Aspalathus linearis (Rooibos) – a functional food targeting cardiovascular disease

Carine Smith *a and Amanda Swart *b
aDept Physiological Sciences, Science Faculty, Stellenbosch University, Stellenbosch, South Africa. E-mail: csmith@sun.ac.za
bDept Biochemistry, Science Faculty, Stellenbosch University, Stellenbosch, South Africa. E-mail: acswart@sun.ac.za

Received 22nd May 2018 , Accepted 20th August 2018

First published on 5th September 2018


Increasing consumer bias toward natural products and the considerable wealth of indigenous knowledge has precipitated an upturn in market-driven research into potentially beneficial medicinal plants. In this context, Aspalathus linearis (Rooibos) has been identified to be a promising candidate which may impact cardiovascular disease (CVD), which is one of the most widely studied chronic diseases of modern times. Despite these efforts, ischemic heart disease remains the number one cause of mortality globally. Apart from genetic predisposition and other aetiological mechanisms specific to particular types of CVD, co-factors from interlinked systems contribute significantly to disease development and the severity of its clinical manifestation. The bioactivity of Rooibos is directed towards multiple therapeutic targets. Experimental data to date include antioxidant, anti-inflammatory and anti-diabetic effects, as well as modulatory effects in terms of the immune system, adrenal steroidogenesis and lipid metabolism. This review integrates relevant literature on the therapeutic potential of Rooibos in the context of CVD, which is currently the most common of non-communicable diseases. The therapeutic value of whole plant extracts versus isolated active ingredients are addressed, together with the potential for overdose or herb–drug interaction. The body of research undertaken to date clearly underlines the benefits of Rooibos as both preventative and complementary therapeutic functional food in the context of CVD.


Introduction

South Africa is a country known for its richness and diversity in terms of both indigenous flora and traditional, indigenous use of plants for their medicinal properties and as functional foods. One plant in particular – Aspalathus linearis (Rooibos) – is consumed globally as tisane or herbal tea. Apart from its distinct refreshing taste, the potential medicinal benefits of Rooibos which adds to its popularity and ever increasing demand, has been widely researched.

The global shift towards natural product supplementation for healthy ageing coincides with the launch of the “Traditional Medicine Strategy: 2014–2023” initiative by the WHO, which is aimed at prioritizing traditional medicines and their use for the improvement of health. According to the most recent World Health Organisation (WHO) fact sheet,1 ischemic heart disease remains the number one cause of mortality globally. Despite the efforts of the large cohort of scientists dedicated to studying cardiovascular disease (CVD), this condition has claimed 8.76 million lives in 2015 alone – far more than the 6.24 million ascribed to stroke, the second leading cause of death.2 These statistics suggest that remedial therapy is largely inefficient and that preventative and/or complimentary medicine should be considered in order to prevent progression of the disease to a point of no return.

The aim of this review is to provide a holistic overview of the medicinal potential of Rooibos, which is classified as a traditional African medicine,3 specifically in the context of cardiovascular health. To this end, an update on the characterization of Rooibos is followed by an in depth overview of peer-reviewed data on Rooibos, integrated with relevant aetiological factors implicated in cardiovascular pathology. A comprehensive discussion of all mechanisms implicated in CVD is beyond the scope of this review – rather, we have limited ourselves to only the mechanisms most relevant to facilitate contextualization and interpretation of available Rooibos data, together with reference to its impact on therapeutic approaches in the treatment of CVD.

The character of Rooibos

The characterization of the compounds in Rooibos is an ongoing process, expanding as analytical tools improve, with a vast number of Rooibos compounds having been identified to date. Rooibos constituents comprise a wide range of polyphenolic compounds including dihydrochalcones, flavanones, flavones, flavonols, monomeric and oligomeric flavan-3-ols, lignans, hydroxycinnamic acid and derivatives, phenolic carboxylic acids, etc. Recently, Walters et al.4 reported 39 phenolic compounds in Rooibos of which 18 were identified for the first time using a comprehensive 2D method – normal phase high performance countercurrent chromatography (NP-HPCCC) in the first dimension and a reverse phase ultra-high pressure liquid chromatography (RP-UHPLC) in the second dimension. Separation in the first dimension enabled the chromatographic separation of both polar and less polar polyphenolic di-C-glycosides and mono- and di-O-glycosides while the UHPLC in the second dimension enabled the separation of compounds co-eluting in NP-HPCCC chromatographic fractions. They successfully identified scolymoside (a flavone), hesperidin (a flavanone) and phloretin-3,5-di-C-D-glucopyranoside (a dihydrochalcone) for the first time using quadrupole time-of-flight MS (Q-TOF-MS) to identify compounds and confirm peak purity.4 Later in the same year, Stander et al.5 profiled 59 compounds in Rooibos using ultra-performance liquid chromatograph (UPLC) (Waters, Milford, MA, USA) coupled to a Waters Synapt G2 Quadrupole time-of-flight (QTOF) mass spectrometer (MS). Of these, 25 were novel of which seven remain unknown. Their profile analyses eloquently confirmed that Rooibos constituents exhibit characteristic regional variation and they identified the main polyphenol compounds to be aspalathin, nothofagin, orientin, iso-orientin, vitexin, isovitexin, isoquercetin, quercetin, and rutin (Table 1). Aspalathin was again confirmed as being unique to Rooibos together with phenylpropenioc acid glucoside (PPAG) and two additional dihydrochalcones, phloridzin and a sieboldin analogue.5
Table 1 Major phenolic compounds and oxidized derivatives in Rooibos
Structure Compound
image file: c8fo01010b-u1.tif Phenylpyruvic acid-2-O-glucopyranoside (PPAG)
R = β-D-glucopyranosyl
Flavonols
image file: c8fo01010b-u2.tif Isoquercitrin
R = β-D-glucopyranosyloxy
Hyperoside
R = β-D-galactopyranosyloxy
Rutin
R = α-L-rhamnopyranosyl-(1 → 6)-β-D-glucopyranosyloxy
Quercetin-3-O-robinobioside
R = α-L-rhamnopyranosyl-(1 → 6)-β-D-galactopyranosyloxy
Dihydrochalcones
image file: c8fo01010b-u3.tif Aspalathin
R1 = OH, R2 = C-β-D-glucopyranosyl
Nothofagin
R1 = H, R2 = C-β-D-glucopyranosyl
Flavanones
image file: c8fo01010b-u4.tif Dihydro-orientin ((S)/(R)-eriodictyol-8-C-glucopyranoside)
R1 = β-D-glucopyranosyl, R2 = H
Dihydro-iso-orientin ((S)/(R)-eriodictyol-6-C-glucopyranoside)
R1 = H, R2 = β-D-glucopyranosyl
Flavones
image file: c8fo01010b-u5.tif Luteolin-7-O-glucopyranoside
R1 = OH, R2 = R4 = H, R3 = β-D-glucopyranosyl
Orientin
R1 = R3 = OH, R2 = β-D-glucopyranosyl, R4 = H
Iso-orientin
R1 = R3 = OH, R2 = H, R4 = β-D-glucopyranosyl
Vitexin
R1 = R4 = H, R2 = β-D-glucopyranosyl, R3 = OH
Iso-vitexin
R1 = R2 = H, R3 = OH, R4 = β-D-glucopyranosyl


Green vs. fermented Rooibos – what is the difference?

Rooibos is cultivated primarily for the purposes of producing an herbal tea for both the South African and international markets and is sold as either fermented Rooibos or green Rooibos, with the former being the more commonly available product. In the processing of Rooibos after harvesting, plant material is subjected to a fermentation process during which polyphenolic compounds are oxidized – briefly, plant material is bruised and allowed to undergo oxidation in the open air to develop its characteristic colour, aroma and flavour.

Numerous reports have published analyses of Rooibos constituents, with the most recent analyses conducted by Walters et al.6 illustrating the effects of the degree of processing on major polyphenols. Table 2 depicts the LC-MS analyses of aqueous extracts of green, semi-fermented and fermented plant material from 10 sub-divided Rooibos bushes. Changes to the phenolic composition during the fermentation process showed the greatest decrease to be in aspalathin, unique to Rooibos, which is converted to eriodictyol-glucopyranoside isomers, with the fermentation process resulting in higher levels of these flavanones. These isomers may be further oxidized to the flavone derivatives, orientin and iso-orientin. The level of nothofagin, which is oxidized to vitexin and isovitexin, is also significantly reduced during processing. It is interesting to note that PPAG is one of the more stable constituents present in Rooibos and remains unaffected by the fermentation process.6

Table 2 Phenolic composition (g per 100 g extract) and extract yield (g per 100 g plant material) of green, semi-fermented and fermented rooibos aqueous extracts. Data are presented as mean ± SD (n = 10) and different subscript capital letters indicate significant (p < 0.05) differences between treatments
Compound Unfermented (green) Semi-fermented Fermented
<LOQ compounds below limit of accurate quantification.a Quantified as eriodictyol-7-O-glucopyranoside equivalents.b Quantified as rutin equivalents. (Adapted from Walters et al.4 reproduced with permission.).
(S)-Eriodictyol-6-C-glucopyranosidea <LOQ 0.152 ± 0.015B 0.400 ± 0.041A
(R)-Eriodictyol-6-C-glucopyranosidea <LOQ 0.148 ± 0.014B 0.398 ± 0.043A
(S)-Eriodictyol-8-C-glucopyranosidea <LOQ 0.092 ± 0.017B 0.139 ± 0.024A
(R)-Eriodictyol-8-C-glucopyranosidea <LOQ 0.080 ± 0.006B 0.131 ± 0.018A
PPAG 0.835 ± 0.622A 0.834 ± 0.644A 0.927 ± 0.724A
Aspalathin 8.410 ± 0.998A 4.280 ± 0.877B 1.370 ± 0.419C
Nothofagin 0.572 ± 0.257A 0.260 ± 0.117B 0.105 ± 0.054C
Iso-orientin 0.890 ± 0.210A 0.691 ± 0.141B 0.698 ± 0.131B
Orientin 0.820 ± 0.181A 0.643 ± 0.113B 0.815 ± 0.130A
Luteolin-7-O-glucopyranoside 0.061 ± 0.027B 0.071 ± 0.035A 0.057 ± 0.027B
Isovitexin 0.157 ± 0.047A 0.111 ± 0.032B 0.119 ± 0.03B
Vitexin 0.130 ± 0.039A 0.105 ± 0.026B 0.121 ± 0.024AB
Quercetin-3-O-robinobiosideb 0.674 ± 0.287A 0.563 ± 0.242B 0.562 ± 0.274B
Hyperoside 0.128 ± 0.062A 0.095 ± 0.048B 0.078 ± 0.051B
Rutin 0.246 ± 0.100A 0.212 ± 0.092B 0.161 ± 0.085C
Isoquercitrin 0.092 ± 0.047A 0.064 ± 0.033B 0.070 ± 0.037B
Extract yield 11.80 ± 1.46A 10.60 ± 1.00B 9.570 ± 0.905C


Rooibos: an evidence-based solution to cardiac health

The characterization of Aspalathus linearis directly led to a large volume of work focused on the potential antioxidant capacity of Rooibos due to its high polyphenol content.7 In one of the first studies of this nature, Rooibos extract (both green and fermented) supplementation (4 g dL−1 administered ad lib via drinking water) was associated with an increased glutathione to oxidized glutathione (GSH[thin space (1/6-em)]:[thin space (1/6-em)]GSSG) ratio in livers of supplemented (normally healthy) rats,8 indicative of lower oxidative stress levels. This is particularly significant when considering that in comparison, black (Camilla sinensis) tea decreased the liver oxygen radical absorbance capacity (ORAC) in the same study, suggesting a relative depletion of endogenous antioxidant systems. Several subsequent studies confirmed the antioxidant effect of Rooibos in ethnopharmacological studies,9 as well as in in vitro studies,10–12 animal studies13–18 and in a human model.19 Most notable was the latter study by Marnewick and colleagues, which was the first to provide clinical proof that daily Rooibos consumption has a beneficial role in the context of CVD prevention. Rooibos was shown to reduce oxidative stress in adults at risk for developing CVD – their redox status improved, as reflected by significantly increased glutathione (GSH) and GSH[thin space (1/6-em)]:[thin space (1/6-em)]GSSG ratios (1.35- and 1.85-fold respectively) after a 6-week period of Rooibos consumption. GSH is an intracellular thiol antioxidant and decreased levels have been shown to result in increased ROS production, implicated in inflammation and perturbing the immune response.20 Rooibos consumption also significantly improved the blood lipid status biomarker profile, linking the health promoting properties of Rooibos and CVD: serum LDL-cholesterol and triacylglycerol levels were decreased significantly while HDL-cholesterol was significantly increased.19

Although the interconnected nature of oxidative stress-related damage and inflammation has been the topic of a number of reviews in the context of cardiovascular and metabolic disease,21–26 not many Rooibos-related studies have focused on the anti-inflammatory effects of the plant. The limited available data in this context seems to be in agreement though, with Rooibos treatment associated with both a limitation of pro-inflammatory cytokine signalling16,27 and enhanced secretion of the anti-inflammatory cytokine, IL-10.27,28 For example, in a rodent model of LPS-induced liver damage, 4 weeks of pre-injury Rooibos supplementation resulted in lower TNF-α and IL-6 levels in hepatic tissue.16 In terms of the active constituents of Rooibos responsible for these effects, in a murine model of sepsis induced by administration of the ubiquitous nuclear protein, high mobility group box 1 (HMGB1), the Rooibos constituents nothofagin and aspalathin specifically, were shown to reduce levels of TNF-α, IL-6 and NF-kB.29 Similarly, in a rat model of colorectal cancer, supplementation with orientin – another constituent of Rooibos – was again linked to lower TNF-α, IL-6 and NF-kB, as well as lower levels of inflammatory enzymes iNOS and COX-2.30 In terms of anti-inflammatory outcome, Rooibos exposure was reported to significantly increase IL-10 production by ovalbumin-challenged murine splenocytes in culture,31 while daily supplementation increased adrenal IL-10 levels in a rat model of stress. More specifically in the context of CVD, Rooibos supplementation in rats were reported to increase IL-10 levels both in circulation and in myocardial tissue.32

As mentioned, the association of inflammation and oxidative stress is well documented and the fact that the two are inextricably linked to chronic diseases is generally accepted. From this literature, there is also overwhelming evidence for obesity being an additional confounding aetiological factor in this context, as it serves to exacerbate both oxidative stress and inflammation, ultimately resulting in cardiometabolic diseases such as insulin resistance and diabetes.21–23,25,33–35

In a recent longitudinal study spanning 15 years, the onset age of diabetes was reported to inversely correlate with CVD-related mortality risk. Data indicated that there is an increased risk of premature death resulting from CVD with the onset of type 2 diabetes at an earlier age. Authors concluded therefore that delaying the onset of the disease may reduce CVD related deaths. These findings, which were suggested to be applicable to Western countries,36 together with childhood obesity being associated with increased levels of atherosclerotic lipoprotein-low density lipoprotein cholesterol, or LDL-C,37 highlight the urgent requirement for preventative measures to be implemented early in life as childhood obesity is on the increase globally.

Inactivity, or a sedentary lifestyle, which often goes hand in hand not only with obesity but also with unhealthy diet, is another well-established contributor to the development of CVD. Interestingly, according to the United States 2015 Behavioral Risk Factor Surveillance System study, a highly active lifestyle in obese women seemed to effectively counter the maladaptive effects associated with obesity, decreasing their risk for CVD.38 This report is in line with the known anti-inflammatory effects of habitual exercise39,40 and expands by suggesting that exercise may be an avenue for disease prevention even in obese individuals. The practicality of this approach is however questionable: the cross-sectional European Action on Secondary Prevention through Intervention to Reduce Events (EUROASPIRE) surveys reported adequate levels of physical activity in only 17% of Belgian coronary patient participants. In addition, 47% of the cohort exhibited central obesity and 21% suffered from diabetes.41 Data reported by Pharr et al.38 indicate that appropriate interventions that counter the maladaptive sequelae of obesity implicated in the aetiology of CVD, may limit the progression of CVD.

The detrimental effects of a sedentary lifestyle on cardiovascular health, especially when combined with unhealthy dietary habits is well documented. Of particular interest and relevance to this review, dietary intake of advanced glycation end products (AGEs, formed via the Maillard reaction in high-heat food processing and employed to enhance taste and flavour of food42) are associated with the promotion of atherosclerosis43 and inflammation,43,44 even in apparently healthy subjects.45 More specifically, dietary AGEs are reported to deplete endogenous antioxidant defense systems such as vitamin C and E,42 AGE-receptor 1 and the pro-survival factor sirtuin-1.46 Reduced levels of sirtuin-1 have also been linked to accelerated ageing – the pro-inflammatory, high oxidative stress syndrome characteristic of modern chronic diseases such as diabetes and CVD.24,47 Furthermore, in a crossover study in 20 patients with type II diabetes, a high AGE meal (when compared to a low AGE meal) was associated with a relatively greater impairment in macro- and microvascular function, with significant evidence of endothelial dysfunction and oxidative stress.48

Vascular complications further account for disabilities associated with diabetes. High mortality rates in diabetic patients are attributed to CVD, with AGEs playing an important role in the pathogenesis of CVD due to the deleterious effects on endothelial cell function.49 AGEs decrease elasticity of vasculatures which is associated with AGEs increasing oxidative stress together with nitric oxide being inactivated to form peroxynitrite.50,51 Nitric oxide production and its bioavailability and activity in vascular endothelium is particularly important in the regulation of blood flow with abnormal production of nitric oxide resulting in vasoconstriction, thrombosis, inflammation and vascular hypertrophy in various disease states. Since AGEs impede endothelial nitric oxide expression and activity52,53 and inhibit nitric oxide platelet activation and aggregation,54 these macroproteins may contribute to abnormalities linked to endothelial dysfunction in diabetes. Rooibos has been shown to increase nitric oxide production in cultured endothelial cells while having a marginal inhibitory effect on angiotensin-converting enzyme (ACE)10 which catalyses the conversion of angiotensin I to angiotensin II, which in turn stimulates the adrenal to produce aldosterone which regulates blood pressure. To note, in vivo studies reported ramipril, an ACE inhibitor, to reduce blood pressure as well as AGEs while angiotensin receptor blockers in hypertensive diabetics decreased AGE levels significantly.55 In a prior in vitro study by Persson et al. in 2012, Rooibos was shown to exhibit mixed inhibition of ACE similar to that of enalaprilat, an ACE inhibitor.56 The group had previously reported an oral intake of 400 ml Rooibos tea to significantly inhibit ACE activity in healthy volunteers and they suggested that Rooibos may exert cardiovascular effects due to the inhibitory effects on the enzyme's activity.10

Although the antioxidant/radical scavenging activity of Rooibos has been thoroughly investigated,57 assays investigating structure–antioxidant relationship demonstrated the importance of a free hydroxyl group at position 3 on the C-ring. In trolox equivalent antioxidant capacity (TEAC) assays isoquercitrin, rutin and hyperoside for example exhibited 50% lower antioxidant activity compared to quercetin, aspalathin and nothofagin. While the former has a hydroxyl group at position 3, the dihydrochalcones do not. A decoupled low density lipid (LDL) oxidation experiment was subsequently employed to discriminate between radical scavenging and metal chelating properties of the polyphenols which was suggested to reflect in vivo conditions. Isoquercitrin was shown to preferentially complex metal ions while aspalathin's antioxidant activity was attributed to radical scavenging. These data therefore demonstrated that the dihydrochalcones would be more effective than the flavonols in terms of protection against reactive oxygen species (ROS).58 Aspalathin was subsequently shown to not only significantly reduce the basal ROS levels in RIN-5F pancreatic β-cells but also AGE-induced ROS production. In the same study using the ob/ob type 2 diabetic mouse model, aspalathin improved the impaired glucose tolerance and decreased fasting blood glucose levels significantly while not influencing serum insulin levels. These effects were shown to be mediated through AMPK activation resulting in translocation of the insulin-sensitive glucose transporter 4 (GLUT4) to plasma membrane.59 These data corroborated an earlier study in which aspalathin also significantly decreased blood glucose levels in db/db mice.60 In addition in ob/ob mice, aspalathin decreased both circulating and liver triglyceride levels as well as TNF-α levels.59 Interestingly, PPAG, one of the more stable major compounds in Rooibos was reported to increase glucose uptake in vitro, as well as facilitating insulin secretion.60,61 TNF-α is known to decrease the expression of GLUT4 in isolated adipose cells, which are highly responsive to insulin,62 after which it was subsequently reported that TNF-α production was significantly increased in both obese humans63 and in obese insulin-resistant rodents.64 In the latter study, peripheral glucose uptake increased in response to insulin upon exposure to TNF-α-IgG together with GLUT4 expression being restored in adipocytes. While GLUT4 was downregulated in tissue and adipocytes by TNF-α, levels increased in adipocytes after TNF-α-IgG treatment.64

The anti-diabetic potential of Rooibos has to date been recorded in various in vitro models and in obese and diabetic rodent models for aspalathin and for both green and fermented Rooibos. In obese and diabetic rodent models, green and fermented Rooibos,12,13 as well as aspalathin,32 effectively lowered blood glucose levels and associated oxidative stress. Acute aspalathin treatment resulted in improved glucose uptake into cardiomyocytes in young rats, both directly (Akt activation and GLUT4 translocation) and indirectly (increasing insulin sensitivity), while in those from older rats, the effect was limited to increased insulin sensitivity only. In this study, aspalathin also had no effect on glucose uptake into cardiomyocytes from high fat, diet-associated obese insulin-resistant rats.32 In a prior study undertaken by Kamakura et al.12 green Rooibos extracts had increased glucose uptake independent of insulin in obese diabetic (KK-A(y)) mice. The extract also induced phosphorylation of 5′-adenosine monophosphate-activated protein kinase (AMPK) in L6 myotubes, while also promoting phosphorylation of Akt, resulting in GLUT4 translocation. In addition, the extract also suppressed AGEs-induced ROS levels in RIN-5F cells.12 Together, this suggested that aspalathin requires activation of the PI3K/Akt signaling pathway in order to exert its anti-diabetic function.

A number of reviews have associated AGEs with inflammation and oxidative stress in the pathophysiology of cardiovascular disease underlying their role in mediating CV risk in patients with diabetes mellitus. There is a growing body of evidence linking AGEs with endothelial cell dysfunction, hypercoagulability, impaired fibrinolysis and altered platelet activation, indicating that AGEs may mediate CV risk in patients with DM. Higher AGE serum levels in diabetic patients (compared to normal non-diabetics) may be a contributing factor in macro- and microvascular complications characteristic of diabetes as well as the development and progression of the pathogenesis of CVD in general. It has been suggested that AGEs may be a feasible therapeutic target for clinical strategies in the treatment of diabetes and cardiovascular complications.49,55,65–67 It is in this context that Rooibos and in particular aspalathin, which reduced AGE-induced ROS production as discussed above, may have therapeutic applications in diabetic patients at risk for CVD. Cardiac complications in diabetes patients are associated with oxidative stress due to a severe imbalance between the production and clearance of ROS as well as reactive nitrogen species by antioxidant defense systems. Phytochemicals have been reported to reduce oxidative stress in diabetic cardiomyopathy via nuclear factor (erythroid-derived 2)-like 2 (Nrf2), important in the maintenance of oxidative homeostasis. Nrf2 plays a regulatory role in cellular resistance to oxidants, as it modulates basal and induced expression of antioxidant response element-dependent genes encoding protective antioxidant proteins, thus regulating the physiological and pathophysiological outcomes of oxidant exposure. H9c2 cardiomyocytes have been shown to be protected against high glucose-induced oxidative stress by aspalathin, while diabetic mice supplemented with 130 mg kg−1 aspalathin daily for 6 weeks, had enhanced expression of Nrf2 and its associated downstream genes.18 Furthermore, the antioxidant effect had specific significance in the context of cardiovascular health in particular, as the aspalathin-supplemented animals did not suffer the cardiac remodeling and enlargement of left ventricular walls normally observed in diabetes. Significantly, this major component of green Rooibos was even more effective than the anti-diabetic drug metformin in this model. An earlier study by the same group had reported an aqueous Rooibos extract to protect cardiomyocytes isolated from diabetic-induced rats and exposed to hydrogen peroxide against oxidative stress.68 These data link the antioxidant and anti-inflammatory effects of Rooibos directly to cardiac benefits.

Rooibos addressing stress: implications for the heart

Given the central role of oxidative stress and inflammation in the aetiology of CVD, ailments associated with chronic stress such as depression, anxiety and insomnia, which are generally linked to the endocrine system, also have a contributory role in cardiovascular health. Although the impact of depression is less well recognised it is currently considered a significant independent risk factor for heart disease. Depression is also linked with CVD risk factors such as diabetes, obesity and unhealthy lifestyles. Furthermore, both depression and anxiety may increase the risk of developing CVD and vice versa – CVD may increase the risk of developing depression and anxiety. In addition, depression, together with stress and anxiety, increases the risk of death and exacerbate complications.69,70 It is here too that Rooibos has a role to play, albeit an indirect cardioprotective role – the anti-anxiety, soothing properties of Rooibos being one of the oldest anecdotal properties attributed to Rooibos consumption.

The development of cardiovascular disease and dyslipidemia are closely associated with chronic exposure to glucocorticoid excess, which also impacts insulin action, metabolic syndrome and obesity. Excessive glucocorticoid production resulting from chronic stress with the consequential activation of the hypothalamic-pituitary-adrenal (HPA) axis leads to increased adrenal cortisol production and a disrupted cortisol circadian rhythm.71

An aetiological role for anxiety and depression in the high-stress modern lifestyle in CVD have been suggested by De Smedt and colleagues41 in addition to the aforementioned risk factors. In their study, in which they conducted a cross-sectional EUROASPIRE survey including ±600 Belgian coronary patients, 25–30% suffered from anxiety and depression. In line with these findings, a recent study with a cohort of more than 9000 middle-aged individuals showed that persons displaying adverse psychological factors (such as perceived stress, anxiety, depression, low satisfaction) were less likely to have ideal cardiovascular health, even when correcting for age, ethnicity and education.72 In addition, pre-existing depression was associated with an even poorer prognosis if present at the time of diagnosis of coronary heart disease, cancer and stroke.73

A similar profile is evident in even much younger individuals. In a South African study, young females had significantly higher perceived stress and anxiety levels than older counterparts.74 This paints a particularly bleak picture for the future, as a recent review of epidemiological studies investigating the validity of the Developmental Origins of Health and Disease (DOHaD) theory,75 has identified early life stress as independent risk factor of CVD later in life. Although according to the authors, the molecular mechanisms of early-life stress requires further clarification before personalized therapeutic approaches may be formulated, recent research has already given some direction to guide preventative measures which may be broadly applicable. For example, persisting elevated activation of the HPA axis in chronic stress has well-established maladaptive effects via both glucocorticoid and mineralocorticoid receptors,76 as also reported in depression,77 which results in glucocorticoid insufficiency, increased inflammation and oxidative stress, as was recently reported in patients with coronary heart disease.78 A recent study using maternal separation as rodent model for early-life stress, also demonstrated that the stress intervention resulted in significant oxidative stress (assessed by ROS, mitochondrial glutathione, ATP and cytochrome c release) in cardiac tissue specifically.79 Interestingly, voluntary running exercise mitigated the undesirable outcome in this model. Intense exercise was recently reported to improve endogenous anti-oxidant capacity in patients with heart failure.80 Together, these studies suggest that antioxidant therapy can limit the progression of CVD. Indeed, candidates involved in oxidative stress and inflammation – such as superoxide anions, nitric oxide and peroxynitrites – were recently suggested as biomarkers for heart failure, which is the leading cause of morbidity and premature mortality in CVD patients.81

Considering the potential of Rooibos in the context of acute cardioprotection, as in after an acute myocardial infarction, both green and fermented Rooibos significantly improved aortic output recovery after experimental ischaemia/reperfusion in isolated, perfused rat hearts from animals supplemented for 7 weeks. Extracts also limited pro-apoptotic signaling (decreased cleaved caspase-3 and poly ADP ribose polymerase (PARP)), which were ascribed to the high polyphenol content of Rooibos.15 These effects may potentially be ascribed to Rooibos's in vivo inhibition of angiotensin-converting enzyme (ACE),82 which may result in relative vasodilation. We have shown that while Rooibos does not inhibit aldosterone biosynthesis, it does inhibit the biosynthesis of its precursor steroids, deoxycorticosterone, corticosterone and 18-hydroxycorticosterone significantly in forskolin-stimulated H295R cells, a human adrenocortical carcinoma adrenal cell model. Of the flavonoid compounds tested, orientin and vitexin decreased aldosterone levels significantly while aspalathin had no effect on aldosterone or its precursor steroids.83,84 In terms of CVD-associated hypertension specifically, inhibition of aldosterone synthase (CYP11B2), which catalyzes aldosterone biosynthesis, has been reported to mitigate elevated plasma aldosterone levels associated with congestive heart failure and myocardial fibrosis.85–87 In addition, in a genome-wide association study, gene variants near cytochrome P450 17α-hydroxylase/17,20-lyase (CYP17A1) and CYP17A2 genes were associated with hypertension.88 CYP17A1 gene polymorphisms were also associated with increased plasma levels of the inflammation marker C-reactive protein (CRP) – which incidentally is also a recognized cardiovascular risk indicator89 and has been shown to be decreased in humans after consumption of a single fermented Rooibos infusion.90 CYP17A1, together with 3β-hydroxysteroid dehydrogenase (3βHSD2), are at a branch point in adrenal steroidogenesis, with their catalytic activity channeling pregnenolone into mineralocorticoid, glucocorticoid and androgen hormone biosynthesis pathways. Our studies conducted in H295R cells clearly show that Rooibos modulates the steroid shunt in these pathways under basal conditions and significantly so when cells are stimulated with forskolin to mimic adrenocorticotropic hormone (ACTH) action, supporting in vivo studies suggesting that Rooibos may alleviate stress and anxiety via its modulation of glucocorticoid biosynthesis.91 In the context of stress as aetiological factor in CVD, Rooibos may therefore have potential applications.

Rooibos is arguably the medicinal plant that has been most comprehensively assessed in terms of its effects on cortisol biosynthesis in the context of steroidogenesis and modulation of adrenal cytochrome P450 (CYP) enzymes. The adrenal CYP enzymes, together with 3βHSD, catalyse the biosynthesis of the mineralocorticoid, glucocorticoids and androgen steroid hormones. These CYP enzymes are substrate specific and constituitively expressed in the adrenal and steroidogenic tissue whereas the inducible CYP enzymes expressed in the liver, kidney and intestines metabolise a vast array of drugs, xenobiotics and phytochemicals. In the glucocorticoid pathway, pregnenolone is converted to corticosterone and cortisol by CYP17 and 3βHSD together with cytochrome P450 21-hydroxylase (CYP21A2), and cytochrome P450 11β-hydroxylase (CYP11B1). Long-term over-production of cortisol is directly implicated in the progression of diseases such as obesity, diabetes, heart failure and hypertension, amongst others.92

Both CYP17A1 and CYP21A2, transiently expressed in the fibroblast-like monkey kidney COS-1 cells, are inhibited by unfermented Rooibos whole extracts as well as aspalathin and nothofagin.84 The same study also reported steroid production in H295R cells to be modulated in the presence of Rooibos, supporting the inhibitory effects on these key enzymes in adrenal steroidogenesis. Analysis of metabolites showed that, under stimulated conditions, Rooibos extracts decreased the production of cortisol and corticosterone significantly with cortisol levels returning to basal levels – again a positive outcome, given the direct link between cortisol overproduction and CVD aetiology mentioned earlier. A comprehensive study incorporating isolates of the major constituents in Rooibos –aspalathin, nothofagin, orientin, vitexin and rutin – reported that all flavonoids inhibited both branch point enzymes, 3βHSD2 and CYP17A1, significantly reflective of Rooibos's ability to modulate steroid hormone levels in the three pathways. However, only aspalathin and nothofagin inhibited CYP21A2 in COS-1 cells.83 Quercetin, a rutin derivative, is a known inhibitor of 3βHSD93 which could also play a role in the decreased production of adrenal steroids after Rooibos consumption.

The capacity of Rooibos to influence hormone levels as demonstrated in H295R cells, did not translate to significant differences in cortisol levels in humans at risk for CVD supplemented for 6 weeks,91 possibly due to the known large inter-individual and intra-individual variation in these parameters in humans, even in the absence of pathology. The supplementation did however achieve significantly increased cortisone levels in males and significantly decreased cortisol[thin space (1/6-em)]:[thin space (1/6-em)]cortisone ratios in both males and females, indicating that rooibos may indeed affect changes at glucocorticoid level. Further work exploring different doses and larger cohorts is required to fully elucidate this potential effect of Rooibos in human models. Nevertheless, data from an inherently less variable rodent model indeed supported an effect for Rooibos as glucocorticoid modulator, with 10 days of Rooibos supplementation resulting in significantly decreased circulating corticosterone as well as corticosterone[thin space (1/6-em)]:[thin space (1/6-em)]11-dehydrocorticosterone ratios.91 To note –rats do not produce cortisol, with corticosterone being their major glucocorticoid. In vitro experiments elucidated that Rooibos may achieve this beneficial effect on the glucocorticoid system by selectively inhibiting 11βHSD type 1 (11βHSD1) thus preventing the conversion of the inactive metabolites back to cortisol and corticosterone respectively, to which the decreased in vivo active[thin space (1/6-em)]:[thin space (1/6-em)]inactive glucocorticoid ratios can be attributed. 11βHSD isoforms were expressed in CHO-K1 Chinese hamster ovary cells and exposed to Rooibos, resulting in the inhibition of cortisone's conversion to cortisol by 11βHSD1 but not of cortisol's conversion to cortisone by 11βHSD type 2 (11βHSD2).91 These data strongly suggest that Rooibos is able to impact glucocorticoid biosynthesis thereby reducing glucocorticoid production while at the same time favouring the inactivation of glucocorticoids to their inactive keto-metabolites due to its selective inhibitory effect on 11βHSD1.

11β-HSD1 has long been put forward as a target in the treatment of obesity, type 2 diabetes and the metabolic syndrome in order to decrease tissue-specific cortisol production. The metabolic syndrome, identified as a major risk factor for type 2 diabetes and CVD, can best be described as a clinical condition comprising hyperglycemia, insulin resistance, dyslipidemia, obesity and hypertension. Even though 11β-HSD1 is highly expressed in adipose tissue, obesity and the metabolic syndrome are not characterized by cortisol excess.94 Nevertheless, targeting 11βHSD1 activity may impact the metabolic syndrome and obesity favourably as well as type 2 diabetes, by reducing glucocorticoid-mediated hepatic glucose output, improving insulin sensitivity and increasing insulin secretion.

Obesity as such is also considered a chronic metabolic disorder and, while also being an independent risk factor for CVD, is associated with co-morbidities that include hypertension, dyslipidemia, glucose intolerance and type 2 diabetes. Adipose tissue is regarded as an endocrine organ which produces, amongst others, TNF-α, IL-6 and angiotensinogen (precursor of angiotensin). Interestingly adipose tissue produces about a third of circulating IL-6, which modulates CRP production in the liver. It has been suggested that CRP may be a marker of a chronic inflammatory state capable of triggering acute coronary syndrome.95 In addition, numerous steroidogenic enzymes, besides 11βHSD1, are expressed (mRNA) in adipose tissue: steroidogenic acute regulatory protein (StAR), cytochrome P450 side-chain cleavage enzyme (CYP11A1), 3βHSD2 and CYP21A2 amongst others. The presence of StAR suggests de novo steroid biosynthesis although this would be limited to the production of deoxycorticosterone (DOC) from cholesterol catalyzed by CYP11A1, 3βHSD2 and CYP21. DOC is quite possibly the most understudied of the steroid hormones and is considered to be both a glucocorticoid and a potent mineralocorticoid, with hypertension being a well-known effect of DOC. Of interest, DOC has been used to induce hypertension in experimental animals.96 DOC is also a precursor steroid for 3a,5a-tetrahydro-DOC (THDOC), a stress responsive neuroactive steroid. While glucocorticoid levels in the brain are dependent on uptake, the de novo biosynthesis of DOC is possible as the aforementioned steroidogenic enzymes expressed in adipose tissue are also expressed in the human brain.97 THDOC has GABAA receptor-positive modulatory effects which include anxiolytic and sedative actions.98 While neurosteroid increases in response to acute stressors are adaptive, responses are blunted in chronic stress and depression. Failure to mount a satisfactory response due to frequent adaptations to repeated stressors perturbs homeostasis, further contributes to dysregulation of the HPA axis and associated glucocorticoid imbalance.98 It is in this scenario that 11βHSD1 plays an important regulatory role together with 11βHSD2. Acute stressors increase 11βHSD1 expression in the brain while chronic stress decreases expression, a mechanism suggested to compensate the negative feedback of glucocorticoids on the HPA axis in the maintenance of normal circulating glucocorticoid levels.99 The stress response is mediated by both the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR). The MR has a significantly greater affinity for glucocorticoids, corticosterone and cortisol, and would thus be predominantly ligand bound at low glucocorticoid levels, while the GR would remain unoccupied since the affinity of the GR for these steroids is ∼10-fold lower. The GR would become substantially activated upon increased glucocorticoid levels such as those characteristic of a stress response. Since 11β-HSD1 is widely expressed throughout the adult CNS, the conversion of the 11-keto derivatives, which increase concomitantly with cortisol and corticosterone, will further contribute to active glucocorticoid levels and glucocorticoid action in brain cells. It has also been shown that 11β-HSD1 expression increases with ageing and contributes to cognitive decline, corroborated by carbenoxolone administration, a known inhibitor of 11β-HSD1, improving cognitive function in elderly men.100

The GR is widely expressed in neurons and glia and the MR in the hippocampus, septum and scattered nuclei in the brain stem. Although the expression and localization of the MR and GR are different, they are co-localized in neurons of the limbic brain – allowing interdependent downstream glucocorticoid effects exerted at the genomic level by receptors forming MR/MR and GR/GR homodimers as well as MR/GR heterodimers, with the latter enhanced by acute stress. Chronic stress results in MR[thin space (1/6-em)]:[thin space (1/6-em)]GR imbalance with different levels of glucocorticoids binding to the MR and the GR together with dysregulation of the hypothalamic-pituitary-adrenal axis. While persistent activation of the GR results in depression, chronic stress will also increase GR expression.99,101–103 We have reported that Rooibos attenuates cortisol production in humans at risk for CVD while also significantly decreasing corticosterone and DOC levels in rats as well as decreasing the active[thin space (1/6-em)]:[thin space (1/6-em)]inactive glucocorticoid ratios in both humans and rats.91 While the effects of quercetin on the estrogen receptor had been studied in terms of exhibiting phytoestrogenic activity and having anti-cancer properties,104 the interaction of Rooibos and its polyphenolic compounds on steroid receptors has, to our knowledge, not been investigated to date. However, the well characterized anti-oxidant/radical scavenging activity of Rooibos’ constituents may indeed have additional indirect effects on steroid receptors. In vitro studies have shown the genomic function of corticosteroid receptors can be modulated by ROS, with increased ROS having been shown to activate MR independently of ligand. Peroxynitrites induced ligand independent MR transactivation while not affecting genomic GR activity, indicating that oxidative stress may induce a change in physiological MR effects to pathophysiological effects. The inhibition of MR, ROS scavengers, TNF antagonists/inhibition have all been shown to prevent inflammation and excessive ROS to effect over-activation of the sympathetic nervous system and hypertension.103,105 It is therefore possible that bioactivities of Rooibos as discussed in previous sections may be mediated via the MR and/or the GR and this potential target should certainly be explored.

Together, these studies across in vitro and in vivo models, provide strong evidence of a stress-reducing effect of Rooibos via activity on the steroidogenic P450 enzymes involved in especially glucocorticoid biosynthesis. The in vitro models point to Rooibos reducing glucocorticoid production while also reducing aldosterone precursors and favouring the inactivation of glucocorticoids which were corroborated in a rat study and in a human study conducted in subjects at risk for CVD. Given the elucidated mechanisms by which these effects are achieved, it clearly links to CVD aetiology and thus supports a role for Rooibos as a preventative strategy in the management of stress-related conditions, in the context of modulating cortisol levels specifically in terms of stress and anxiety.

It should however be kept in mind that Rooibos effects would be dependent on the absorption and bioavailability of the compounds. Inter-individual variation has been reported, where the flavonoid levels detected in plasma and urine samples upon consumption of Rooibos differed significantly between individuals, despite consuming equal amounts.106 Although data on the metabolism of Rooibos polyphenols are limited, studies have reported on the potential of polyphenolic compounds to affect drug metabolism via the inducible drug metabolizing CYP enzymes. A number of studies have been conducted into the interference of polyphenols with CYP3A4, which is the main CYP isoform involved in drug metabolism. Quercetin and rutin both decreased the bioavailability of the immunosuppressant cyclosporine by activating CYP3A4.107 Luteolin was shown to interfere with CYP3A4 and CYP3A5 when co-administered with drugs metabolized by these enzymes.108 Herb–drug interactions were also investigated in the presence of Rooibos, aspalathin and PPAG, and while PPAG had no effect on CYP2C8, CYP2C9 or CYP3A4 activity, aspalathin exhibited only 20% inhibition of CYP3A4 at 110 and 220 μM.109 These studies were however conducted at very high concentrations of polyphenolic compounds which ranged from 30 to 400 μM. Given the molecular structures of the polyphenols and their interactions with the steroidogenic CYPs they undoubtedly would also bind a number of the inducible CYP enzymes, and may either stimulate or inhibit these enzymes. It is indeed possible that they may be further metabolized by specific liver or kidney CYP enzymes prior to being conjugated by phase II enzymes since the glucuronidated, sulfated and methylated derivatives have been identified in circulation and in urine.110 In the study by Patel et al. 2016,109 fermented and unfermented Rooibos extracts inhibited CYP3A4 significantly at concentrations ranging from 25 to 100 μg ml−1. While aspalathin inhibited the catalytic activity of CYP2C8, albeit 15% in the presence of 220 μM, neither aspalathin nor Rooibos extracts inhibited CYP2C9. Unfortunately polyphenols were not quantitated in the extracts, hampering interpretation of CYP inhibition specific to bioactive compounds.

Although this review has focused largely on the beneficial effects of Rooibos, it is possible that pharmacokinetic interactions may occur when consumed with CVD-treatment drugs. Rooibos may have detrimental effects on CYP3A4 and CYP2C9 as these enzymes are involved in the metabolism of drugs commonly used for treating dyslipidemia and hypertension, as well as diabetes mellitus (targeting pancreatic β-cells stimulating insulin release or those increasing insulin sensitivity). If inhibited, it is possible that Rooibos may compensate due to its specific favourable bioactivities. However, drug effects may be exacerbated since some flavonoids may behave as stimulators. Further investigations into pharmacokinetic interactions would be beneficial to further test the scientific support for advocating Rooibos consumption as a therapeutic aid in the treatment of CVD.

CYP3A4, which metabolises about 50% of all xenobiotics, has also been shown to be expressed in endothelium, endocardium and coronary vessels,111 while CYP2C8 and CYP2C9 are expressed in normal heart tissue112 as well as in failing heart tissue.113 The CYP2 subfamily is ubiquitously expressed in the cardiovascular system with CYP2C8, CYP2C9 as well as CYP2J2 – also constitutively expressed in cardiac tissue, being the main isozymes responsible for epoxygenase activity. CYP2J2 is present in healthy tissue at significantly higher levels than CYP2C8 and CYP2C9,112 with the latter two present mainly in arteries and induced after ischemic injury.114,115 CYP epoxygenase and hydroxylase activities catalyse the production of epoxyeicosatrienoic acids (EETs) and hydroxyeicosatetraenoic acids (HETEs) in the arachidonic acid cascade – modulation of these two activities would either have detrimental effects (upregulation of CYP hydroxylase activity) or favourable effects (upregulation of CYP epoxygenase activity). Limited studies have been reported to date regarding the influence of Rooibos on these enzymes. As discussed above, data suggests that the effect of Rooibos would be insignificant as CYP2C9 was not influenced, while inhibition of CYP2C8 by compounds was negligible at very high concentrations. Further studies are warranted to investigate effects of Rooibos extracts on CYP expoxygenases and hydroxylases together with analyses of Rooibos flavonoid content.

CYP enzymes, and in particular CYP2C, CYP2J and CYP1A2/1B have an important role in cardiovascular health, specifically in terms of the biosynthesis of epoxyeicosatrienoic acids (EETs) and hydroxyeicosatetraenoic acids (HETEs), which are produced in the metabolism of arachidonic acid. Arachidonic acid is formed by the action of phospholipases which catalyses the release of cell membrane phospholipids. The metabolism of arachidonic acid subsequently diverges into three pathways, namely the CYP pathway, the lipoxygenase (LOX) pathway and the cyclooxygenase (COX) pathway The arachidonic acid cascade is central to both inflammation and to more specific symptoms of CVD, such as hypertension and dyslipidemia. The imbalance of excessive quantities of omega-6 fatty acids relative to deficient omega-3 fatty acids, characteristic of the Western diet,116 results in an accumulation of arachidonic acid in cardiomyocyte cell membranes. The flux through this pathway is subsequently upregulated and has been linked to inflammatory disease and cardiovascular risk.117 Similar results have been reported for arachidonic acid – in association with increased oxidative stress – in early stage diabetes in obese Zucker rats fed a high fat diet,118 elucidating yet another molecular link between unhealthy diet, obesity and diabetes in the aetiology of CVD. The CYP and COX pathways are relevant in the context of the current review topic.

EET and HETE levels are determined by the expression and/or activity of the CYP epoxygenase and hydroxylase enzymes and as such regulate vascular tone, extracellular fluid volume and heart contractility. In the CYP hydroxylase pathway, CYP4A and CYP4F convert arachidonic acid to the cardiotoxic HETEs, while CYP2C and CYP2J have the capacity to catalyze the production of cardioprotective EETs.119 In particular, the formation of 20-HETE in vascular smooth muscle (stimulated by angiotensin II and endothelin) is associated with vasoconstriction and hypertension,119 as well as chronic inflammation,120 while EETs are associated with reversal of hypertension and an anti-inflammatory outcome.121 CYP2C8, CYP2C9 and CYP2C19 polymorphisms have been associated with increased CVD susceptibility122 while in contrast, endothelium-specific CYP2J2 over-expression was reported to ameliorate insulin resistance in a mouse model.123 These studies highlight the impact of shifts in the balance between HETEs and EETs and, of course, the importance of regulation by CYP enzymes in cardiovascular health.

Turning attention to the COX pathway – this pathway is mediated by two main enzymes which both catalyse the production of prostaglandins from arachidonic acid: COX-1 is a constitutive enzyme with homeostatic functions, in which prostaglandins activate platelets for blood clotting and which protects the gastric and intestinal lining. In contrast, COX-2 is induced under specific conditions, namely by the pro-inflammatory cytokines, TNF-α and interleukin-1 (IL-1), to enhance inflammatory symptoms such as fever and pain. To note – we have discussed the inhibition of TNF-α by aspalathin, nothofagin and orientin. Up-regulation of both enzymes is implicated in inflammation and their inhibition has proven therapeutic effects in the context of CVD, although not without long term side-effects.124 Of interest, among the side-effects reported for commonly prescribed non-steroidal drugs specifically inhibiting COX-2, is disruption of the CYP pathway to favor HETE production.120 Together these studies highlight the importance of the inhibition of inflammation in limiting disease progression, as well as the requirement for safer drug options than those currently available. Both quercetin and luteolin have also been shown to inhibit COX-2 transcriptional activity with an IC50 of 10.5 and 22 μM respectively.125 To date, limited data has reported on the action of Rooibos on COX enzymes in the arachidonic acid cascade. The only evidence found were reports of Rooibos extracts inhibiting COX-2 expression in skin cells,126 while orientin and luteolin inhibited COX-2 and iNOS with both also suppressing pro-inflammatory NF-kB expression, in an in vivo rat study30 and murine macrophages in vitro.127 Supporting the inhibitory action, luteolin was shown to decrease prostaglandin E2 and nitric oxide production, while orientin reduced TNF-α and IL-6 expression as well.

Considering the literature consulted, the favourable effects on inflammation and insulin sensitivity reported for Rooibos may also be achieved, at least in part, via modulation of the arachidonic acid cascade CYPs, in order to facilitate a more favourable HETE to EET balance. This topic remains to be assessed and approached more directly. Nevertheless, the fact that results from in vivo studies point to a mild modulatory role – which does not compromise the organism's ability to respond acutely to stressful stimuli27 – suggests Rooibos to be a potential alternative for long-term, low-risk, preventative supplementation.

Cocktails for health?

Taking into account medicinal plant products on the market, it is of interest that some products contain whole extracts, while others contain only one purified ingredient from the plant. The latter approach may be the result of following the traditional pharmaceutical approach, in order to develop a standardized, characterized product with minimal (or known) risk to the consumer. In the case of medicinal plants and indigenous knowledge however, anecdotal evidence of function exists only on complete plants or crude extracts containing a cocktail of multiple ingredients. Thus, purification of plant material may result in loss of active compounds, or altered metabolism after ingestion, rendering the natural product less effective in practice and perhaps risky in terms of side-effects.

Evidence from medicinal plant literature suggest that active compounds, and in particular polyphenols, in plants work in synergy128 and that different constituents may – through different molecular targets – have different primary effects.129 This is particularly evident for Rooibos, where supplementation with the complete plant consistently yields better results than purified active constituents in isolation. For example, while 1–100 μM of aspalathin and 100 μM rutin increased glucose uptake into skeletal muscle myotubules, only the complete green Rooibos extract was effective in liver cells.130 Similarly, in the same study, a 1[thin space (1/6-em)]:[thin space (1/6-em)]1 aspalathin[thin space (1/6-em)]:[thin space (1/6-em)]rutin mixture, as well as the complete extract, but not single compounds, were able to lower plasma glucose levels in streptozotocin (STZ)-induced diabetes in rats and to sustain it for at least 6 hours. Furthermore, in the context of its effects on adrenal steroidogenesis, whole Rooibos extracts were more potent than purified aspalathin or nothofagin.84 More specifically, different polyphenols in Rooibos were shown to have different CYP enzymes as primary targets. The inhibition (and degree of inhibition) of the catalytic activity of CYP enzymes by particular polyphenols were also dependent on the substrate assayed83 – for example, even if such a polyphenol can inhibit the enzyme, it can only do so for specific reactions catalyzed by that enzyme. Thus, in the absence of intolerance to a particular substance, consumption of the whole plant may have best therapeutic effect. This is particularly applicable to Rooibos, as the polyphenols contained in Rooibos have been shown to have additive, and not opposing, effects.

Although orientin, another compound present in abundance in Rooibos, has not often been included as pure compound in Rooibos-related research, recent papers have also reported other cardiovascular benefits for this polyphenol. For example, orientin was shown to prevent intracellular triglyceride build-up in mouse adipocytes (3T3-L1) by increasing expression of the Ppar-γ gene through repression of C/ebpδ and inhibition of the PI3K/Akt-FOXO1 signaling pathway.131 Similarly, the antioxidant signaling of this flavone was recently demonstrated to activate the eNOS/nitric oxide signaling cascades, mitigating the progression of cardiac remodeling after experimental myocardial infarction in mice.132 Similarly, while nothofagin is mostly known for its antioxidant and anti-inflammatory effects in the context of vascular inflammation,29,133 recent emerging evidence suggests it to also possess diuretic, natriuretic and potassium-sparing properties.134,135

Several non-major constituents of Rooibos also has proven anti-diabetic and/or cardioprotective effects. In this context, the multifunctional nature of rutin as antioxidant, anti-inflammatory and organ protective compound has been reported in a review on its potential as therapeutic in diabetes,136 while other reviews have highlighted cardioprotective effects of quercetin137 and luteolin.138 Vitexin has been known for some time to protect the heart against ischaemia-reperfusion injury in the context of myocardial infarction139,140 and was recently also shown to reduce endoplasmic reticulum (ER) stress after infarction.141 Perhaps the take-home message in this regard is to not disregard components present in smaller quantities, as constituents do not necessarily require equally high doses to be equally effective.

Taken together, these studies suggest that the complete Rooibos extract may facilitate cardiovascular benefits achieved via multiple modes of action effected by different plant constituents. Thus, using Rooibos as an infusion or extracted form may be even more beneficial than an isolated compound or a cocktail thereof. Of course, consumption of isolated compounds or a cocktail of ingredients may, on the other hand pose a higher risk for side-effects. Consumer safety and the potential for herb–drug interaction should be addressed.

Potential for undesired effects

Given the potent antioxidant capacity of Rooibos, it is necessary to consider the risk of its constituents to act as pro-oxidants, as previously described for well-known antioxidants such as quercetin, vitamin C, glutathione, resveratrol and even Trolox (the golden standard used in many antioxidant assays).142,143 Briefly, the mechanism implicated here is the ease with which these antioxidants undergo single electron auto-oxidation in the presence of metals such as iron or copper, or peroxidases such as myeloperoxidase (secreted by white cells during inflammation) or lactoperoxidase, resulting in a free radical substance. However, literature suggests that more than one consecutive auto-oxidation reaction is required before DNA damage is incurred, and that auto-oxidation may be prevented in the presence of other antioxidants with the capacity to stabilize the one-electron reductants.

When considering the experimental data related to this potential side-effect, the complexity of the topic and the need for individualized characterization in this context becomes clear. For example, the plant-derived alkaloid antioxidant, delta-7-mesembrenone, was recently reported to show evidence of in vitro pro-oxidant activity only when used at extremely high concentrations.129 This suggests that lowering the dose of the supplement may ameliorate the risk. However, in contrast, both water and ethanol extracts from green tea (Camellia sinensis) were shown to have in vitro pro-oxidant function at low dose, while high doses had a net antioxidant effect.144 Furthermore, the pro-oxidant effect of green tea was dependent on the availability of the polyphenolic antioxidants (−)-epigallocatechin and (−)-epicatechin.

Investigations into these mechanisms have thus far been limited to in vitro experiments, with the relevance of this effect in an in vivo system being less apparent. However, a recent review summarized studies reporting adverse effects associated with use of other antioxidant substances.145 Symptoms observed in healthy population which may have relevance to CVD included increased levels of creatine kinase (which may be indicative of oxidant tissue damage) and inflammatory markers, as well as prevention of intervention-mediated improvement in insulin sensitivity. The authors further concluded that “over-quenching” of free radicals by exogenous antioxidants may down-regulate endogenous antioxidant systems, as well as compromise homeostatic processes dependent on free radicals such as nitric oxide. Although no adverse effects of this nature has been reported in Rooibos literature, the effects of long-term high-dose supplementation with Rooibos has not been investigated. In our opinion, when consuming Rooibos as the traditional herbal tea, there is little risk of consuming mega doses due to the large fluid volume that would be required. However, consumers of supplements enriched with Rooibos extract, should be advised on the potential risks involved.

There has only been a single report (a single case study) suggesting adverse effects of Rooibos – in the context of potential hepatotoxicity.146 The authors described sudden elevations in liver enzymes (ALT, γ-GT and ALP) in a patient following two weeks of flavoured Rooibos tea consumption (1 liter daily), which subsided after discontinuation of consumption. However, the tea consumed was flavoured with extracts from strawberry, chamomile and daisy petals, all of which could have contributed to the symptoms observed. Furthermore, in this case, the patient had pre-existing liver pathology related to Waldenström's macroglobulinemia – a type of lymphoma. In our opinion it is thus unlikely that Rooibos was the cause of these symptoms, as also supported by the rodent study which reported no specific detrimental or adverse effects of green or fermented Rooibos on either liver or kidney enzyme function.8 In fact, a more recent study has suggested Rooibos to have liver protective function.16

In terms of potential herb–drug interaction, no pharmacological studies are available that have directly assessed potential interactions between Rooibos constituents and commonly used cardiovascular medicines. However, a recent review on the use of herbal medicines by cardiovascular patients reported that Rooibos intake should be monitored in these patients, given the multiple reported significant and beneficial cardiovascular effects of Rooibos.147 Thus, in terms of Rooibos – although the possibility of interaction with other herbs or pharmaceutical drugs cannot be excluded at this stage – it seems that the only reason for caution at present, is that Rooibos is so effective on many fronts, that lower doses of conventional medication may be required in consumers of Rooibos, due to its potential in aiding the management of cardiovascular disease.

Conclusions

Abundant scientific evidence in the reviewed literature suggest Rooibos to be of benefit – both as a preventative approach to CVD and as a complementary therapeutic functional food to improve long-term prognosis. Several therapeutic target mechanisms have been identified for Rooibos plant extracts, which have been comprehensively characterized in terms of its composition and changes in constituent levels attributed to fermentation. Despite the fact that – as for most natural products – very few human clinical trials have been conducted on this functional food, the available in vivo data from both human and animals studies highlighting potential health benefits are overwhelmingly positive. It is furthermore of clinical importance that human trials aimed at elucidating potential Rooibos–drug interactions be conducted, specifically due to the fact that regular Rooibos consumers receiving treatment for CVD may require lower doses of prescribed medicine. In our opinion, future research related to Rooibos and its constituents should now progress to the clinical trial phase in earnest, as Rooibos clearly has tremendous potential in alleviating the plight of individuals with CVD, or at risk of developing CVD.

Conflicts of interest

There are no conflicts of interest to declare.

Acknowledgements

The authors would like to thank the South African Rooibos Council for their continued financial support of Rooibos-related research. The authors would like to acknowledge Rooibos LTD. for the use of the photograph of Rooibos in the graphical abstract. The authors would also like to thank Dr Jimmy leGong for technical assistance in the preparation of the manuscript.

Notes and references

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