Rooibos ( Aspalathus linearis ) and Honeybush ( Cyclopia spp.): From Bush Teas to Potential Therapy for Cardiovascular Disease

Cardiovascular disease (CVD) is a leading cause of worldwide deaths. A number of risk factors for cardiovascular disease as well as type 2 diabetes and stroke present as the metabolic syndrome. Metabolic risk factors include hypertension, abdominal obesity, dyslipidaemia and increased blood glucose levels and may also include risk factors such as vascular dysfunction, insulin resistance, low high density lipoprotein (HDL) cholesterol levels and inflammation. Rooibos ( Aspalathus linearis ) and honeybush ( Cyclopia spp.) are indigenous South African plants whose reported health benefits include anti-tumour, anti-inflammatory, anti-obesity, antioxidant, cardioprotective and anti-diabetic properties. The last two decades have seen worldwide interest and success for these plants, not only as health beverages but also as preservatives, flavourants and skincare products. This review will focus on the current literature supporting the function of these plants as nutraceuticals capable of potentially reducing the risk of cardiovascular disease.


Introduction
Cardiovascular disease is the leading cause of deaths worldwide, killing 17.9 million people in 2016 [1]. While the number of cardiovascular disease related morbidity and mortality in the developed world has decreased or remained steady, the developing world has seen an increase. Limited resources, poverty, poor access to affordable healthcare, poor implementation of health policies, as well as poor education may be some of the reasons for the increase in cardiovascular diseases in low to middle income countries [2]. A number of risk factors for cardiovascular disease as well as type 2 diabetes and stroke present as metabolic syndrome. Metabolic risk factors include hypertension, abdominal obesity, dyslipidaemia, increased blood glucose levels, and may also include risk factors such as vascular dysfunction, insulin resistance, low levels of high density lipoprotein cholesterol (HDL-C) and inflammation. Natural products could play a significant role in drug discovery and development with examples including morphine, isolated from the opium poppy (Papaver somniferum) and artemisinin, from Artemisia afra [3][4][5]. Nutraceuticals are foods or supplements with health benefits [6]. To this end, a

Risk factors for cardiovascular disease
A multitude of risk factors predispose to the onset of cardiovascular disease. This includes unmodifiable risk factors, such as increasing age, male gender, ethnicity, family history and genetics [15]. Modifiable risk factors include tobacco smoking, an unhealthy diet, a sedentary lifestyle, high alcohol intake, high blood pressure, being overweight or having central obesity, dyslipidaemia, impaired glucose tolerance or diabetes [15]. Diabetes not only quadrupled from 1980 to 2014 but approximately 57% of diabetic women and 67% of diabetic men are likely to present with cardiovascular disease by the age of 50 [1,16]. Metabolic syndrome is largely preventable and includes a number of clinical findings which when occurring together, increase the risk of diabetes and cardiovascular disease. These include central obesity with any of the following risk factors including increased triglycerides, fasting plasma glucose, blood pressure and reduced HDL cholesterol levels [17]. Metabolic syndrome is also accompanied by changes in neuroendocrine and autonomic function [18]. It is known that early life stressors can predispose to disease outcome in later life, including cardiovascular disease [19]. Chronic stress influences cardiovascular outcome and anxiety and depression are also risk factors for cardiovascular disease [20][21][22]. This leads to changes in glucocorticoids and mineralocorticoids via modulation of the hypothalamic pituitary axis (HPA) [18]. Xenobiotics, including drugs and herbal infusions are metabolised by drug metabolising enzymes such as the phase I, cytochrome P450 system, which also influences the formation of steroid hormones [23]. CYP21A2 are precursors to both mineralocorticoids such as aldosterone and glucocorticoids such as cortisol and cortisone. Interestingly, rooibos flavonoids aspalathin and nothofagin inhibits CYP21A2 but not CYP11B1, which is responsible for converting 11-deoxycortisol to cortisol [24]. Substrate conversion of CYP117A1 and CYP21A2 was also inhibited by rooibos but other flavonoids such as rutin, orientin and vitexin were unable to inhibit CYP21A2. Rutin, under forskolininduced stress, was the best inhibitor of steroid production followed by nothofagin

Origin, distribution and markets for rooibos and honeybush
Rooibos [Aspalathus linearis L. (Burm.f.) R. Dahlgren (Leguminosae)] is a member of the fynbos biome, which contains needle-like leguminous plants. It occurs in the Cederberg area of the Western Cape, and in South Africa it is one of the most widely consumed herbal teas or tisanes. Its marketing potential was realized by Benjamin Gunzberg in 1904 and since then its popularity has steadily risen worldwide [40]. The top five export markets for rooibos are Germany, Japan, the Netherlands, the United Kingdom and the United States of America [41]. Rooibos is used as herbal infusion, health beverage, an ingredient in skin care products and cosmetics as well as a flavourant and colouring agent in a number of food applications. Honeybush (Cyclopia spp.), another member of the fynbos biome, is a bushy shrub found between the Piketberg area in the West, and Port Elizabeth in the East of South Africa (Figure 1). The year 1996 welcomed the first commercial harvests for honeybush, followed by the establishment of the South African Honeybush Tea Association (SAHTA) to manage the farming and sustainability practices as well as commercial interests of honeybush. After harvesting of rooibos or honeybush crops, leaves and stems are cut into small pieces, moistened and are fermented, either on open heaps or alternatively for honeybush also using an oven or fermentation tank. This is followed by drying of the fermented rooibos or honeybush. Fermentation of these plants is however associated with a change in phenolic composition as well as colour compared, to the green or unfermented plants which undergoes considerably less oxidation [42,43]. The main contributors to the commercial market out of the 24 species of Cyclopia are C. intermedia, C. genistoides and C. subternata. C. intermedia has the largest market share; however, it is harvested from the wild, making the future sustainability and profitability of the crop problematic [44]. Honeybush is a budding commercial interest, used mainly as a tisane with great potential for development and is exported to countries such as the Netherlands, Germany and Japan [45].

Polyphenols that may be responsible for beneficial effects
Polyphenols are secondary plant metabolites commonly occurring in the diet in tea, coffee, wine, fruit, vegetables and cereals. The four main types of polyphenols, namely stilbenes, phenolic acids, flavonoids and lignins, can be classified according to the number of polyphenol rings and various chemical groups associated with the rings [46]. Flavonoids share a C3-C6-C3 backbone and the classification system includes groups such as the flavonols, flavones, isoflavones, flavanones, antho-cyanidins, and flavanols [46]. A number of studies have reported a reduction in the risk of cardiovascular disease with the intake of polyphenols [47]. The most prevalent polyphenols in rooibos include aspalathin, nothofagin, orientin, iso-orientin, vitexin and isovitexin, isoquercitrin and rutin [48,49]. Aspalathin and aspalalinin are two unique dihydrochalcones in rooibos, with the former having been widely researched to date for its antioxidant and other health promoting properties [50,51]. The flavonoid precursor in rooibos, Z-2-(β-ᴅ-glucopyranosyloxy)-3-phenylpropenoic acid (PPAG), has also received considerable attention for its anti-diabetic properties [52]. In Cyclopia species, the xanthones mangiferin, isomangiferin and the flavanone hesperidin are predominant [43,53]. Mangiferin is not unique to honeybush, and also occurs in mangoes (Mangifera indica) and plants such as Pyrrosia sheareri and Anemarrhena asphodeloides [54][55][56]. Bioavailability refers to the amount of the substance that is ingested that is available for metabolism [46]. It involves a number of processes, including intestinal absorption, plasma kinetics, metabolism, binding to plasma albumin and excretion  by the liver and kidneys [46]. Factors affecting bioavailability include isomeric form, processing methods, the type of compound and matrices surrounding the compound. The bioavailability of rooibos and honeybush is poor [57,58]. The potential benefits attributed to their intake may therefore be hampered by poor bioavailability and also affect their maximal efficacy. Recently, the use of nanoencapsulation methods have been explored in order to increase the bioavailability and stability of aspalathin which could possibly promote the use of more effective nutraceuticals [59]. Since fermentation reduces polyphenol content, a number of studies have explored the benefits of unfermented, green rooibos or so called aspalathin rich extracts of rooibos in an attempt to elucidate the health promoting properties of the tisane [60,61]. Others have looked at the effects of single isolate polyphenols from rooibos and honeybush such as aspalathin and mangiferin, to explore their functional benefits and identify a more targeted therapeutic option (Figure 2) [63,64].

Antioxidant effects
Oxidative stress occurs due to an imbalance in the production of reactive oxygen species (ROS) and the availability of ROS scavengers. This may occur due to an excess of unstable reactive species including free radicals, such as reactive oxygen, reactive chlorine, reactive nitrogen and non-radical species, all of which may interact with cells, causing cellular damage [65]. Oxidative stress contributes to the pathobiology of cancer, cardiovascular disease, ageing, diabetes and atherosclerosis; therefore, it is plausible that dietary sources of antioxidants may be useful in reducing oxidative stress. Aerobic organisms are therefore reliant on innate antioxidant defences, e.g., superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), reduced glutathione (GSH), uric acid, albumin and peroxiredoxins, to deal with large quantities of ROS in an attempt to reduce oxidative stress. Mitochondria are key generators of ROS in aerobic organisms, producing them as they generate energy; however, ROS are also important in cell signalling and released by macrophages to promote immunological attacks [66]. Xenobiotics, including rooibos and honeybush, are metabolised by the cytochrome P450 family and in the process, radicals are also produced and further detoxification processes facilitate their removal [67]. In cardiovascular disease, oxidative stress may manifest as dysfunction in the vascular endothelium or cardiac myocytes, and may arise due to increased intracellular Ca 2+ as a result of ROS [68]. Oxidative stress associated with hyperglycaemia may occur from the glycation of proteins and the formation of advanced glycation end products, the auto-oxidation of glucose and the polyol pathway. In rodent models of streptozotocin (STZ)-induced diabetes, rooibos exerted antioxidant effects through increases in the activity of superoxide dismutase, catalase, glutathione peroxidase and decreasing lipid peroxidation [64,69,70]. Rooibos also decreased advanced glycation end products but clinical markers of diabetes such as fructosamine, glycated haemoglobin and glucose were unaffected [70]. Nuclear factor erythroid 2-related factor 2 (Nrf2), a critical regulator of the antioxidant response of the cell, facilitates the removal of oxidants through increased antioxidant enzyme activity [67]. The regulator of Nrf2, Kelch-like ECH-associated protein 1 (Keap 1), has oxidative and electrophilic sensitive cysteine residues which upon activation allows for dissociation and activation of Nrf2 [71]. In H9c2 cardiomyocytes, aspalathin (1 μM) increased the expression of Nrf2, and antioxidant genes and enzymes, including SOD, CAT, GPX and peroxiredoxins [64]. The expression of cytoprotective genes including heme oxygenase 1 (H0-1), NAD(P)H dehydrogenase (quinone 1), uncoupling protein 2 and apoptotic genes such as B-cell lymphoma 2 (Bcl-2) were also increased after aspalathin treatment. Uncoupling protein (UCP)3 and caspase 8, were however decreased. This suggests cellular survival due to reduced caspase 8, an important trigger for cell death [72]. UCP3 and UCP2 act in concert in the mitochondrial antioxidant response with UCP2 appearing to play a greater cytoprotective role in cardiomyocytes [73]. In the diabetic (db/db) mouse model, aspalathin increased Nrf2 expression as well as its downstream gene targets [74]. High dose aspalathin (130 mg/kg) also ameliorated the effects of hyperglycaemia in the heart by reducing the expected left ventricular enlargement. It could not however reduce fasting plasma glucose levels compared to metformin in these mice. The study confirmed the role of Nrf2 in the antioxidant response of the cell, strengthened the case for the potential use of nutraceuticals such as aspalathin in the treatment of diabetes, and also provides evidence for the differential effects observed with isolates compared to whole extracts of plants. Oxidative stress and inflammation co-exists in a number of diseases. The relationship between the two appears complex and is suggested to be a possible reason why antioxidant supplements in clinical trials have been unsuccessful [75]. In humans, no clear evidence of the antioxidant effects of flavonoids or pro-oxidant effects exist [76]. Supplementation of six cups of rooibos per day over a 6 week period in adults with increased cardiovascular risk increased plasma total polyphenols, reduced glutathione (GSH), and increased the GSH: oxidized glutathione (GSSG) ratio compared to the control period [77]. Furthermore, TBARS and conjugated dienes were reduced, indicating a reduction in oxidative stress after rooibos consumption. Ferric reducing antioxidant power (FRAP), oxygen radical absorbance capacity (ORAC) and 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate (ABTS) were unaffected. It is however known that assays for the measurement of antioxidant status can be difficult to compare and may also be non-specific and complicated by the instability of the species they are measuring [78]. Flavanoids and other polyphenols in honeybush are responsible for its antioxidant effects [79]. Fermentation of honeybush and rooibos however reduces antioxidant activity [80]. Mangiferin was the most effective scavenger of ABTS˙+ and in terms of its ability to reduce ferric ion, than the flavanone eriocitrin or the flavone luteolin [43]. This could be due to the hydrophilic nature of mangiferin, which is a glucoside. In an in vitro study using skin cells, aqueous extracts of Cyclopia subternata sp. exhibited the highest ABTS˙+ scavenging ability compared to other unfermented species of rooibos and Camellia sinensis [53]. In addition, rooibos, honeybush and Chinese green tea demonstrated ferric reducing antioxidant power (FRAP) and oxygen radical absorbance capacity (ORAC) abilities. Rooibos and honeybush also had better ORAC values than green tea, while rooibos aqueous extracts had the highest FRAP. In another model using hairless SKH-1 mice, unfermented honeybush and mangiferin had the highest FRAP compared to the fermented honeybush and hesperidin, and also the highest total antioxidant capacity [63]. Fermented and unfermented honeybush as well as mangiferin and hesperidin also protected against ultraviolet (UV) B-induced lipid peroxidation. Fermented and unfermented extracts of honeybush as well as hesperidin increased SOD and CAT activity, while mangiferin increased SOD but not catalase activity [63]. When Chinese green tea, rooibos and honeybush were evaluated for their ability to reduce lipid peroxidation, unfermented extracts generally offered better protection against lipid peroxidation but green tea offered the highest level of protection compared to the other infusions [53]. Mangiferin activated phosphatidylinositol 3-kinase (PI3K) induced protein kinase b (PKB/Akt) and Nrf2 signalling pathways, thus decreasing oxidative stress in an in vivo model using human kidney cells exposed to tert-butyl hydroperoxide [81]. The activities of SOD, CAT, GPX and the non-enzymatic intracellular antioxidant GSH were also enhanced and lipid peroxidation was decreased. When compared to vitamin C, ROS scavenging ability as measured by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method and FRAP was decreased at lower concentrations compared to vitamin C, which was used as the positive control. The expression of Nrf2, HO-1, SOD, Akt and GSK-β and the mechanistic target of rapamycin (mTOR) and cyclin D were also increased by exposure to mangiferin. In an STZ-induced diabetic Wistar rat model, mangiferin improved antioxidant status and reduced apoptosis and inflammation [82]. This could be due to modulation of the AGE-RAGE/MAPK signalling pathways. Advanced glycation end products (AGEs), which are increased in diabetes, lead to upregulation and activation of the receptor for advanced glycation end products (RAGE). This interaction causes inflammation and oxidative stress, through increases in the production of ROS, leading to lipid peroxidation. AGEs can also inhibit peroxisome proliferator activated receptor (PPAR)γ, a regulator in inflammation as well as the metabolism of lipids and glucose [83]. In the study mangiferin also displayed potential as a therapeutic in preventing AGE mediated lipogenesis. Antioxidants such as β-carotene, α-tocopherol and ascorbate were unsuccessful in reducing incidences of cardiovascular [84] and other diseases such as cancer [85], in fact appearing to increase risk in some cases, e.g., with vitamin C, x-tocopherol and beta-carotene [86][87][88]. Antioxidant supplementation to alleviate oxidative stress could however be affected by other factors such as the dose of the antioxidant, timing of the intervention, interactions with other antioxidants, whether it as administered as an isolate or a whole extract, the type of extract, the model as well as the methods that are used to detect oxidative stress. Rooibos and honeybush exert antioxidant effects by scavenging free radicals, chelating metal ions, or upregulating indigenous antioxidant enzymes. The aromatic ring structures of polyphenols also contain free hydroxyl groups which contribute to their antioxidant ability. The structural differences between rooibos polyphenols may thus also explain differences in antioxidant activity of these compounds [89]. The antioxidant activity of rooibos and honeybush polyphenols may also be explained by their ability to increase the activity of antioxidant enzymes, through upregulation of Nrf2, PI3K and other signalling pathways involved in cell survival. It may also be explained by their ability to act as antioxidants themselves, exerting differential physiological effects. The antioxidant effects of rooibos and honeybush and their polyphenols have been extensively studied in detail elsewhere and some of these effects are discussed here under the anti-inflammatory, anti-obesity, anti-diabetic and cardiovascular effects of these nutraceuticals (Tables 1-4).

Anti-inflammatory effects
Inflammation and oxidative stress form a common thread in the metabolic syndrome, type 2 diabetes and cardiovascular disease [132,133]. The rooibos flavonoid orientin from rooibos reduced the number of mast cells in colon sections as well as

[101]
Extracts are considered fermented unless otherwise indicated.   NFK-β, TNF-α, IL-6, iNOS and COX2, reflecting its anti-inflammatory potential [92]. Aspalathin and nothofagin inhibited LPS-mediated expression of the lipopolysaccharide (LPS) receptor (TLR4) and LPS-mediated barrier disruption. This occurred by increasing barrier integrity and inhibiting the expression of cell adhesion molecules (CAMs) and reducing neutrophil adhesion and migration in human umbilical vein endothelial cells [95]. The barrier protective effects of aspalathin and nothofagin were confirmed in a mouse model, in which the dihydrochalcones reduced LPS-induced mortality. This suggests that aspalathin and nothofagin can be regarded as potential therapeutics against vascular inflammation. The protective effects of aspalathin and nothofagin in a systemic inflammatory response induced by caecal ligation included decreases in inflammatory markers, including TNFα-expression NF-κB, NO and IL-6 production, enhanced antioxidant activity and decreased lipid peroxidation. [91]. The anti-inflammatory effects of rooibos and honeybush may involve a reduction in inflammatory cytokines such as IL-6, TNF-α, COX2 [91,92,94]. It may also involve a reduction in neutrophil adhesion, and other mechanisms [96], Table 1.

Anti-obesity effects
Obesity and overweight are major risk factors for cardiovascular disease and type 2 diabetes. Insulin resistance, a key finding in the metabolic syndrome, is characterised by alterations in glucose uptake in insulin sensitive tissues such as the liver, skeletal muscle and adipose tissue [134]. Non-alcoholic fatty liver disease (NAFLD), characterised by fat accumulation in the liver, is an independent predictor of the metabolic syndrome which influences the progression of cardiovascular disease [135]. Since NAFLD is complex, utilising therapeutic strategies with multiple targets may be beneficial. The PI3K/Akt pathway is a complex insulin regulated pathway involved in glucose and lipid metabolism, as well as other cellular processes, including protein synthesis, cell signalling cell growth and apoptosis [136]. In type 2 diabetes and obesity, inhibition of the pathway interferes with beta cell function and insulin secretion, exacerbating insulin resistance. A green rooibos extract reduced lipid accumulation as well as lipolysis in C3A liver cells [102]. Rooibos also ameliorated palmitate-induced insulin resistance, by activating phosphorylated PKB/Akt and 5′ adenosine monophosphate-activated protein kinase (AMPK), as well as increasing glucose transporter (GLUT)2 expression. Furthermore, fatty acid oxidation was enhanced by increasing FOXO1, decreasing malonyl-CoA decarboxylase, increasing carnitine palmitoyl transferase 1 (CPT1) and increasing acetyl CoA carboxylase in insulin deficient cells. In the accompanying obese insulin resistant rat model, only a high dose (195 mg/kg) of green rooibos extract (GRE) could reduce insulin levels and the HOMA-IR index. No significant differences were detected in blood glucose, body weights or food intake. Insulin receptor and insulin receptor substrates 1, and 2 were however upregulated by GRE, suggesting that GRE may be beneficial in ameliorating obesity-induced insulin resistance. In 3T-L1 adipocytes, palmitate-induced insulin resistance was ameliorated by both aspalathin, as well as GRE [104]. This was accompanied by increases in Akt activation and decreases in nuclear factor (NF)-κB, IRS1 and AMP phosphorylation. GLUT4 expression was enhanced by the GRE but not aspalathin, suggesting that the whole extract rather than the isolated compound may be beneficial in providing a more multi-targeted therapeutic approach in improving palmitate-induced effects on glucose and lipids. A number of other rooibos polyphenols, such as the C-glycosidic flavones orientin, iso-orientin and luteolin, have anti-obesity effects, as reflected by their inhibition of pancreatic lipase, which is responsible for digesting and absorbing triglycerides [137]. Fermented rooibos also inhibited lipid accumulation in 3T3-L1 adipocytes [105]. Z-2-(β-ᴅ-glucopyranosyloxy)-3-phenylpropenoic acid (PPAG), increases glucose uptake, as demonstrated in Chang cells [52]. In their in vivo model of obese insulin resistant rats, basal fasting glucose decreased and glucose tolerance was improved by PPAG. Increases in mRNA expression of genes involved in glucose (GLUT1 and GLUT4) and insulin metabolism (IR) and others such as PPARα and SOCS3 in the liver were also seen. PPARα regulates the action of fatty acids in the liver, suggesting that insulin signalling, glucose and lipid metabolism may be altered by PPAG. SOCS3 associates with various proteins to inhibit cytokine signals, e.g., leptin, growth hormone, IL-6, leukaemia inhibitory factor (LIF) as well as insulin [138]. It may therefore mediate leptin resistance, which occurs in obesity but is also regulated by leptin, which may partially explain these findings. Administration of rooibos to LDL receptor deficient (Ldlr − / − ) mice reduced liver steatosis and white adipose tissue and increased brown adipose tissue in high fat diet rats. Macrophage recruitment was decreased but no difference was seen in adipocyte size or number in the rooibos supplemented group. Furthermore, there appeared to be no liver toxicity, and free fatty acids, triglycerides and cholesterol were reduced by rooibos in the high fat diet group [108]. In humans, rooibos reduced oxidative stress, improved HDL-C, and triacylglycerol and low density lipoprotein cholesterol (LDL-C) in adults at risk of developing cardiovascular disease. [77]. These results suggest that rooibos and flavonoids in rooibos, such as aspalathin, may be potential adjuvants in the management of the metabolic syndrome. The anti-obesity effects of Cyclopia intermedia, C. subternata and C. maculata are limited but have been studied in 3T3-L1 adipocytes [106,107,139]. C. maculata and C. subternata decreased intracellular lipid and triglycerides and increased PPARγ, a regulator of glucose and lipid metabolism [107]. Their antiobesity effects also include increased release of glycerol [106]. Hormone sensitive lipase (HSL) expression, which is regulated by SIRT1a, a rate limiter of lipolysis, was also increased by fermented C. maculata. Furthermore, perilipin expression, which appears to play a key role in the metabolism of lipids and lipolysis of adipose tissue was upregulated [140]. C. intermedia increased HSL, SIRT1, UCP3 as well as PPARγ expression in 3T3-L1 adipocytes [139]. The authors attributed increases in SIRT1 and PPARγ to be indicative of changes from white to brown adipose tissue, however the expression of UCP1, which characterises brown adipose tissue was not measured in the study [141]. While the exact function is unclear, UCP3 may protect against lipotoxicity of the mitochondria [142]. In the parallel obese db/db mouse model, no increases in body weight gain were seen with C. intermedia treatment, and neither food nor water consumption was affected [139]. Extracts of mangiferin and other polyphenols isolated from plants containing components similar to that found in honeybush have proven useful in understanding their mechanisms of action and how they could be used as nutraceuticals in the treatment of cardiovascular disease. In a double-blind randomised placebo controlled clinical trial, mangiferin (150 mg/day) from mangoes, was given to 97 overweight, hyperlipidaemic patients over a period of 12 weeks. Increased high density lipoprotein cholesterol (HDL-C), L-carnitine, as well as decreases in total cholesterol, low density lipoprotein cholesterol (LDL-C) and triglycerides were obtained in the patients [143]. No alterations in liver and kidney function markers were seen, suggesting that chronic treatment over 12 weeks was safe in human participants. Since the metabolic syndrome is so complex, providing a multi-targeted approach, such as through affecting the PI3K/Akt pathway may be somewhat beneficial. The anti-obesity effects are further described in Table 2. The limited available information on the anti-obesity effects of rooibos and honeybush however, suggests that more research is needed to fully understand and enable the translation of these effects from animals to man.

Anti-diabetic effects
Diabetic hyperglycaemia involves macro-and microvascular complications [144]. Free radicals are also produced, promoting oxidative stress [145]. Evidence suggests that rooibos and its polyphenols may reduce oxidative stress associated with the pathogenesis of diabetes [34,69]. A meta-analysis and systematic review narrowed down to 12 peer-reviewed studies reported a reduction in blood glucose levels in diabetic rodent models that had received either rooibos or its polyphenols, while no clinical trials have investigated the effects of rooibos in the context of type 2 diabetes [146]. Rooibos decreases fasting blood glucose and improves glucose tolerance [117]. The anti-diabetic actions may be related to the expression of genes responsible for glucose uptake (GLUT1, GLUT2), insulin signalling (IR), as well as other genes such as PPRα in the liver in a model of obese insulin resistant rats treated with PPAG [52]. The involvement of insulin dependent GLUT4, in skeletal muscle has also been reported [111]. Rooibos may be exerting anti-diabetic effects by affecting the metabolism and uptake of glucose. Aspalathin influences key genes involved in the metabolism of lipids, insulin resistance, inflammation and apoptosis, possibly reversing metabolic abnormalities by involving PPARγ, Adipoq, IL-6/Jak2 pathway and Bcl-2 [74]. PPAG increased beta cell mass and delayed hyperglycaemia in STZ-diabetic mice [147]. This was accompanied by increases in anti-apoptotic B-cell lymphoma 2 (Bcl-2), but no antioxidant effect, and in human islet cells PPAG also decreased cell death. This suggests that PPAG increases beta cell mass by decreasing apoptosis and increasing Bcl-2 as reflected in both STZ diabetic and obese mouse models [114,148]. In the absence of insulin, GRE also increased phosphorylation of both AMPK and Akt, in L6 skeletal myotubules. Activation of AMPK could be a therapeutic strategy in the treatment of obese and type 2 diabetics, as animal studies suggest a dysregulation of AMPK in these states. The mechanism for the amelioration of diabetic complications in rodents by rooibos and honeybush may also be due α-glucosidase inhibition; or SGLT2 inhibiting potential of rooibos and honeybush or their respective flavonoids [119,[149][150][151]. α-Glucosidase is an enzyme present on the intestinal brush border where it digests starch, increasing blood glucose [152]. Anti-diabetic drugs, therefore commonly utilize strategies such as α-glucosidase inhibition or SGLT2 inhibition to facilitate their actions [152,153]. The dihydrochalcone phlorizin, an SGLT2 inhibitor, provides a basis to explore natural plant based agents for use in the management of diabetes [154]. Strategies to target SGLT2 rather than SGLT1 is also associated with reduced drug toxicity as found in studies of phlorizin and anti-diabetic drugs [153]. In an STZ-diabetic model, unfermented extracts of Cyclopia maculata improved glucose tolerance, fasting glucose, β-cell area, triglyceride levels as well as the insulin: glucagon ratio. Plasma nitrite was reduced but no alterations were seen in other markers of nitrotyrosine and lipid peroxidation or the serum antioxidant enzymes [155]. Mangiferin and naringenin from Salacia oblonga also reduced blood glucose, normalised AST and ALT levels, improved antioxidant status and decreased protein carbonyl levels, glycogen and TBARS in the liver. Beta cell damage in the pancreas was also ameliorated by the compounds. It was proposed that activation of PPARγ and GLUT4, which was also accompanied by insulin sensitisation, may be partially responsible for the anti-diabetic effects [156]. Since naringenin is also found in honeybush, it suggests that extracts from honeybush could possibly also produce similar effects. Mangiferin from Anemarrhena asphodeloides Bunge reduced blood glucose levels and ameliorated hyperinsulinemia, the anti-diabetic properties possibly due to a reduction in insulin resistance in these rats [157]. Furthermore, mangiferin and naringenin isolated from Salacia oblonga reduced blood glucose levels, and increased GLUT4 and PPARγ expression, suggesting increased insulin sensitisation and demonstrating the anti-diabetic properties of the polyphenols. In conclusion, the anti-diabetic effects of rooibos and honeybush ( Table 3) are mediated by complex signalling pathways that affect glucose and lipid metabolism as well as oxidative stress.

Cardiovascular effects
Angiotensin converting enzyme (ACE) inhibitors reduce blood pressure by inhibiting the conversion of angiotensin to angiotensin II, the latter being a potent vasoconstrictor which increases blood pressure through a variety of mechanisms [158]. Angiotensin II can also interact with the angiotensin I receptor, enhancing ROS production, which contributes to endothelial dysfunction by inactivating vasodilatory nitric oxide [158]. Endothelial dysfunction is commonly associated with risk factors of cardiovascular disease, including obesity and type 2 diabetes [159]. It also precedes atherosclerosis, which involves a number of complex processes such as the release of pro-inflammatory cytokines and inflammation, oxidation of LDL as well as macrophage recruitment and platelet adhesion [158]. Cardiovascular protective effects of rooibos have also been established in a number of studies ( Table 4). In an in vitro human umbilical vein endothelial cell (HUVEC) study, rooibos could not inhibit ACE but increases in nitric oxide were detected [125]. ACE-inhibition was also determined in 17 healthy volunteers receiving a single oral dose of rooibos [124]. Inhibition of ACE occurred up to 60 min after ingestion but no change in blood pressure or nitric oxide was detected. ACE-inhibitory effects have also been reported for rooibos and honeybush and some of their flavonoids [131]. This effect on ACE could be due to the presence of the C2〓C3 double bond, the catechol group at the B-ring and the cetone group on C4 on the C-ring of flavonoids [131]. The best ACE-inhibitor in HUVEC's was luteolin, followed by quercetin, rutin, kaempferol, rhoifolin and apigenin K. The honeybush flavonoids genistein and hesperetin also displayed ACE-inhibitory effects but these were less potent than luteolin. Rooibos probably inhibits ACE using a mixed type of inhibition, which binds to the enzyme's active and allosteric sites [126]. Organic fractions of, as well as flavonoids in rooibos such as chrysoeriol and rutin inhibited spontaneous and low and/or high K + -induced contractions in models utilising jejunum and bronchial smooth muscle [130,160]. Anti-contractile effects were reportedly due to the opening of K + channels as well as a Ca 2+ antagonistic action. These studies were not done in vascular smooth muscle, but it is possible that rooibos could exert antihypertensive effects via similar mechanisms in vascular smooth muscle. It also caused mild reduction of atrial force and rate of spontaneous contractions in the Guinea pig and decreased blood pressure in anaesthetised rats [130]. In comparison, isolated compounds of rooibos tested in jejunal, tracheal and aortic preparations showed variable effects. In adults with cardiovascular risk factors, six cups of rooibos was able to increase antioxidant status by increasing the GSH: GSSG ratio and decrease lipid peroxidation by decreasing TBARS and conjugated dienes [77]. Furthermore, increases in the healthy HDL cholesterol were also seen. In a recent study using a high fat diet to induce obesity in Wistar rats, treatment with an enriched GRE, containing large quantities of aspalathin, improved glucose tolerance, vascular function, as determined by aortic vascular contraction-relaxation studies and enhanced the antioxidant status. Modest improvements in blood pressure were also seen in a recent study in obese rats, suggesting cardiovascular benefits with the intake of the rooibos [161]. Fermented rooibos also improved vascular function in nicotine exposed rats [162]. Since tobacco smoking is a major risk factor for cardiovascular disease, this suggests a possible therapeutic role for rooibos in the reduction of cardiovascular complications associated with tobacco smoking, such as vascular dysfunction or oxidative stress. Cardioprotective effects of rooibos and its flavonoids typically appear to involve a reduction in lipid peroxidation and upregulation of the antioxidant enzymes through Nrf2 activation, and a reduction in apoptosis which may also be associated with © 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.  [77,122,129]. The anti-inflammatory effects of rooibos, also support its role as a cardioprotective agent ( Table 1). No literature investigating the vascular modulating effects of honeybush are currently available and anti-inflammatory effects on HUVECs are discussed above. Decreases in VLDL, LDL-C and increases in HDL-C by naringenin and mangiferin from Salacia sp. however suggests the potential of similar isolates from honeybush to ameliorate endothelial dysfunction [156].

Conclusions and perspectives
Rooibos and honeybush have a number of health benefits. The evidence for their use as functional nutraceuticals is reflected in anti-diabetic, anti-inflammatory, antioxidant, anti-obesity, and vascular effects in animal and in vitro studies. The translation of these results into human medical care has however been limited. The inability to translate animal studies to that of humans may be due to the result of the controlled genetic background and environmental conditions of animals used as models to study disease [163]. Given the unstable nature of ROS and the limitations in assays used to detect oxidative stress, it is noteworthy to remember that intervention studies may only as good as the biomarkers used to measure the intervention. As we investigate the therapeutic role of rooibos or honeybush in the treatment of cardiovascular disease, it is evident that we are only but scratching the surface. These plants have complex chemical compositions and while isolating single components could be beneficial, the interplay and additive effects of other components in whole extracts could be more beneficial [111]. The mechanisms behind these benefits involve numerous signalling pathways that regulate their effects. Understanding these complexities may be what is needed to promote their transition from bush teas to nutraceutical therapy against cardiovascular disease but until then we may as well enjoy a cuppa.

Conflict of interest
There is no conflict of interest to disclose.

Thanks
Thanks go to Professor H. Kuivaniemi for proofreading this manuscript and to the SAHTA for allowing the use of the honeybush image. and JNK pathways are involved in luteolin-mediated protection of rat hearts and cardiomyocytes following ischemia/reperfusion. PLoS One. 2013;8 (12)