Afriplex GRTTM extract attenuates hepatic steatosis in an in vitro model of NAFLD.

BACKGROUND: Currently, it is acknowledged that vitamin E, insulin sensitizers and anti-diabetic drugs are used to manage non-alcoholic fatty liver disease (NAFLD), however, these therapeutic interventions harbour adverse side effects. Pioglitazone, an anti-diabetic drug, is currently the most effective therapy to manage NAFLD. The use of natural medicines is widely embraced due to the lack of evidence of their negative side effects. Rooibos has been previously shown to decrease inflammation and oxidative stress in experimental models of diabetes, however, this is yet to be explored in a setting of NAFLD. This study was aimed at investigating the effects of an aspalathin-rich green rooibos extract (Afriplex GRTTM) against markers of hepatic oxidative stress, inflammation and apoptosis in an in vitro model of NAFLD. METHODS: Oleic acid [1 mM] was used to induce hepatic steatosis in C3A liver cells. Thereafter, the therapeutic effect of Afriplex GRTTM, with or without pioglitazone, was determined by assessing its impact on cell viability, changes in mitochondrial membrane potential, intracellular lipid accumulation and the expression of genes and proteins (ChREBP, SREBF1, FASN, IRS1, SOD2, Caspase-3, GSTZ1, IRS1 and TNF-α) that are associated with the development of NAFLD. RESULTS: Key findings showed that Afriplex GRTTM added to the medium alone or combined with pioglitazone, could effectively block hepatic lipid accumulation without inducing cytotoxicity in C3A liver cells exposed oleic acid. This positive outcome was consistent with effective regulation of genes involved in insulin signaling, as well as carbohydrate and lipid metabolism (IRS1, SREBF1 and ChREBP). Interestingly, in addition to reducing protein levels of an inflammatory marker (TNF-α), the Afriplex GRTTM could ameliorate oleic acid-induced hepatic steatotic damage by decreasing the protein expression of oxidative stress and apoptosis related markers such as GSTZ1 and caspase-3. CONCLUSION: Afriplex GRTTM reduced hepatic steatosis in oleic acid induced C3A liver cells by modulating SREBF1, ChREBP and IRS-1 gene expression. The extract may also play a role in alleviating inflammation by reducing TNF-α expression, suggesting that additional experiments are required for its development as a suitable therapeutic option against NAFLD. Importantly, further research is needed to explore its antioxidant role in this model.

A high fat, high sugar diet induces hepatic Peroxisome proliferator-activated receptor gamma coactivator 1-alpha promoter hypermethylation in male Wistar rats.

Previously we reported that a high fat, high sugar (HFHS) diet induced adiposity, hyperinsulinaemia, hyperleptinaemia, hypertriglyceridaemia and increased liver mass in male Wistar rats. In the present study, the mechanisms underlying the increased liver mass were further elucidated by assessing hepatic lipid accumulation and the expression and methylation status of key metabolic genes using histology, quantitative real-time PCR and pyrosequencing, respectively. The HFHS diet induced hepatic steatosis, increased hepatic triglycerides (1.8-fold, p < 0.001), and increased the expression of sterol regulatory element-binding transcription factor 1 (Srebf1) (2.0-fold, p < 0.001) and peroxisome proliferator-activated receptor gamma (Pparg) (1.7-fold, p = 0.017) in the liver. The expression of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (Pgc1a) was decreased (2.6-fold, p < 0.010), which was accompanied by hypermethylation (p = 0.018) of a conserved CpG site in the promoter of Pgc1a in HFHS fed rats compared to controls. In silico analysis identified putative binding sites for CCAAT/enhancer-binding protein beta (C/EBPß) and hepatocyte nuclear factor 1 (HNF1) within proximity to the hypermethylated CpG. As Pgc1a is a co-activator of several transcription factors regulating multiple metabolic pathways, hypermethylation of this conserved CpG site in the promoter of Pgc1a may be one possible mechanism contributing to the development of hepatic steatosis in response to a HFHS diet. However, further work is required to confirm the role of Pgc1a in steatosis.

The Effect of Rooibos (Aspalathus linearis), Honeybush (Cyclopia intermedia) and Sutherlandia (Lessertia frutescens) on Testicular Insulin Signalling in Streptozotocin-Induced Diabetes in Wistar Rats.

BACKGROUND: Testicular insulin signalling is altered in diabetic (DM) males. While unravelling the mechanism through which DM exert these detrimental effects, studies have shown the importance of insulin regulation in glucose homeostasis, and how a lack in insulin secretion indirectly led to reduced male fertility. The current study aimed to investigate the role of rooibos, honeybush and Sutherlandia on insulin signalling in the testicular tissue of type I diabetic rats. METHODS: Animals (n=60) were randomly divided into six groups. The groups include a control group, a vehicle group, and diabetes was induced in the remainder of animals via a single intraperitoneal injection of STZ at 45mg/kg. The remaining four groups included a diabetic control (DC), diabetic + rooibos (DRF), diabetic + honeybush (DHB) and diabetic + Sutherlandia group (DSL). Animals were sacrificed after seven weeks of treatment, and blood and testes were collected. RESULTS: All diabetic groups (DC, DRF, DHB, DSL) presented with a significant increase in blood glucose levels after diabetes induction compared to the control and vehicle (p<0.001). The DC animals presented with decreased testicular protein expression of IRS-1, PkB/Akt and GLUT4 compared to controls. DRF and DHB animals displayed an acute upregulation in IRS-1, while the DSL group showed improvement in IRS-2 compared to DC. Although, DRF animals presented with a decrease in PkB/Akt, DHB and DSL animals displayed upregulation (22.3%, 48%) compared to controls, respectively. CONCLUSION: The results taken together, it can be suggested that these infusions may enhance insulin signalling through diverse pathways.

The modulating effects of green rooibos (Aspalathus Linearis) extract on vascular function and antioxidant status in obese Wistar rats.

PURPOSE: Obesity is associated with the development of risk factors for cardiovascular disease (CVD) and polyphenols have been shown to possess ameliorative effects against obesity-induced CVD risk factors. Rooibos (Aspalathus linearis) is rich in polyphenols, therefore we investigated the cardioprotective effects of aspalathin-rich green rooibos (GRT) on obesity-induced CVD risk factors in obese Wistar rats. METHODS: Adult male Wistar rats (n = 20 per group) were fed a control or a high-fat diet (HFD) for 16 weeks and treated with GRT (60 mg/kg/day) for six weeks. Blood pressure was monitored throughout. Vascular reactivity was measured and Western blots of cell-signalling proteins (eNOS, AMPK and PKB) were performed in aortic tissues. Effects on oxidative stress were determined by measuring antioxidant enzyme activity and thiobarbituric reactive substance (TBARS) levels in the liver. RESULTS: HFD animals had (1) increased blood pressure, (2) impaired vasodilation, (3) attenuated PKB and AMPK expression, (4) decreased antioxidant enzyme activity, (5) increased malondialdehyde (MDA) levels, and (6) increased phosphorylated eNOS levels. Treatment with GRT extract significantly alleviated these obesity-induced CVD risk factors. CONCLUSIONS: Supplementation with GRT extract alleviated cardiovascular risk factors in the HFD animals, suggesting a therapeutic potential for GRT in obesity-induced cardiovascular risk.

Effect of Rooibos (Aspalathus linearis) extract on atorvastatin-induced toxicity in C3A liver cells.

Rooibos (Aspalathus linearis) has various health benefits. Two case studies have associated chronic Rooibos consumption with conventional prescription medications, including atorvastatin (ATV), with hepatotoxicity. Statins act by inhibiting hydroxymethylglutaryl-coenzyme A reductase, a rate-limiting enzyme in cholesterol synthesis. Although rare, statins are potentially hepatotoxic. The aim was to investigate interactions between aspalathin-rich Rooibos extract GRT™ and ATV-induced hepatotoxicity in C3A liver cells cultured with and without palmitate. Effects of co-treatment of GRT + ATV on cell viability, oxidative stress, apoptosis, mitochondrial integrity, and cellular reactive oxygen species (ROS) production were assessed. Significantly increased ROS production was observed in cells exposed to ATV and palmitate. Combination therapy of GRT + ATV also showed significant increases in ROS production. Under palmitate-treated conditions, ATV-induced significant apoptosis which was not ameliorated by GRT + ATV co-treatment. Despite studies purporting hepatoprotection from Rooibos, our study showed that GRT was unable to modulate ATV-induced hepatotoxic effects in this model.

Rooibos (Aspalathus linearis) protects against nicotineinduced vascular injury and oxidative stress in Wistar rats.

BACKGROUND: Rooibos (Aspalathus linearis) is an indigenous South African plant, traditionally used by the local population as a remedy against several ailments. More recently, rooibos was shown to exhibit potent antioxidant properties, attributed to its polyphenols. We assessed whether treatment with fermented rooibos (RF), unfermented rooibos (RUF) and melatonin (Mel), a well-documented antioxidant included for comparison, could counter the harmful vascular and pro-oxidant effects of nicotine. METHODS: Vascular function, antioxidant enzyme activity and lipid peroxidation were assessed in male adult rats treated with nicotine (5 mg/kg body weight/day) and 2% RF, 2% RUF or 4% Mel co-administration. Nitric oxide (NO) production and cell viability were measured in nicotine-exposed rat aortic endothelial cells (AECs) pre-treated with RF (0.015 mg/ml). RESULTS: Vascular studies showed that co-administration with RF or Mel exerted anti-contractile and pro-relaxation responses in aortic rings, and increased hepatic superoxide dismutase and catalase activity in nicotine-exposed animals. Co-treatment with Mel additionally decreased lipid peroxidation in nicotine-exposed rats. RUF exerted anti-contractile responses in aortic rings of nicotine-treated animals, while in nicotine-exposed AECs, RF pre-treatment increased intracellular NO levels. CONCLUSIONS: For the first time, we have shown that rooibos co-treatment exerted beneficial vascular effects in nicotine-exposed rats, and that this was associated with increased antioxidant enzyme activity.