The combination of red palm oil and rooibos show anti-inflammatory effects in rats

Male Wistar rats weighing 150–200 g were randomly divided into 8 groups and supplemented with fermented/traditional rooibos, red palm oil (RPO) or their combination for 28 days. The four groups were further subdivided into two groups, either receiving 1) No-LPS or 2) LPS injection. Group 1 which is the NO-LPS group consisted of the control group receiving standard rat chow and water, rooibos group receiving standard rat chow and rooibos, RPO group receiving standard rat chow supplemented with RPO 0.2 mL (7 g/kg diet) daily and water. The RPO concentrate was supplied by Carotino SND BHD (Company no. 69046-T) Malaysia. The composition of RPO consumed by the rats is shown in (Table 1).

The rooibos + RPO group received a combination of rooibos and RPO (without LPS treatment). Group 2 which is the LPS group consisted of the control group receiving standard rat chow and water, rooibos group receiving standard rat chow and rooibos, RPO group receiving standard rat chow supplemented with RPO 0.2 mL (equivalent to 7 g/kg diet) daily and rooibos + RPO group receiving the combination of rooibos and RPO (with LPS treatment). Superior grade fermented rooibos was provided by Rooibos Ltd (Clanwilliam, South Africa). The rooibos aqueous extract was prepared by the addition of 100 ml of freshly boiled water to 10 g of tea leaves, filtered and stored at −40°C, and diluted 5 times, a concentration customarily used for tea consumption purposes, before being given to the rats [56]. Phenolic content, antioxidant capacity and flavonoids composition of the rooibos consumed by the animals are as analyzed by Ajuwon et al. [9]. The animals were given 100 ml of the freshly diluted rooibos every second day. The rooibos and water consumption was monitored throughout the feeding period and there were no statistical differences observed in either rooibos or water consumption among the experimental groups (data not shown). At the end of the feeding period (28 days), 18 hours prior to sacrificing, animals in the LPS group were injected (intraperitoneal) with lipopolysaccharide (Escherichia coli serotype) to induce inflammation. The LPS was dissolved in sterile filtered phosphate buffered saline (PBS) to obtain 0.5 mg/kg body weight in 0.1 ml [51]. The animals in the NO-LPS were injected (intraperitoneal) with 0.1 ml of PBS (Table 2).

At the end of the feeding period and inflammation injection protocol, rats were fasted for 16 hours before sacrificed and anaesthetized with an intraperitoneal injection of 2 mg/kg intraval sodium (sodium pentobarbital). Blood was collected from the abdominal aorta (approximately 5–8 ml) and placed into plain tubes for cytokine analysis. Serum was then separated immediately by centrifuging at 5000 g for 5 min at 4°C, the samples were then stored at −80°C till analysis were performed. Hearts were rapidly excised and placed in ice-cold Krebs-Henseleit buffer and transferred to the Langendorff perfusion system. Hearts were perfused with a Krebs-Henseleit buffer equilibrated with 95% O₂ and 5% CO₂ at 37°C (118.5 mM NaCl; 4.75 mM KCl; 1.2 mM MgCl 6 H₂O; 1.36 mM CaCl₂; 25.0 mM NaHCO₃; 1.2 mM KH₂PO₄; 11.0 mM glucose) and a perfusion pressure of 100cmH2O was maintained throughout the protocol. Hearts were mounted to the Langendorff system and perfused for 15 minutes. Coronary flow, heart rate, LVDevP, RPP, ±dp/dt max derivatives, EDLVP were documented at baseline phase. LVDevP was measured with the aid of a balloon made from transparent sandwich wrap film inserted into the left ventricle through the opening of the left atrium. The balloon was connected to a power lab system (AD Instruments Pty Ltd., Castle Hill, Australia). After insertion, the balloon was inflated to 2 mmHg, and the contraction force of the heart against the balloon caused water displacement that was converted to pressure. The systolic and diastolic pressures as well as the heart rate and minimum and maximum derivatives were documented on the computer. At the end of the perfusion protocol hearts were removed from the system and stored at −80°C till biochemical analysis were performed.

Analyses of samples were performed on undiluted myocardial tissue homogenates which were originally prepared in phosphate buffer at a dilution of 1:4. In order to analyze the myocardial cytokines, hearts from all the 8 groups were freeze-clamped with Wollenberger clamp pre-cooled in liquid nitrogen. The heart samples were then grinded into powder and 100 mg of heart tissue powder was diluted with 500 μl of phosphate buffer. The mixture was homogenized by ultrasonic homogenizer at maximum power (2×20 sec), and the homogenate was centrifuged at 4°C for 20 minutes at 5000 g. The supernatant was collected and stored at −80°C till analyses were carried out. Protein tissue content was determined using Bradford technique [57]. Plasma and myocardial IL-1 beta, IL-6 and IL-10 levels were measured using the Bio-Plex bead array system (Bio Rad Laboratories, USA). Assays were carried out in 96-well filter plates, while the rat cytokine kits, (Cat#: RCYTO-80 K) were obtained from Millipore (USA). Samples were evaluated in duplicate. All levels of analytes in quality control reagents included in the kits were within the expected references ranges.

The increased levels of plasma IL-1β in the LPS-induced rats indicate that there was induction of inflammation in response to the presence of the endotoxin (LPS), (Figure 1a). IL-1β is one of the initial pro-inflammatory cytokines to be released in response to the invading microbial pathogens (specifically the lipopolysaccharide, an endotoxin embedded within the bacterial membrane) and it plays a crucial role in the induction of inflammation [58]. Therefore, increased levels of circulating IL-1β are indicative of a systemic inflammatory response [59],[60]. The response to LPS is initiated upon the recognition of LPS by the LPS-binding protein, following the binding of LPS to its binding protein a series of multiple complex signalling pathways is initiated. This will ultimately result in activation of the Toll-like Receptor (TLR) 4 through various adaptor proteins leading to NF-κB activation and eventual induction of inflammatory cytokines [61]–[64]. LPS triggers the release of inflammatory cytokines from various cells of the immune system, the released cytokines leads to an acute inflammatory response directed towards the invading pathogen [65]–[67]. The finding of elevated plasma levels of IL-1β therefore confirms that inflammation was induced in the current model. Our results are in agreement with previous reports by Ohsaki and co-workers [51] which used a similar dose of LPS and showed increased IL-6 mRNA levels indicating that inflammation was induced. Our results also show that supplementation of either rooibos or red palm oil, together and separate, could not prevent an increase in plasma IL-1β.

However, in the myocardium, our results show that consumption of rooibos in the LPS-induced rats was associated with increased myocardial levels of IL-1β compared to the positive control, while RPO and rooibos + RPO did not affect the induction of myocardial IL-1β (Figure 2a). Myocardial and endothelial cells have the capacity to respond to LPS via activation of the TLRs leading to induction of inflammatory cytokines [67]. The role of cytokines in inflammation is complex and is determined by various factors such as the magnitude of cytokine induction, the presence of receptors to cytokines and also by the presence of antagonist mediators such as anti-inflammatory cytokines. The increased levels of myocardial levels of IL-1β in the LPS-induced rats consuming rooibos may represent a normal cellular response to the presence of the endotoxin [68], especially because the observed increases in myocardial levels of IL-1β in LPS-induced rats consuming rooibos were not associated with alterations cardiac function.

Dietary intervention with rooibos, RPO or their combination did not have any effect on plasma IL-6 levels in LPS-treated animals and their non-treated counterparts (Figure 1b). There was differential modulation of myocardial IL-6 by the dietary supplements in the LPS-induced supplemented rats. Consumption of rooibos and that of the combination of rooibos and RPO in LPS-induced rats caused an increased in myocardial IL-6 compared to the positive control level comparative to that of the negative control animals (Figure 2b). Even though IL-6 is classically characterized as a pro-inflammation cytokine, it has been shown to have both pro-inflammatory and anti-inflammatory features [69],[70]. IL-6 can evoke an anti-inflammatory environment by inducing the production of anti-inflammatory cytokines, such as IL-10 and IL-1ra in humans [70]. Our results show that the increase in myocardial IL-6 in the LPS-induced rats consuming rooibos and rooibos + RPO was associated with enhanced up-regulation of myocardial IL-10. Therefore the elevation of both IL-6 and IL-10 indicates that in this instance, IL-6 might be acting as an anti-inflammatory cytokine leading to enhancement of IL-10 production. There is evidence showing that in some instances acute elevation of IL-6 may be beneficial, especially following exercise in humans [70]. Xing and co-workers [71] showed that endogenous IL-6 plays an anti-inflammatory role in both local and systemic acute inflammatory responses in mice. This mechanism acts by controlling the level of pro-inflammatory, but not anti-inflammatory, cytokines [72]. Others have also shown that blockade of IL-6 in patients with rheumatoid arthritis led to enhanced cholesterol and plasma glucose levels, indicating a role for IL-6 in modulation of glucose and lipid metabolism [73],[74]. Results in the current study would therefore indicate that endogenous IL-6 rather protected than harmed the heart against induction of LPS, especially in the local organ region as is presented in Figure 2b. The results further show that dietary intervention can influence the levels of IL-6 in cardiac tissue. There is also a difference between systemic and local response to IL-6 levels with LPS induction in the presence of dietary supplements such as red palm oil and rooibos. This needs to be further investigated and clarified.

The current results report that plasma IL-10 levels were significantly elevated in the two LPS-treated groups consuming either rooibos or RPO when compared to the LPS control. However, the LPS-induced rats consuming the combination of rooibos and RPO did not show any effect on plasma IL-10 levels indicating that there was no additional benefit on plasma IL-10 levels when rooibos and RPO were given in combination (Figure 1c). The results indicate that dietary supplementation with rooibos and RPO up-regulated the production of IL-10 in response to the presence of inflammation. IL-10 is a potent anti-inflammatory cytokine whose role is to counteract the effects of pro-inflammatory mediators in various forms of shock and inflammation [75]. Inflammatory cells in the circulation are activated in response to invasion of LPS and the initial induction of inflammatory cytokines in response to LPS is aimed at clearing local effect of the invading pathogen [76]. However, the body has also evolved regulatory systems to maintain the balance between the levels of pro-inflammatory mediators and anti-inflammatory mediators in order to sustain cellular homeostasis and immune system integrity. We have shown that rooibos and RPO, when supplemented individually, enhanced production of IL-10 in the blood, suggesting a potential anti-inflammatory effect at systemic level. Therefore the concomitant release of IL-1 β and IL-10 in plasma of LPS-induced rats consuming rooibos and RPO indicate that dietary intervention with rooibos and RPO modulated the inflammatory response in the model of inflammation by enhancing systemic production of the anti-inflammatory cytokine. Rooibos is rich in various polyphenolic compounds, some unique to the plant as well, of which flavonoids are the most predominant [77]. Various polyphenolic molecules have been shown to exhibit anti-inflammatory activity [78],[79]. Polyphenols have a wide range of biological effects which include antioxidant and anti-inflammatory effects [80]–[83]. In the current study we have shown that rooibos consumption was associated with increased levels of plasma IL-10. This is in line with previous studies where polyphenols were associated with enhanced production of IL-10 and suppression of IL-1β [81].

Just as polyphenols form a vital part of a healthy diet, vitamins are also equally essential for human health. RPO is rich in various forms of vitamin E and carotenoids which function as cellular antioxidants [84],[13]. Inflammation and oxidative stress are closely related and are usually common features underlying etiological and pathological mechanisms for most chronic diseases including cardiovascular diseases [85],[86]. Both tocotrienols and tocopherol are potent antioxidants and have also been shown to possess potential anti-inflammatory properties [19],[22].

Dietary supplementation with the combination of rooibos and RPO resulted in increased myocardial levels of IL-10 in the LPS-induced rats compared to the positive control while, when supplemented individually, rooibos and RPO in the presence of LPS, did not have any effect on myocardial IL-10 levels (Figure 2c), this result could be indicative of a threshold needed to exert the effect, i.e. that of increased IL-10 levels. To our knowledge this is the first evidence showing that the combination of rooibos and RPO resulted in up-regulation of myocardial IL-10 levels. Elevated myocardial levels of IL-10 have been linked to cardio-protection [87]. Endogenous production of IL-10 has been shown to play an important role in maintaining myocardial integrity during ischaemia-reperfusion via modulation of the inflammation response. In this regard Yang et al. [88], reported that genetic deletion of IL-10 was associated with enhanced inflammation and increased myocardial infarction and necrosis.

LPS treatment and dietary supplementation with rooibos and RPO did not have an effect on baseline cardiac functional parameters. The increases in plasma IL 1β levels and in myocardial tissue of the LPS-induced rooibos group were not associated with ventricular dysfunction or reduction in coronary flow (Table 3). This is contrary to reports that IL-1β has a negative inotropic effect and that it also leads to endothelial dysfunction [89],[54]. The reason for this could be that the dose of LPS that we used in this study was sufficient to induce inflammation but not high enough to induce cardiac dysfunction. In previous studies where ventricular dysfunction was reported, higher doses of LPS were used [90],[91]. Lew et al. [92], also reported that sub-lethal dose of LPS had minimal effect on cardiac function. Another plausible reason could be that low doses or sub-lethal doses of LPS have been shown to have a pre-conditioning effect [93]–[95].