Evaluation of Lippia scaberrima Sond. and Aspalathus linearis (Burm.f.) R. Dahlgren extracts on human CYP enzymes and gold nanoparticle synthesis: implications for drug metabolism and cytotoxicity.

BACKGROUND: Metabolism is an important component of the kinetic characteristics of herbal constituents, and it often determines the internal dose and concentration of these effective constituents at the target site. The metabolic profile of plant extracts and pure compounds need to be determined for any possible herb-drug metabolic interactions that might occur. METHODS: Various concentrations of the essential oil of Lippia scaberrima, the ethanolic extract of Lippia scaberrima alone and their combinations with fermented and unfermented Aspalathus linearis extract were used to determine the inhibitory potential on placental, microsomal and recombinant human hepatic Cytochrome P450 enzymes. Furthermore, the study investigated the synthesis and characterization of gold nanoparticles from the ethanolic extract of Lippia scaberrima as a lead sample. Confirmation and characterization of the synthesized gold nanoparticles were conducted through various methods. Additionally, the cytotoxic properties of the ethanolic extract of Lippia scaberrima were compared with the gold nanoparticles synthesized from Lippia scaberrima using gum arabic as a capping agent. RESULTS: All the samples showed varying levels of CYP inhibition. The most potent inhibition took place for CYP2C19 and CYP1B1 with 50% inhibitory concentration (IC50) values of less than 0.05 µg/L for the essential oil tested and IC50-values between 0.05 µg/L-1 µg/L for all the other combinations and extracts tested, respectively. For both CYP1A2 and CYP2D6 the IC50-values for the essential oil, the extracts and combinations were found in the range of 1 - 10 µg/L. The majority of the IC50 values found were higher than 10 µg/L and, therefore, were found to have no inhibition against the CYP enzymes tested. CONCLUSION: Therefore, the essential oil of Lippia scaberrima, the ethanolic extract of Lippia scaberrima alone and their combinations with Aspalathus linearis do not possess any clinically significant CYP interaction potential and may be further investigated for their adjuvant potential for use in the tuberculosis treatment regimen. Furthermore, it was shown that the cytotoxic potential of the Lippia scaberrima gold nanoparticles was reduced by twofold when compared to the ethanolic extract of Lippia scaberrima.

Rooibos tea waste binary oxide composite: An adsorbent for the removal of nickel ions and an efficient photocatalyst for the degradation of ciprofloxacin.

In this study, rooibos tea waste (RTW) incorporated with a binary oxide (BO; Fe2O3-SnO2) has been reported for the first time as a highly efficient adsorbent material for the elimination of Ni(II) ions. The as-synthesised rooibos tea waste-binary oxide (RWBO) composite adsorbent was characterised using miscellaneous techniques such as FTIR, XRD, SEM, EDX, TGA, BET, and XPS. The RWBO was then tested for the removal of Ni(II) in a batch adsorption experiment. The composite adsorbent showed a great removal efficiency of about 99.75% for Ni(II) ions at 45 °C, 180 min agitation time, pH 7, and dosage of 250 mg. The adsorption process was found to be endothermic and spontaneous. Also, the spent adsorbent [RWBO-Ni(II)] was found to be solar light active with a narrow band gap of 1.4 eV. It was further used as a photocatalyst for the photocatalytic abatement of 10 mg/L ciprofloxacin with an extent of degradation of 83% obtained after 150 min. In addition, the extent of mineralisation of the ciprofloxacin by the spent adsorbent as obtained from the TOC data was found to be 64%. Overall, the RWBO composite adsorbent lends itself as an efficient, eco-friendly and promising material for environmental remediation.

Fermented Rooibos tea (Aspalathus linearis) Ameliorates Sodium Fluoride-Induced Cardiorenal Toxicity, Oxidative Stress, and Inflammation via Modulation of NF-κB/IκB/IκKB Signaling Pathway in Wistar Rats.

High dose of fluoride intake is associated with toxic effects on kidney and cardiac tissues. This study evaluated the potential protective effect of fermented rooibos tea (RTE) on sodium fluoride (NaF)-induced cardiorenal toxicity in rats. Male Wistar rats (n = 56) were randomly allocated into one of seven equal groups: control, NaF (100 mg/kg orally), NaF + RTE (2%, w/v), NaF + RTE (4%, w/v), NaF + lisinopril (10 mg/kg orally), 2% RTE, and 4% RTE. The experiment lasted for 14 days and RTE was administered to the rats as their sole source of drinking fluid. NaF induced cardiorenal toxicity indicated by elevated level of urea, creatinine, LDH, creatinine kinase-MB, and cardiac troponin I in the serum, accompanied by altered histopathology of the kidney and heart. Furthermore, levels of H2O2, malondialdehyde, and NO were elevated, while GSH level was depleted in the kidney and heart due to NaF intoxication. Protein levels of c-reactive protein, TNFα, IL-1B, and NF-κB were increased by NaF in the serum, kidney, and heart. RTE at 2% and 4% (w/v) reversed cardiorenal toxicity, resolved histopathological impairment, attenuated oxidative stress and inhibited formation of pro-inflammatory markers. RTE at both concentrations down-regulates the mRNA expression of NF-κB, and upregulates the mRNA expression of both IκB and IκKB, thus blocking the activation of NF-κB signaling pathway. Taken together, these results clearly suggest that the protective potential of rooibos tea against NaF-induced cardiorenal toxicity, oxidative stress, and inflammation may be associated with the modulation of the NF-κB signaling pathway.

Rooibos tea-in the cross fire of ROS, mitochondrial dysfunction and loss of proteostasis-positioned for healthy aging.

Impaired mitochondrial function and loss of cellular proteostasis control are key hallmarks of aging and are implicated in the development of neurodegenerative diseases. A common denominator is the cell's inability to handle reactive oxygen species (ROS), leading to major downstream oxidative damage that exacerbates neuronal dysfunction. Although we have progressed in understanding the molecular defects associated with neuronal aging, many unanswered questions remain. How much ROS is required to serve cellular function before it becomes detrimental and how does the cell's oxidative status impact mitochondrial function and protein degradation through autophagy? How does ROS regulate autophagy? Aspalathus linearis, also commonly known as rooibos, is an endemic South African plant that is gaining globally acclaim for its antioxidant properties and its role as functional medicinal beverage. In this article we dissect the role of rooibos in the context of the cell's ROS handling capacity, mitochondrial function and autophagy activity. By addressing the dynamic relationship between these critical interconnected systems, and by evaluating the functional properties of rooibos, we unravel its position for preserving cell viability and promoting healthy aging.

Aspalathus linearis suppresses cell survival and proliferation of enzalutamide-resistant prostate cancer cells via inhibition of c-Myc and stability of androgen receptor.

Enzalutamide, a nonsteroidal antiandrogen, significantly prolonged the survival of patients with metastatic castration-resistant prostate cancer (CRPC). However, patients receiving enzalutamide frequently develop drug resistance. Rooibos (Aspalathus linearis) is a shrub-like leguminous fynbos plant endemic to the Cedarberg Mountains area in South Africa. We evaluated the possibility of using a pharmaceutical-grade green rooibos extract (GRT, containing 12.78% aspalathin) to suppress the proliferation and survival of enzalutamide-resistant prostate cancer (PCa) cells. Treatment with GRT dose-dependently suppressed the proliferation, survival, and colony formation of enzalutamide-resistant C4-2 MDV3100r cells and PC-3 cells. Non-cancerous human cells were more resistant to GRT treatment. GRT suppressed the expression of proteins involved in phosphoinositide 3-kinase (PI3K)-Akt signaling, androgen receptor (AR), phospho-AR (Ser81), cyclin-dependent kinase 1 (Cdk1), c-Myc and Bcl-2 but increased the expression of apoptotic proteins. Overexpression of c-Myc antagonized the suppressive effects of GRT, while knockdown of c-Myc increased the sensitivity of PCa cells to GRT treatment. Expression level of c-Myc correlated to resistance of PCa cells to GRT treatment. Additionally, immunofluorescence microscopy demonstrated that GRT reduced the abundance of AR proteins both in nucleus and cytoplasm. Treatment with cycloheximide revealed that GRT reduced the stability of AR. GRT suppressed protein expression of AR and AR's downstream target prostate specific antigen (PSA) in C4-2 MDV3100r cells. Interestingly, we observed that AR proteins accumulate in nucleus and PSA expression is activated in the AR-positive enzalutamide-resistant PCa cells even in the absence of androgen. Our results suggested that GRT treatment suppressed the cell proliferation and survival of enzalutamide-resistant PCa cells via inhibition of c-Myc, induction of apoptosis, as well as the suppression of expression, signaling and stability of AR. GRT is a potential adjuvant therapeutic agent for enzalutamide-resistant PCa.

Therapeutic effects of an aspalathin-rich green rooibos extract, pioglitazone and atorvastatin combination therapy in diabetic db/db mice.

Oral therapeutics used to treat type 2 diabetes and cardiovascular disease often fail to prevent the progression of disease and their comorbidities. Rooibos (Aspalathus linearis), an endemic South African plant used as an herbal tea, has demonstrated positive effects on glycemia and hypercholesterolemia. However, the treatment efficacy of rooibos extract in combination with conventional hypoglycemic and hypolipidemic medications on blood glucose and lipid profiles has not been established. This study aimed to investigate the effects of combining an aspalathin-rich green rooibos extract (Afriplex GRT™) with pioglitazone and atorvastatin, on blood glucose and lipid levels in obese diabetic (db/db) mice. Six-week-old male db/db mice and their nondiabetic lean littermate controls (db+) were divided into 8 experimental groups (n = 6/group). Db/db mice were treated daily either with pioglitazone (25 mg/kg), atorvastatin (80 mg/kg) and GRT (100 mg/kg), a combination of either drug with GRT or a combination of GRT-pioglitazone and atorvastatin for 5 weeks. Untreated vehicle controls were given dimethyl sulfoxide (0.1%) and phosphate buffered saline solution. At termination, serum and liver tissue were collected for lipid and gene expression analysis. Treatment with GRT, pioglitazone and atorvastatin combination effectively lowered fasting plasma glucose (FPG) levels in db/db mice (p = 0.02), whilst increasing body weight, liver weight, and reducing retroperitoneal fat weight. Atorvastatin monotherapy was effective at reducing cholesterol (from 4.00 ± 0.12 to 2.93 ± 0.13, p = 0.0003), LDL-C (from 0.58 ± 0.04 to 0.50 ± 0.00, p = 0.04), HDL-C (from 2.86 ± 0.05 to 2.50 ± 0.04, p = 0.0003) and TG (from 2.77 ± 0.50 to 1.48 ± 0.23, p = 0.04), compared to the untreated diabetic control. The hypotriglyceridemic effect of atorvastatin was enhanced when used in combination with both GRT and pioglitazone. The addition of pioglitazone to GRT significantly lowered FPG and TG. In db/db mice, Apoa1 was significantly downregulated in the liver, whilst Pparγ was significantly upregulated compared to their db+ counterparts. GRT monotherapy downregulated Apoa1 expression (p = 0.02). Atorvastatin combined with GRT significantly downregulated mRNA expression of Apoa1 (p = 0.03), whilst upregulating the expression of Pparγ (p = 0.03), Pparα (p = 0.002), Srebp1 (p = 0.002), and Fasn (p = 0.04). The GRT-pioglitazone-atorvastatin combination therapy downregulated Apoa1 (p = 0.006), whilst upregulating Fasn (p = 0.005), Pparα (p = 0.041), and Srebp1 (p = 0.03). Natural products can improve the efficacy of current drugs to prevent diabetes-associated complications. GRT in combination with pioglitazone enhanced the reduction of FPG, whilst the addition of atorvastatin to the combination, significantly lowered triglyceride levels. However, when GRT was used in combination with atorvastatin only cholesterol levels were affected. Although these results confirm both glucose- and lipoprotein-lowering biological effects of GRT in combination with pioglitazone and atorvastatin, increased expression of genes involved in lipogenesis, cholesterol, and fatty acid transport, ß-oxidation, and synthesis and storage of fatty acids, may exacerbate the hepatotoxic effects of atorvastatin.

Aspalathus linearis infusion affects hole-board test behaviour and amino acid concentration in the brain.

Rooibos tea, brewed using Aspalathus linearis leaves, is a popular South African herbal infusion, but its everyday intake is not fully described in terms of the neuropsychopharmacological outcomes. The cell-protective activity of A. linearis is connected with the ability of reducing glycaemia, inflammation as well as oxidative stress. It was already shown that "fermented" rooibos herbal tea (FRHT), which is rich in phenolic compounds, improves the cognitive performance of rats in the water maze and impacts dopaminergic striatal transmission. The present research was taken to extend the knowledge about the feasible behavioural and neurochemical implications of sustained oral FRHT consumption. We hypothesized that it might affect brain amino acid content and thus induce behaviour and neuroprotection. FRHTs of different leaf to water ratios (1:100, 2:100 and 4:100), analysed by chromatographic methods as regards their flavonoid characteristics, were given to rats as only liquid for 3 months. Their behaviour was evaluated in the hole-board test (HBT). Brain amino acids concentration was analysed in the striatum, hippocampus and prefrontal cortex by HPLC-ECD. The rats drinking rooibos tea presented increased motor activity defined as time spent on moving in the HBT. Their exploration measured by head-dipping and rearing was enhanced. Longer time of the testing-box central zone occupation indicated to reduction in anxiety-related behaviour. Excitatory amino acids (aspartate and glutamate) content was decreased in the striatum of animals drinking the infusions whereas taurine level was increased both in the striatum and hippocampus. In conclusion we suggest that long-term FRHT intake affects exploration and anxiety-related behaviour of the rats as well as exerts biochemical outcomes in the brain that support the neuroprotective impact of rooibos tea.

Photosynthetic metabolism during phosphate limitation in a legume from the Mediterranean-type Fynbos ecosystem.

Phosphorus (P) is an essential mineral, required for crucial plant genetic, metabolic and signaling functions. Under P deficiency, normal physiological function can be disrupted, especially photosynthetic metabolism. The majority of photosynthetic studies of P stress has been on model organisms, and very little is known about plants that evolved on P deficient soils. Aspalathus linearis (Burm.f.) R.Dahlgren, a native to the Mediterranean ecosystem of South Africa was used to study the photosynthetic responses during short-term P limitation. A. linearis seedlings were cultured under glasshouse conditions and exposed to short-term P stress. Leaf photosynthetic gas exchange was coupled with metabolic analyses. In spite of the decline in leaf cellular Pi, the photosynthetic rates remained unchanged. These leaves also maintained their levels of light harvesting and reaction center pigments. The efficiency of the light reactions' utilization of ATP and NADPH increased during P-stress. Leaf glucose levels decreased during P-stress, while sucrose concentrations remained unaffected. These results show that during short-term P-stress, A. linearis can maintain its photosynthetic rates by altering the structural and functional components of the light reactions.

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.

Safety Assessment of Phytochemicals Derived from the Globalized South African Rooibos Tea ( Aspalathus linearis) through Interaction with CYP, PXR, and P-gp.

Rooibos tea ( Aspalathus linearis) is a well-known South African herbal tea enjoyed worldwide. Limited reports indicate the potential of rooibos tea to alter the activity of certain cytochrome P450 (CYP450) isozymes. In this study, the phytochemical investigation of MeOH extract of A. linearis (leaves and stems) resulted in the isolation and characterization of 11 phenolic compounds. The MeOH extract exhibited significant inhibition of the major human CYP450 isozymes (CYP3A4, CYP1A2, CYP2D6, CYP2C9, and CYP2C19). The strongest inhibition was observed by the extract for CYP3A4 (IC50 1.7 ± 0.1 µg/mL) followed by CYP2C19 (IC50 4.0 ± 0.3 µg/mL). Among the tested phytochemicals, the most potent inhibitors were isovitexin on CYP3A4 (IC50 3.4 ± 0.2 µM), vitexin on CYP2C9 (IC50 8.0 ± 0.2 µM), and thermopsoside on CYP2C19 (IC50 9.5 ± 0.2 µM). The two major, structurally related compounds aspalathin and nothofagin exhibited a moderate pregnane-X receptor (PXR) activation, which was associated with increased mRNA expression of CYP3A4 and CYP1A2, respectively. These results indicate that a high intake of nutraceuticals containing rooibos extracts may pose a risk of herb-drug interactions when consumed concomitantly with clinical drugs that are substrates of CYP enzymes.

Electrospraying as a suitable method for nanoencapsulation of the hydrophilic bioactive dihydrochalcone, aspalathin.

The bioactive hydrophilic dihydrochalcone, aspalathin, has poor stability and bioavailability hampering its use in functional food ingredients with standardised aspalathin content. The aim of the study was to produce nanoparticles with controlled release to overcome these obstacles. Nanoencapsulation was investigated using both natural (chitosan and lecithin) and synthetic (poly(lactide-co-glycolide) and Eudragit S100® (ES100)) polymers by suitable conventional methods and electrospraying for all polymers. All polymer-method combinations produced particles smaller than 1.1 µm. Electrospraying produced more favourable results than conventional methods for the synthetic polymers, resulting in spherical particles with higher (p < 0.05) encapsulation efficiencies (>50%) and loading capacities (>10%). Opposite trends were observed for natural polymers. An in vitro release study revealed biphasic aspalathin release profiles at pH 7.4 with ES100 electrosprayed nanoparticles having the slowest (p < 0.05) release rate (1.67 h-1). Overall, ES100 electrosprayed nanoparticles showed the most favourable combination of parameters.

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.

Chemistry of color formation during rooibos fermentation.

Nonenzymatic oxidative degradation of aspalathin, a dihydrochalcone unique to green rooibos (Aspalathus linearis), resulted in formation of the characteristic red-brown color of processed rooibos tea. As recently reported, two colorless dimers were formed by oxidative coupling. Incubations of aspalathin showed further distinct signals. Isolation by multilayer countercurrent chromatography (MLCCC) followed by preparative high-performance liquid chromatography (HPLC) led to pure substances. Subsequent analysis by NMR and MS techniques identified a third colorless dimeric compound. In addition for the first time, two colored structures with dibenzofuran skeleton, (S)- and (R)-3-(7,9-dihydroxy-2,3-dioxo-6-ß-d-glucopyranosyl-3,4-dihydrodibenzo[b,d]furan-4a(2H)-yl) propionic acid, and their corresponding mechanistic precursors were unequivocally established. Color-dilution analysis revealed these compounds as the key chromophores of the incubated aspalathin solutions, ultimately being degraded to unknown, more stable tannin-like structures. Their mechanistic importance to color formation was further underlined by detection of the dibenzofurans also in fermented rooibos tea after trapping with o-phenylenediamine as their corresponding quinoxaline derivatives.

African legumes: a vital but under-utilized resource.

Although nodulated legumes have been used by indigenous peoples in Africa for centuries, their full potential has never been realized. With modern technology there is scope for rapid improvement of both plant and microbial germplasm. This review gives examples of some recent developments in the form of case studies; these range from multipurpose human food crops, such as cowpea (Vigna unguiculata (L.) Walp.), through to beverages (teas) that are also income-generating such as rooibos (Aspalathus linearis (Burm. f.) R. Dahlgren, honeybush (Cyclopia Vent. spp.), and the widely used food additive gum arabic (Acacia senegal (L.) Willd.). These and other potential crops are well-adapted to the many different soil and climatic conditions of Africa, in particular, drought and low nutrients. All can nodulate and fix nitrogen, with varying degrees of effectiveness and using a range of bacterial symbionts. The further development of these and other species is essential, not only for African use, but also to retain the agricultural diversity that is essential for a changing world that is being increasingly dominated by a few crops such as soybean.

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.

Regeneration of coenzyme Q9 redox state and inhibition of oxidative stress by Rooibos tea (Aspalathus linearis) administration in carbon tetrachloride liver damage.

The effect of rooibos tea (Aspalathus linearis) on liver antioxidant status and oxidative stress was investigated in rat model of carbon tetrachloride-induced liver damage. Synthetic antioxidant N-acetyl-L-cysteine (NAC) was used for comparison. Administration of carbon tetrachloride (CCl4) for 10 weeks decreased liver concentrations of reduced and oxidized forms of coenzyme Q9 (CoQ9H2 and CoQ9), reduced -tocopherol content and simultaneously increased the formation of malondialdehyde (MDA) as indicator of lipid peroxidation. Rooibos tea and NAC administered to CCl4-damaged rats restored liver concentrations of CoQ9H2 and alpha-tocopherol and inhibited the formation of MDA, all to the values comparable with healthy animals. Rooibos tea did not counteract the decrease in CoQ9, whereas NAC was able to do it. Improved regeneration of coenzyme Q9 redox state and inhibition of oxidative stress in CCl4-damaged livers may explain the beneficial effect of antioxidant therapy. Therefore, the consumption of rooibos tea as a rich source of natural antioxidants could be recommended as a market available, safe and effective hepatoprotector in patients with liver diseases.