acid, and xylitol) on the stability of GRE, microencapsulated with inulin for production of a powdered beverage. The stability of the powder mixtures stored in semi-permeable (5 months) and impermeable (12 months) single-serve packaging at 30 °C and 40 °C/65% relative humidity was assessed. More pronounced clumping and darkening of the powders, in combination with higher first order reaction rate constants for dihydrochalcone degradation, indicated the negative effect of higher storage temperature and an increase in moisture content when stored in the semi-permeable packaging. These changes were further increased by the addition of crystalline ingredients, especially citric acid monohydrate. The sensory profile of the powders (reconstituted to beverage strength iced tea solutions) changed with storage from a predominant green-vegetal aroma to a fruity-sweet aroma, especially when stored at 40 °C/65% RH in the semi-permeable packaging. The change in the sensory profile of the powder mixtures could be attributed to a decrease in volatile compounds such as 2-hexenal, (Z)-2-heptenal, (E)-2-octenal, (E)-2-nonenal, (E,Z)-2,6-nonadienal and (E)-2-decenal associated with "green-like" aromas, rather than an increase in fruity and sweet aroma-impact compounds. Green rooibos extract powders would require storage at temperatures ≤ 30 °C and protection against moisture uptake to be chemically and physically shelf-stable and maintain their sensory profiles.

Identification of a novel di-C-glycosyl dihydrochalcone and the thermal stability of polyphenols in model ready-to-drink beverage solutions with Cyclopia subternata extract as functional ingredient.

Heat processing of ready-to-drink beverages is required to ensure a microbiologically safe product, however, this can result in the loss of bioactive compounds responsible for functionality. The objective of this study was to establish the thermal stability of a novel dihydrochalcone, 3',5'-di-ß-d-glucopyranosyl-3-hydroxyphloretin (2), 3',5'-di-ß-d-glucopyranosylphloretin (3) and other Cyclopia subternata phenolic compounds, in model solutions with or without citric acid and ascorbic acid. The solutions were heated at 93, 121 and 135 °C, relevant to pasteurisation, commercial sterilisation and ultra-high temperature (UHT) pasteurisation, respectively. For most compounds, the acids decreased the second order reaction rate constants, up to 27 times. Compound 2 (46.29 ± 0.53 (g/100 g)-1 h-1), and to a lesser extent compound 3 (5.94 ± 0.01 (g/100 g)-1 h-1) were the most thermo-unstable compounds when treated at 135 °C without added acids. Even though differential effects were observed for compounds at different temperatures and formulations, overall, the phenolic compounds were most stable under UHT pasteurisation conditions.

Comprehensive off-line CCC × LC-DAD-MS separation of Cyclopia pubescens Eckl. & Zeyh. phenolic compounds and structural elucidation of isolated compounds.

INTRODUCTION: The minor phenolic constituents of Cyclopia pubescens Eckl. & Zeyh. are unknown and one dimensional (1D) liquid chromatography (LC) is unable to provide sufficient separation. METHODOLOGY: A two-dimensional (2D) LC method incorporating normal-phasehigh performance countercurrent chromatography (NP-HPCCC) in the first dimension (1 D) and reversed-phase ultra-high-performance liquid chromatography (RP-UHPLC) as the second dimension (2 D) was developed. The analytical HPCCC method was subsequently scaled up to semi-preparative mode and fractions pooled based on phenolic sub-groups. The phenolic compounds in selected fractions were subsequently isolated using RP-HPLC on a C18 column. Isolated compounds were identified by nuclear magnetic resonance (NMR) spectroscopy. The absolute configurations of compounds were determined by optical rotation and electronic circular dichroism spectra. Sugars were identified by gas chromatography-mass spectrometry (GC-MS) analysis. RESULTS: The comprehensive off-line 2D CCC × LC method gave a good spread of the phenolic compounds. Orthogonality calculated using both the convex hull and conditional entropy methods were 81%. High-resolution mass spectrometric fragmentation spectra obtained from a quadrupole-time-of-flight instrument and ultraviolet-visible (UV-vis) spectral data were used to (tentatively) identify 32 phenolic compounds from the analytical CCC fractions. Of the seven isolated compounds, (2S)-5-O-[α-l-rhamnopyranosyl-(1 → 2)-ß-d-glucopyranosyl]eriodictyol (3) and (2S)-5-O-[α-l-rhamnopyranosyl-(1 → 2)-ß-d-glucopyranosyl]-5,7,3',4'-tetrahydroxyflavan (4) were newly identified in all plants. The other isolated compounds were identified as (2S)-5-O-[α-l-rhamnopyranosyl-(1 → 2)-ß-d-glucopyranosyl]naringenin (1), R-neo-eriocitrin (2), 3-O-α-l-arabinopyranosyl-3,4-dihydroxybenzoic acid (5), 4-O-ß-d-glucopyranosyl-Z-4-hydroxycinnamic acid (6) and 4-(4'-O-ß-d-glucopyranosyl-4'-hydroxy-3'-methoxyphenyl)-2-butanone (7). CONCLUSIONS: Among the 32 compounds (tentatively) identified, only six were previously identified in Cyclopia pubescens using 1D LC. Most of the isolated compounds were also identified for the first time in Cyclopia spp., improving the knowledge of the minor phenolic compounds of this genus.

Honeybush Extracts (Cyclopia spp.) Rescue Mitochondrial Functions and Bioenergetics against Oxidative Injury.

Mitochondrial dysfunction plays a major role not only in the pathogenesis of many oxidative stress or age-related diseases such as neurodegenerative as well as mental disorders but also in normal aging. There is evidence that oxidative stress and mitochondrial dysfunction are the most upstream and common events in the pathomechanisms of neurodegeneration. Cyclopia species are endemic South African plants and some have a long tradition of use as herbal tea, known as honeybush tea. Extracts of the tea are gaining more scientific attention due to their phenolic composition. In the present study, we tested not only the in vitro mitochondria-enhancing properties of honeybush extracts under physiological conditions but also their ameliorative properties under oxidative stress situations. Hot water and ethanolic extracts of C. subternata, C. genistoides, and C. longifolia were investigated. Pretreatment of human neuroblastoma SH-SY5Y cells with honeybush extracts, at a concentration range of 0.1-1 ng/ml, had a beneficial effect on bioenergetics as it increased ATP production, respiration, and mitochondrial membrane potential (MMP) after 24 hours under physiological conditions. The aqueous extracts of C. subternata and C. genistoides, in particular, showed a protective effect by rescuing the bioenergetic and mitochondrial deficits under oxidative stress conditions (400 µM H2O2 for 3 hours). These findings indicate that honeybush extracts could constitute candidates for the prevention of oxidative stress with an impact on aging processes and age-related neurodegenerative disorders potentially leading to the development of a condition-specific nutraceutical.

Left ventricular mass and urinary metabolomics in young black and white adults: The African-PREDICT study.

BACKGROUND AND AIMS: Increased left ventricular mass is an independent predictor for cardiovascular events, and shown to be higher in black than white populations. To gain a better understanding of early factors contributing to increased left ventricular mass in young black adults, we investigated metabolomic profiles, identified and compared metabolites that associated with left ventricular mass index in healthy black and white adults. METHODS AND RESULTS: We included normotensive black and white participants from the African-PREDICT study, with data on urinary metabolomics and echocardiography. Urinary metabolites were measured using three different analytical platforms. Univariate statistical analyses, including independent t-test (adjusted for multiple comparisons), effect size (d ≥ 0.3) and single regression analyses were used to identify metabolites. When comparing the black and white groups, the black group had higher central systolic blood pressure (p > 0.005), whereas left ventricular mass index was similar between the groups (p = 0.97). Three from a total of 192 metabolites were identified to be more abundant (p < 0.046) and inversely associated with left ventricular mass index in the black group only: hydroxyproline (ß = -0.22; p = 0.045), glycine (ß = -0.20; p = 0.049) and trimethylamine (ß = -0.21; p = 0.037). CONCLUSION: Higher urinary levels of hydroxyproline, glycine and trimethylamine were inversely associated with left ventricular mass index in the black adults only. Hydroxyproline and glycine are important in maintaining healthy collagen turnover and stability in the heart. Our results may reflect an increase in collagen biosynthesis and collagen deposition in the left ventricle due to higher central systolic blood pressure in the black population.

Model development for predicting in vitro bio-capacity of green rooibos extract based on composition for application as screening tool in quality control.

Mounting evidence of the ability of aspalathin to target underlying metabolic dysfunction relevant to the development or progression of obesity and type 2 diabetes created a market for green rooibos extract as a functional food ingredient. Aspalathin is the obvious choice as a chemical marker for extract standardisation and quality control, however, often the concentration of a single constituent of a complex mixture such as a plant extract is not directly related to its bio-capacity, i.e. the level of in vitro bioactivity effected in a cell system at a fixed concentration. Three solvents (hot water and two EtOH-water mixtures), previously shown to produce bioactive green rooibos extracts, were selected for extraction of different batches of rooibos plant material (n = 10). Bio-capacity of the extracts, tested at 10 µg ml-1, was evaluated in terms of glucose uptake by C2C12 and C3A cells and lipid accumulation in 3T3-L1 cells. The different solvents and inter-batch plant variation delivered extracts ranging in aspalathin content from 54.1 to 213.8 g kg-1. The extracts were further characterised in terms of other major flavonoids (n = 10) and an enolic phenylpyruvic acid glucoside, using HPLC-DAD. The 80% EtOH-water extracts, with the highest mean aspalathin content (170.9 g kg-1), had the highest mean bio-capacity in the respective assays. Despite this, no significant (P≥ 0.05) correlation existed between aspalathin content and bio-capacity, while the orientin, isoorientin and vitexin content correlated moderately (r≥ 0.487; P < 0.05) with increased glucose uptake by C2C12 cells. Various multivariate analysis methods were then applied with Evolution Program-Partial Least Squares (EP-PLS) resulting in models with the best predictive power. These EP-PLS models, based on all quantified compounds, predicted the bio-capacity of the extracts for the respective cell types with RMSECV values ≤ 11.5, confirming that a complement of compounds, and not aspalathin content alone, is needed to predict the in vitro bio-capacity of green rooibos extracts. Additionally, the composition of hot water infusions of different production batches of green rooibos (n = 29) at 'cup-of-tea' equivalence was determined to relate dietary supplementation with the extract to intake in the form of herbal tea.

Impact of Cold versus Hot Brewing on the Phenolic Profile and Antioxidant Capacity of Rooibos (Aspalathus linearis) Herbal Tea.

Consumption of rooibos (Aspalathus linearis) as herbal tea is growing in popularity worldwide and its health-promoting attributes are mainly ascribed to its phenolic composition, which may be affected by the brewing conditions used. An aspect so far overlooked is the impact of cold brewing vs regular brewing and microwave boiling on the poly(phenolic) profile and in vitro antioxidant capacity of infusions prepared from red ('fermented', oxidized) and green ('unfermented', unoxidized) rooibos, the purpose of the present study. By using an untargeted metabolomics-based approach (UHPLC-QTOF mass spectrometry), 187 phenolic compounds were putatively annotated in both rooibos types, with flavonoids, tyrosols, and phenolic acids the most represented type of phenolic classes. Multivariate statistics (OPLS-DA) highlighted the phenolic classes most affected by the brewing conditions. Similar antioxidant capacities (ORAC and ABTS assays) were observed between cold- and regular-brewed green rooibos and boiled-brewed red rooibos. However, boiling green and red rooibos delivered infusions with the highest antioxidant capacities and total polyphenol content. The polyphenol content strongly correlated with the in vitro antioxidant capacities, especially for flavonoids and phenolic acids. These results contribute to a better understanding of the impact of the preparation method on the potential health benefits of rooibos tea.

Potential of benzophenones and flavanones to modulate the bitter intensity of Cyclopia genistoides herbal tea.

Variation in the bitter taste of Cyclopia genistoides (honeybush) herbal tea and reported modulation between its major xanthones, mangiferin and isomangiferin, prompted further investigation into the potential modulatory effects of honeybush phenolics. Combinations of crude benzophenone (BF)-, xanthone (XF)-, and flavanone (FF)-rich fractions and their major individual phenolic compounds were analysed by descriptive sensory analysis. The fractions were prepared from a bitter, hot water extract of green C. genistoides. Fraction BF, which is below the bitter threshold (intensity 10 on 100-point scale), enhanced the bitter intensity of XF and FF slightly (p < 0.05), although none of the major individual benzophenones retained this bitter enhancing effect. On the contrary, 3-ß-d-glucopyranosyl-4-ß-d-glucopyranosyloxyiriflophenone, the major benzophenone in BF, significantly (p < 0.05) decreased the bitter taste of XF, at a low concentration, whereas FF suppressed the bitter intensity of XF and mangiferin, the major xanthone present in XF. Hesperidin, however, had no effect on the bitter intensity of XF. In contrast, (2S)-5-[α-L-rhamnopyranosyl-(1→2)-ß-d-glucopyranosyloxy]-naringenin, the major compound of FF, significantly (p < 0.05) enhanced the bitter taste of XF when added at concentrations comparable to that of 'fermented' honeybush tea infusions. The concentration-dependence of these bitter taste interactions may be responsible for the variable bitter intensity of C. genistoides herbal tea.

Phenolic composition of rooibos changes during simulated fermentation: Effect of endogenous enzymes and fermentation temperature on reaction kinetics.

Phenolic compounds of Aspalathus linearis (rooibos) are susceptible to oxidation during "fermentation", a process characterized by the formation of a red-brown leaf color. The role of enzymes in this process is not yet understood. An experiment with dried green rooibos plant material pre-treated at 170 °C for 30 min to denature and "inactivate" endogenous enzymes was conducted to confirm the role of oxidative enzymes. The phenolic composition of "enzyme inactivated" plant material was not significantly (p ≥ .05) affected by simulated fermentation, compared to control samples, as determined using piece-wise multivariate analysis of variance for successive time intervals. This proves that rooibos enzymes participate in the oxidation of phenolic compounds during fermentation of the plant material. A kinetic modeling approach was subsequently used to establish reaction kinetic parameters for selected rooibos phenolic compounds. The degradation of aspalathin and nothofagin and formation of eriodictyol glucosides during simulated fermentation at four temperatures from 37 to 50 °C were best described by the fractional conversion model based on first-order kinetics (r2 > 0.98), which allows for non-zero equilibrium concentrations. The extent of degradation for other compounds was too low to enable kinetic modeling. Reaction rates for the degradation/formation of phenolic compounds during fermentation followed the Arrhenius law. Less phenolic degradation (higher equilibrium concentration), but a higher reaction rate constant, was observed at higher temperatures, which could possibly be attributed to inactivation of enzymes.

Membrane selection and optimisation of tangential flow ultrafiltration of Cyclopia genistoides extract for benzophenone and xanthone enrichment.

Ultrafiltration of Cyclopia genistoides extract was optimised to increase its benzophenone and xanthone content as quantified using HPLC-DAD. Regenerated cellulose (RC) and polyethersulphone membranes with molecular weight cut-offs of 10 and 30 kDa were evaluated in terms of compound enrichment, permeate flux and permeate yield, using dead-end ultrafiltration. Compound enrichment was subsequently optimised using the 10 kDa RC membrane and tangential flow ultrafiltration (TFU). The effect of extract composition on compound enrichment, due to natural variation in the source material, was assessed using extracts from different batches of plant material (n = 11). Transmembrane pressure and feed flow rate affected (p < 0.05) process efficiency (mean permeate flux, compound enrichment and membrane fouling). TFU achieved ≥20% enrichment of the target compounds, proving its suitability for preparation of a nutraceutical extract of C. genistoides.