Rooibos Plant Material Research Articles

Application of direct injection-ion mobility spectrometry-mass spectrometry (DI-IMS-MS) for the analysis of phenolics in honeybush and rooibos tea samples

Consumer awareness of the health benefits of herbal teas such as rooibos and honeybush has led to an increase in production demand and to inevitable accidental or deliberate adulteration. In this study, direct injection-ion mobility spectrometry-mass spectrometry (DI-IMS-MS) is proposed as a rapid methodology for the differentiation of rooibos and honeybush blends (adulterations). Analytical data was obtained from 100 rooibos, 130 honeybush and 7 blend samples. Principal component analysis (PCA) showed differences between samples of rooibos and honeybush blends, and further showed the diagnostic value of marker compounds in distinguishing Cyclopia intermedia, C. genistoides and C. subternata, mainly used for honeybush production. Markers such as eriodictyol-C-hexose isomers, orientin, aspalathin, and vitexin were characteristic of blends containing more than 80 % rooibos plant material, and quinic acid, iriflophenone-3-C-glucoside-4-O-glucoside, C-glycosylxanthones isomers (mangiferin and isomangiferin), and eriodictyol were characteristic of blends containing more than 80 % C. intermedia plant material. Furthermore, the C-glycosylxanthones isomers (mangiferin and isomangiferin) were prominent in C. genistoides, quinic acid was prominent in C. subternata and piscidic acid was prominent in C. intermedia plant material. Phenolic compounds in the herbal teas were further characterised based on an array of complementary descriptors (e.g., retention time, arrival times, accurate mass information, tandem MS fragmentation data, and TWCCSN2) to strengthen compound identification.

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.

Untargeted analysis of chromatographic data for green and fermented rooibos: Problem with size effect removal

While analyzing chromatographic data, it is necessary to preprocess it properly before exploration and/or supervised modeling. To make chromatographic signals comparable, it is crucial to remove the scaling effect, caused by differences in overall sample concentrations. One of the efficient methods of signal scaling is Probabilistic Quotient Normalization (PQN) [1]. However, it can be applied only to data for which the majority of features do not vary systematically among the studied classes of signals. When studying the influence of the traditional “fermentation” (oxidation) process on the concentration of 56 individual peaks detected in rooibos plant material, this assumption is not fulfilled. In this case, the only possible solution is the analysis of pairwise log-ratios, which are not influenced by the scaling constant. To estimate significant features, i.e., peaks differentiating the studied classes of samples (green and fermented rooibos plant material), we propose the application of rPLR (robust pair-wise log-ratios) as proposed by Walach et al. [2]. It allows for fast computation and identification of the significant features in terms of original variables (peaks) which is problematic, while working with the unfolded pair-wise log ratios. As demonstrated, it can be applied to designed data sets and in the case of contaminated data, it allows proper conclusions.

Minimising variation in aspalathin content of aqueous green rooibos extract: optimising extraction and identifying critical material attributes

High levels of aspalathin, an antidiabetic dihydrochalcone, in green rooibos underpins interest in the production of a standardised extract. Elements of a quality-by-design approach were applied to optimise extraction conditions, aiming at the delivery of a dry matter yield (DMY) ≥ 160 g kg-1 and an extract with an aspalathin content (AC) ≥ 80 g kg-1 . Hot water extraction parameters, namely extraction time, extraction temperature and water-to-plant material ratio, were optimised for DMY and aspalathin extraction efficiency (AEE) using Design of Experiments. Good polynomial prediction models were obtained and multiresponse desirability plots indicated 37 min, 93 °C and 23:1 as optimal conditions. Even when using 30 min and 10:1 instead for practical reasons, the target DMY and AC values could be achieved with the caveat that plant material with an AC ≥ 30 g kg-1 is used. Particle size distribution and stem content were identified as contributing to variation in the AC of raw material. By setting raw material specifications in terms of AC, as well as applying practical optimum extraction conditions, 160 g kg-1 extract with an AC ≥ 80 g kg-1 could be consistently achieved from green rooibos plant material. © 2017 Society of Chemical Industry.

Multivariate analysis of variance of designed chromatographic data. A case study involving fermentation of rooibos tea

An ultimate goal of investigations of rooibos plant material subjected to different stages of fermentation is to identify the chemical changes taking place in the phenolic composition, using an untargeted approach and chromatographic fingerprints. Realization of this goal requires, among others, identification of the main components of the plant material involved in chemical reactions during the fermentation process. Quantitative chromatographic data for the compounds for extracts of green, semi-fermented and fermented rooibos form the basis of preliminary study following a targeted approach. The aim is to estimate whether treatment has a significant effect based on all quantified compounds and to identify the compounds, which contribute significantly to it. Analysis of variance is performed using modern multivariate methods such as ANOVA-Simultaneous Component Analysis, ANOVA – Target Projection and regularized MANOVA. This study is the first one in which all three approaches are compared and evaluated. For the data studied, all tree methods reveal the same significance of the fermentation effect on the extract compositions, but they lead to its different interpretation.

Inhibitory Interactions of Aspalathus linearis (Rooibos) Extracts and Compounds, Aspalathin and Z-2-(β-d-Glucopyranosyloxy)-3-phenylpropenoic Acid, on Cytochromes Metabolizing Hypoglycemic and Hypolipidemic Drugs.

Rooibos extract, due to its glucose and lipid lowering effects, has potential as a nutraceutical for improvement of metabolic dysfunction. Potential herb-drug interactions as a result of the use of natural products are of increasing concern. Cytochrome P450 enzymes, CYP2C8, CYP2C9, and CYP3A4, are important in the metabolism of hypoglycemic drugs, such as thiazolidinediones (TZDs) and sulfonylureas, and hypocholesterolemic drugs, such as atorvastatin. This study investigated the effects of rooibos extracts, prepared from “unfermented” and “fermented” rooibos plant material and two of the major bioactive compounds, Z-2-(β-d-glucopyranosyloxy)-3-phenylpropenoic acid (PPAG) and aspalathin (ASP), on Vivid® recombinant CYP450 enzymes. Unfermented (GRT) and fermented (FRE) rooibos extracts inhibited the activity of CYP2C8 (7.69 ± 8.85 µg/mL and 8.93 ± 8.88 µg/mL, respectively) and CYP3A4 (31.33 ± 4.69 µg/mL and 51.44 ± 4.31 µg/mL, respectively) based on their respective IC50 concentrations. Both extracts dose- and time-dependently inhibited CYP2C8 activity, but only time-dependently inhibited CYP2C9. CYP3A4 showed concentration-dependent inhibition by ASP, GRT, and FRE at 25, 50, and 100 µg/mL concentrations. ASP, GRT, and FRE time-dependently inhibited CYP3A4 activity with GRT and FRE showing a more potent time-dependent inhibition, comparable to erythromycin. These findings suggest that herb-drug interactions may occur when nutraceuticals containing rooibos extracts are co-administered with hypoglycemic drugs such as TZDs, sulfonylureas, and dyslipidemic drug, atorvastatin.

Improved HPLC method for rooibos phenolics targeting changes due to fermentation

The phenolic composition of rooibos (Aspalathus linearis) herbal tea is inherently linked to its health-promoting properties. “Fermentation” (oxidation) develops the characteristic flavour and colour of the herbal tea product. Changes to the phenolic composition during fermentation include conversion of aspalathin, a dihydrochalcone unique to rooibos and the major bioactive compound, to eriodictyol-glucopyranoside isomers. These compounds have not been quantified in rooibos plant material to date, due to separation challenges. In this study an improved HPLC method using a core shell column was developed to provide effective separation of a phenolic precursor (enolic phenylpyruvic acid-2-O-glucopyranoside, PPAG) and 15 rooibos tea phenolics (aspalathin, four eriodictyol-glucopyranoside isomers and 11 other major phenolic compounds) in a reasonable time. Compounds were tentatively identified using authentic reference standards and/or tandem mass spectrometry. The HPLC method utilising diode-array detection for quantitation was validated. After evaluation of different extraction solvents, a 40% aqueous acetonitrile solution was deemed optimal for extraction of phenolic compounds from rooibos plant material. Green, semi-fermented and fermented plant material from 10 sub-divided rooibos bushes were extracted and analysed. Water extracts, representing food ingredient extracts, were also analysed. The largest decreases with fermentation were observed for the dihydrochalcones, with moderate to small decreases for flavonols. Eriodictyol-glucopyranoside isomer concentrations increased following fermentation.

High-dimensional nested analysis of variance to assess the effect of production season, quality grade and steam pasteurization on the phenolic composition of fermented rooibos herbal tea

A nested analysis of variance combined with simultaneous component analysis, ASCA, was proposed to model high-dimensional chromatographic data. The data were obtained from an experiment designed to investigate the effect of production season, quality grade and post-production processing (steam pasteurization) on the phenolic content of the infusion of the popular herbal tea, rooibos, at ‘cup-of-tea’ strength. Specifically, a four-way analysis of variance where the experimental design involves nesting in two of the three crossed factors was considered.For the purpose of the study, batches of fermented rooibos plant material were sampled from each of four quality grades during three production seasons (2009, 2010 and 2011) and a sub-sample of each batch was steam-pasteurized. The phenolic content of each rooibos infusion was characterized by high performance liquid chromatography (HPLC)-diode array detection (DAD). In contrast to previous studies, the complete HPLC-DAD signals were used in the chemometric analysis in order to take into account the entire phenolic profile.All factors had a significant effect on the phenolic content of a ‘cup-of-tea’ strength rooibos infusion. In particular, infusions prepared from the grade A (highest quality) samples contained a higher content of almost all phenolic compounds than the lower quality plant material. The variations of the content of isoorientin and orientin in the different quality grade infusions over production seasons are larger than the variations in the content of aspalathin and quercetin-3-O-robinobioside. Ferulic acid can be used as an indicator of the quality of rooibos tea as its content generally decreases with increasing tea quality. Steam pasteurization decreased the content of the majority of phenolic compounds in a ‘cup-of-tea’ strength rooibos infusion.

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, –MS2, 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 0.4mg/l in water.

Aspalathin, a flavonoid in Aspalathus linearis (rooibos), is absorbed by pig intestine as a C-glycoside

Aspalathin, a dihydrochalcone and C-glycoside, is the most abundant flavonoid in rooibos (Aspalathus linearis), which is well known as an herbal tea in many countries. Aspalathin appears to have in vitro antioxidative and antimutagenic effects. To understand the effects of aspalathin in the body, research on the absorption in the intestinal tract, metabolism in the body, and identification of circulating metabolites in vivo is required. We investigated the metabolism of aspalathin to identify the parent compound and related metabolites in urine and plasma after orally administering a rooibos extract (16.3% aspalathin by 96 g rooibos extract, which equates to 1.1 kg dried rooibos material), produced from unfermented rooibos plant material, to pigs over a period of 11 days (oral dosage, 157-167 mg aspalathin per kg body weight daily). On days 7 and 11 of the study and days 1 and 2 after termination, urine was collected in 24-hour fractions, and plasma samples were collected at various time points. To our knowledge, this is the first time aspalathin metabolites have been identified in vivo, by presenting evidence of the absorption of aspalathin. Six substances identified in the urine by liquid chromatography-mass spectrometry were identified; these represent aspalathin and the metabolites methylated aspalathin, glucuronidated aspalathin glucuronidated and methylated aspalathin, a glucuronidated aglycone of aspalathin, as well as a metabolite of eriodictyol. The latter compound was methylated and contained 2 glucuronic acid moieties. This study showed that aspalathin can be absorbed by the intestine as C-glycoside as well as being cleaved in an aglycone and sugar moiety. The major metabolite in the enzymatically treated samples was methylated aspalathin. Between 0.1% and 0.9% of the administered dose of aspalathin could be detected in the urine on days 7 and 11 of the feeding study. No metabolites or aspalathin were found in plasma samples. The identification of the metabolites in vivo enables investigations to determine the biological potential of rooibos extracts.

Enhancement of Rooibos (Aspalathus linearis) Aqueous Extract and Antioxidant Yield with Fungal Enzymes

The leaves and stems of the Rooibos plant ( Aspalathus linearis) are used for the production of an herbal tea known for its health promoting properties, which have been linked to its flavonoid content but which is substantially reduced by the traditional processing method employed. Selected food-grade fungi were screened for their potential to improve the yield of soluble matter extracted from rooibos plant material. Fungal cocktails of hydrolyzing enzymes enhanced either the yield of soluble solids ( Lentinula edodes and Rhizopus oryzae cultured in yeast peptone-wheat straw medium) or the yield in antioxidants from fermented rooibos ( R. oryzae cultured in potato dextrose or yeast peptone-wheat straw medium). When applied to green rooibos, L. edodes (cultured in yeast peptone-wheat straw medium) enhanced the release of soluble solids as well as color formation, leading to semifermented rooibos with a relatively high aspalathin content, compared to fermented rooibos.

Quantification of the Major Phenolic Compounds, Soluble Solid Content and Total Antioxidant Activity of Green Rooibos (Aspalathus Linearis) by Means of near Infrared Spectroscopy

Near infrared (NIR) spectroscopy was used to develop calibration models to predict aspalathin, nothofagin and dihydrochalcone contents of dried, green rooibos plant material, as well as to predict the aspalathin content of the respective water extracts. Calibration models were also developed to predict the total polyphenol content (TPC), soluble solid content (SSC) and total antioxidant activity (TAA) of the water extracts. Results showed that NIR spectroscopy can effectively predict the aspalathin content of dried, green rooibos with a standard error of prediction ( SEP) and coefficient of determination ( r2) of 0.45 g 100 g−1 and 0.85, respectively and the dihydrochalcone content of rooibos with an SEP of 0.49 g 100 g−1 and r2 of 0.87. After extending the aspalathin content range of the sample set, by adding varying amounts of a dried rooibos extract powder with high concentrations of aspalathin (15.95 g 100 −1) and nothofagin (1.94 g 100 g−1) to some samples, slightly less accurate, but more robust, models were obtained for the aspalathin ( SEP = 0.53 g 100 g−1; r 2 = 0.87) and dihydrochalcone ( SEP = 0.57 g 100 g−1; r 2 = 0.88) contents. The NIR calibration model developed for nothofagin content ( SEP = 0.10 g 100 g−1; r 2 = 0.71) (extended range: SEP = 0.10 g 100 g−1; r 2 = 0.77) of dried, green rooibos is suitable for screening purposes. The current transmittance NIR calibration models developed for aspalathin content, TPC, SSC and the TAA of green rooibos water extracts did not give acceptable results.