The cardioprotective effect of an aqueous extract of fermented rooibos (Aspalathus linearis) on cultured cardiomyocytes derived from diabetic rats.
Diabetic cardiomyopathy (DCM) is a disorder of the heart muscle that contributes to cardiovascular deaths in the diabetic population. Excessive generation of free radicals has been directly implicated in the pathogenesis of DCM. The use of antioxidants, through dietary supplementation, to combat increased cellular oxidative stress has gained popularity worldwide. Aspalathus linearis (rooibos) is a popular herbal tea that contains a novel antioxidant, aspalathin. Literature has reported on the antidiabetic, anti-inflammatory and free radical scavenging effects of rooibos. However, its protective effect against DCM has not been established. Therefore, this study investigated whether chronic exposure to an aqueous extract of fermented rooibos (FRE) has an ex vivo cardioprotective effect on hearts obtained from streptozotocin (STZ) induced diabetic rats. Adult Wistar rats were injected with 40 mg/kg of STZ. Two weeks after STZ injection, cardiomyocytes were isolated and cultured. Cultured cardiomyocytes were treated with FRE (1 and 10 [micro]g/ml), vitamin E (50 [micro]g/ml), and n-acetyl cysteine (1 mM) for 6h, before exposure to either hydrogen peroxide ([H.sub.2][O.sub.2]) or an ischemic solution. Cardiomyocytes exposed to [H.sub.2][O.sub.2] or an ischemic solution showed a decrease in metabolic activity and glutathione content with a concomitant increase in apoptosis and intracellular reactive oxygen species. Pretreatment with FRE was able to combat these effects and the observed amelioration was better than the known antioxidant vitamin E. This study provides evidence that an aqueous extract of fermented rooibos protects cardiomyocytes, derived from diabetic rats, against experimentally induced oxidative stress and ischemia.
The International Diabetes Federation estimates that the prevalence of diabetes mellitus (DM) will increase to 552 million by the year 2030, with type 2 diabetes (T2D) contributing 95% to the epidemic in industrialized countries (Garg et al., 2012). In diabetics, the risk of developing cardiovascular disease (CVD), especially diabetic cardiomyopathy (DCM), increases up to 4-fold compared to nondiabetic individuals (Voulgari et al., 2010).
The first association between DM and CVDs was made in the Framingham heart study, where chronic hyperglycemia was identified as an independent risk factor for the development of DCM (Rubier et al., 1972). In uncontrolled diabetes, chronic hyperglycemia is implicated in the augmented production of free radicals (King and Loeken, 2004). Increase in free radical production is associated with altered cardiac energy substrate metabolism resulting in myocardial damage. The exacerbation of myocardial damage may eventually lead to cardiomyocyte apoptosis and impaired myocardial function. Thus, investigation into new research areas focusing on suppression of oxidative stress, during chronic hyperglycemia, may be a reasonable therapeutic strategy to protect the diabetic heart at risk from developing DCM.
Improving the quality of life for individuals with diabetes through optimal glycemic control is important. However, optimal control of blood glucose levels still remains a big challenge. Current therapies used to control blood glucose levels do not offer much protection against DCM. The use of plant derived antioxidants as an adjunct to current therapies in ameliorating metabolic disturbances continues to show promise. In recent years, research has highlighted the strong antioxidant properties of polyphenols and their potential to ameliorate diabetic complications (Kumarappan and Mandal, 2008; Zang et al., 2006). Tea consumption is one of the major sources of polyphenolic dietary intake and the relationship between drinking tea and a reduced risk of CVD has been reported (Bohn et al., 2012). The herbal tea, rooibos, produced from the leaves and stems of Aspalathus linearis, is a well known beverage with a high phenolic content. It is mostly consumed as an infusion prepared from the "fermented" (oxidised) plant material. Studies investigating its efficacy have reported on its antioxidant, anti-inflammatory and antidiabetic properties (Joubert et al., 2008; Joubert and De Beer, 2011). Furthermore, rooibos has been shown to prevent oxidative stress in rats and in humans (Ulicna et al., 2006; Marnewick et al., 2011).
To date, little is known about the beneficial potential that rooibos could have on DCM. Therefore, the purpose of the present study was to elucidate whether an aqueous extract of fermented rooibos could protect cardiomyocytes isolated from diabetic rats against experimentally induced oxidative stress and ischemia.
Materials and methods
Chemicals and reagents
CellTracker Blue CMAC dye was purchased from Invitrogen (Carlsbad, CA, USA) and taurine from Acros Organics (Geel, Belgium). Fetal bovine serum (FBS), Hank's balanced salt solution (HBSS), Dulbecco's modified Eagle's medium (DMEM), phosphate buffered saline (PBS), Media 199 and penicillin/streptomycin amphotericin B were from Lonza BioWhittaker (Verviers, Belgium) and hydrogen peroxide (H202) was from Merck (Whitehouse Station, NJ, USA). All other consumables as well as reagents were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA).
Preparation and analysis of rooibos extract
Fermented rooibos was batch extracted on industrial scale (600 kg) using a percolator type extraction vessel as described for unfermented C. subternata extract (Dudhia et al., 2013). High performance liquid chromatography with diode--array detection (HPLC-DAD) analysis was used to determine the major phenolic/phenyl compounds present in the fermented rooibos extract (FRE) (Beelders et al., 2012).
Induction of diabetes
Six-month-old adult male Wistar rats (350-450 g) were used for the study. The animals were housed at the Primate Unit and Delft Animal Centre of the South African Medical Research Council (MRC) in a controlled environment with a 12 h light/dark cycle in a temperature range of 23-25[degrees]C (relative humidity: ~50%). The rats received standard laboratory chow pellets (Afresh Vention, Cape Town, South Africa) ad libitum and had free access to drinking water. Ethical approval for this study was granted by the MRC Ethics Committee for Research on Animals (ECRA no. 11/03/G). Diabetes was induced by a single intraperitoneal injection of streptozotocin (STZ) (40mg/kg body weight). Five days later (following a 4h fast), tail pricks were performed to measure blood glucose. Rats with a fasting blood glucose concentration of [greater than or equal to] 14mmol/l were classified as diabetic and included in the study. Rat hearts were harvested 2 weeks after induction of diabetes.
Radioimmunoassay for insulin determination
Rat insulin was determined using a radioimmunoassay kit as per manufacturer's instruction (Linco Research, Inc., St. Charles, MO, USA).
Ex vivo cardiomyocyte culture
Rat hearts were removed and cardiomyocytes isolated by using a previously described method (Fischer et al., 1991). Cardiomyocytes isolated from the perfused rat heart were resuspended in 10 ml of supplemented Media 199 (5mM carnitine, 5mM taurine, 0.1 mM bromodeoxyuridine, 5 mM creatine monohydrate, 5% FBS and 0.5% penicillin/streptomycin amphoterin B) and incubated in 100 mm tissue culture dishes for a period of 1 h under standard tissue culture conditions to eliminate nonmyocytic cells. The nonadherent cardiomyocytes were harvested and seeded onto laminin coated 6 well tissue culture plates at a density of 5.94 x [10.sup.5] cells/well. Cell viability count was determined by trypan blue assay (Invitrogen, Carlsbad, CA, USA) and cell viability counts of >70% were used for subsequent experiments.
The study consisted of 2 experimental conditions (H202 and ischemic solution exposure) and each experimental condition had 6 experimental groups pretreated with either: (i) negative control (experimental control), (ii) 1 [micro]g/ml FRE, (iii) 10 [micro]g/ml FRE, (iv) 1 mM n-acetyl cysteine (NAC), (v) 50 [micro]g/ml vitamin E and (vi) untreated controls. Cardiomyocytes were pretreated for 6h before exposure to either 32 [micro]M [H.sub.2][O.sub.2] or an ischemic solution (116mM NaCl, 50 mM KCl, 1.8 mM Ca[Cl.sub.2], 2mM Mg[Cl.sub.2] x 6[H.sub.2]O, 26 mM NaHC[O.sub.3], 1 mM Na[H.sub.2]P[O.sub.4] x 2[H.sub.2]O) for 24 h and 2h, respectively. Cells that served as negative controls were treated with media only.
Measurement of metabolic activity
The adenosine triphosphate (ATP) assay was done as a measurement of metabolic activity using a ViaLight Plus kit (Lonza, Basel, Switzerland), following manufacturer's instructions.
Annexin V and propidium iodide (PI)
Apoptosis was evaluated by labelling cells with a combination of annexin V and PL The annexin V conjugate was prepared according to manufacturer's instructions (Invitrogen, Carlsbad, CA, USA) and PI was prepared in PBS (20 [micro]g/ml). The cells were incubated at room temperature for 15 min with annexin V/PI before the solution was removed and cells resuspended in PBS for microscopic analysis.
Determination of membrane leakage
The myocardial membrane leakage was detected using a rat heart fatty acid binding protein (HFABP) ELISA kit according to manufacturer's instructions (HycultBiotech, Uden, Netherlands).
Determination of intracellular reactive oxygen species (ROS)
The ROS concentration of cardiomyocytes was estimated using 2',7'-dichlorfluorescein-diacetate (DCFH-DA) dye following the manufacturer's instructions (Biolabs, San Diego, CA, USA).
Total glutathione content
Total glutathione (GSH) content of cardiomyocytes was detected using CellTracker Blue CMAC dye according to a previously described protocol (King et al., 2004).
Statistical analysis between groups was performed using one-way ANOVA analysis followed by a Tukey post hoc test or an unpaired Student's t-test where appropriate. A p-value of [less than or equal to] 0.05 was deemed as statistically significant.
Composition of FRE
HPLC-DAD analysis of the extract showed high levels of the flavonoids isoorientin (0.92g/100g), orient in (0.72g/100g), quercetin-3-O-robinobioside (0.45g/100g) and aspalathin (0.36g/100g), as well as the phenylpropenoic acid glycoside, phenylpyruvic acid-2-0-(3-D-glucoside (PPAG) (0.71 g/100g) (Table 1). Other flavonoids i.e. nothofagin, vitexin, hyperoside, rutin, isovitexin, isoquercitrin and luteolin-7-O-glucoside were present at less than 0.2 g/100 g.
Confirmation of hyperglycemia
Determination of fasting plasma glucose and random serum insulin levels showed that STZ administration induced hyperglycemia (18.25 [+ or -] 0.85 mmol/1) and hypoinsulinemia (0.39 [+ or -] 0.06 ng/ml) thereby confirming the diabetic status of the rats 2 weeks after STZ injection.
Effect of FRE against cardiomyocyte apoptosis
A significant increase (from 46 [+ or -] 3% to 74 [+ or -] 5%, p < 0.0003) in apoptosis was observed in cardiomyocytes after 24 h exposure to [H.sub.2][O.sub.2] when compared to the negative control (Fig. 1a). Pretreatment with 1 [micro]g/ml of FRE (56 [+ or -] 5%, p < 0.03) protected cardiomyocytes Against [H.sub.2][O.sub.2] induced apoptosis when compared to the [H.sub.2][O.sub.2] control. This protective effect of 1 [micro]g/ml of FRE was comparable to the known antioxidant, NAC (positive control) (55 [+ or -] 4%, p<0.005) (Fig. 1a). It was also noted that treatment with FRE at 10 [micro]g/ml and vitamin E failed to significantly reduce the [H.sub.2][O.sub.2] induced apoptosis (Fig. 1a). Exposing cardiomyocytes for 2h to an ischemic solution resulted in a significant increase in apoptosis when compared to the negative control (from 54 [+ or -] 2% to 74 [+ or -] 2%, p < 0.0001) (Fig. 1b). Pretreatment of cardiomyocytes with 1 [micro]g/ml and 10 [micro]g/ml of FRE (64 [+ or -] 2% and 66 [+ or -] 3%, respectively), vitamin E (66 [+ or -] 3%) and NAC (67 [+ or -] 2%) ameliorated this increase (Fig. 1 b).
Myocardial membrane leakage
Exposure of the cardiomyocytes to [H.sub.2][O.sub.2] did not have a marked effect on FIFABP leakage compared to negative control levels (supplemental Fig. 1a). However, pretreatment of cardiomyocytes with FRE appeared to slightly lower the [H.sub.2][O.sub.2] induced effect (supplemental Fig. 1a). Under ischemic conditions, the levels of HFABP leakage were increased, although not statistically determined, in the ischemic control when compared to the negative control (increased from 5 [+ or -] 3 to 22 [+ or -] 3 [micro]g/l) (supplemental Fig. 1b). Pretreatment with FRE at a dose of 1 and 10 [micro]g/ml appeared to have a slight ameliorative effect on HFABP leakage (decrease of 18 [+ or -] 2 and 18 [+ or -] 3 [micro]g/l, respectively) (supplemental Fig. 1b).
Cardiomyocyte intracellular ROS detection
Exposing cardiomyocytes to H202 resulted in an increased production of intracellular ROS generated DCFH-DA fluorescence (from 100 [+ or -] 4% to 149 [+ or -] 4%, p < 0.05) (Fig. 2a). Pretreatment with 1 [micro]g/ml FRE, NAC and vitamin E was able to reduce this effect (108 [+ or -] 2%, p<0.02; 111 [+ or -] 1%, p<0.02 and 113 [+ or -] 2%, p<0.05, respectively) (Fig. 2a). However, FRE at a higher dose of 10 [micro]g/ml was ineffective (Fig. 2a). Elevated levels of intracellular ROS were detected after exposing cardiomyocytes to ischemic conditions (166 [+ or -] 7%, p< 0.032) (Fig. 2b). Pretreatment with 1 and 10 [micro]g/ml of FRE (129 [+ or -] 9% and 137 [+ or -] 7%, respectively), NAC (138 [+ or -] 15%) and vitamin E (146 [+ or -] 10%) reduced this effect though not significantly (Fig. 2b).
Metabolic activity of cardiomyocytes
Exposure of cardiomyocytes to exogenous H202 resulted in a significant depletion of the intracellular ATP concentration (5 [+ or -] 1%, p<0.0001) (Fig. 3a). Pretreatment with 1 and 10 [micro]g/ml of FRE, as well as NAC and vitamin E ameliorated the H202 induced intracellular ATP depletion (increased from 5 [+ or -] 1% to 87 [+ or -] 9%, p<0.0001; 74 [+ or -] 8%, p<0.0001; 87 [+ or -] 8%, p<0.0001 and 81 [+ or -] 11%, p<0.0001, respectively) (Fig. 3a). A decrease in ATP concentration was observed after the cardiomyocytes were exposed to an ischemic solution (10 [+ or -] 2%, p< 0.0002) (Fig. 3b). This effect was negated by FRE, NAC and vitamin E pretreatment (Fig. 3b). At the lower dose, FRE (92 [+ or -] 2%, p< 0.0001) was more effective than the higher dose of FRE, NAC and vitamin E (64 [+ or -] 5%, p < 0.0002; 77 [+ or -] 5%, p < 0.0002 and 69 [+ or -] 7%, p < 0.0002, respectively) (Fig. 3b).
Preservation of glutathione content
Glutathione content was significantly decreased by [H.sub.2][O.sub.2] (37 [+ or -] 17%, p<0.02) (Figs. 4a and 5(I)b). Pretreatment with 1 [micro]g/ml of FRE was able to preserve the GSH content (99 [+ or -] 7%, p<0.02) (Figs. 4a and 5(I)c). This dose of FRE was more effective than its higher dose, NAC or vitamin E (Figs. 4a and 5(I)c-f). Exposure of cardiomyocytes to an ischaemic solution decreased GSH content (18 [+ or -] 3%, p<0.05) (Figs. 4b and 5(II)b). FRE pretreatment at both concentrations of 1 and 10 [micro]g/ml protected against this ischaemic effect (72 [+ or -] 13%, p <0.01 and 71 [+ or -] 3%, p < 0.0003, respectively) (Figs. 4b and 5(II)c, d). A similar effect was observed after pretreatment with NAC and vitamin E (88 [+ or -] 9% and 70 [+ or -] 11%, respectively) (Figs. 4b and 5(II)e, f).
The prevalence of T2D continues to increase at an alarming rate throughout the world (Garg et al., 2012). In DCM, chronic hyperglycemia, dyslipidemia, hyperinsulinemia and other factors results in myocardial remodelling (Karnik et al., 2007). Myocardial infarction accompanied by contractile dysfunction is the end result of these diabetic complications. Streptozotocin is a well established method to chemically induce diabetes in a dose dependent manner (Nacci et al., 2009). At a dose of 40 mg/kg the adult Wistar rats developed stable non-ketoacidotic diabetes thus making this an effective model to study DCM.
HPLC-DAD analysis of FRE showed that our extract contained high levels of PPAG and the flavonoids isoorientin, orientin, quercetin-3-O-robinobioside and aspalathin. Most of the identified compounds in our extract have been shown to ameliorate metabolic disturbances associated with DM and CVDs. PPAG has been shown to improve glucose uptake and fatty acid oxidation in insulin resistant rats, and also its hypoglycemic potential is evident when tested in obese or diabetic monkeys (Muller et al., 2013; Fey et al., 2011). Aspalathin and isoorientin have been shown to exhibit significant hypoglycemic potential in T2D db/db mice (Kawano et al., 2009; Sezik et al., 2004) while rutin and quercetin have been shown to improve cardiac function in STZ induced diabetic rats (Krishna et al., 2005). During periods of ischemia glucose utilization by cardiomyocytes is increased. The increased transport of glucose uptake is facilitated by both facilitated-diffusion glucose transporters and sodium-dependent glucose transporters (SGLT) (Banerjee et al., 2009). Although the role of SGLT1 in active transport of flavonoids is still controversial, the high levels of SGLT1 expressed by cardiomyocytes could be relevant. Phloridzin (phloretin-2'-0-glucoside), a non-selective SGLTs inhibitor has been shown to protect against the deleterious effects of diabetic cardiomyopathy in db/db mice (Cai et al., 2013). However, Oglycosyl flavonoids are vulnerable to hydrolysis while C-glycosides such as aspalathin, a dihydrochalcone C-glycoside, are generally more metabolically stable and have greater oral bioavailability and bioactivity in vivo (Zhou et al., 2010). In the current study, individual compounds were not tested. Thus, the observed effect could have been an additive effect and not attributed to a single compound. However, to date several studies have reported on the antidiabetic properties of rooibos. Ulicna et al. (2006) showed that an aqueous extract of rooibos ameliorated oxidative stress in STZ induced Wistar rats by significantly lowering malondialdehyde levels in plasma and tissues of rats. Marnewick et al. (2011) showed that chronic consumption of 6 cups of rooibos tea for 6 weeks significantly improved lipid profiles and the redox status of healthy adults at risk for developing CVD.
Results presented in this study showed that pretreatment of cardiomyocytes with FRE for 6h at a dose of 1 or 10 [micro]g/ml reduced intracellular ROS and cell death caused by exogenous [H.sub.2][O.sub.2] and ischemia. The cytotoxic effects of exogenous [H.sub.2][O.sub.2] on cardiomyocytes have been described previously (Wei et al., 2012). The pretreatment also reduced the leakage of HFABP, a protein associated with myocardial damage and more specifically myocardial ischemia, from the cardiomyocytes (Azzazy et al., 2006).
[H.sub.2][O.sub.2] and ischemia further decreased ATP levels and intracellular GSH. This result is in agreement with a study reporting on how increased ROS production inflicts cardiomyocyte injury by an increase in free fatty acids, apoptosis, and depletion of ATP concentrations in the hearts of diabetic patients (Tarquini et al., 2011). Treatment of cardiomyocytes with FRE was able to preserve the ATP as well as GSH content. Depletion of GSH and ATP is strongly associated with generation of oxidative stress and metabolic dysfunction (Amado and Monserrat, 2010). Although vitamin E is a well known antioxidant, in this study it proved to be less effective at protecting cardiomyocytes against cell damage when compared to the lower dose of FRE and NAC. The reduced cardioprotective effect of vitamin E could be explained by the contradictory reports in studies investigating its cardioprotective effects (Cockey, 2005; Shirpoor et al., 2009). An interesting finding in the protective effect of FRE was the relationship between dose and efficacy. Increasing the dose from 1 to 10 [micro]g/ml reduced its protective effect against exogenous [H.sub.2][O.sub.2]. This finding was unexpected, as a dose dependent increase in activity was anticipated. However, it has been reported that polyphenols may have pro-oxidant effects at higher doses which could have been the case in these vulnerable cardiomyocytes (Mennen et al., 2005).
In a normal state, a balance exists in the production of cellular ROS and the natural scavenging effect through cellular antioxidants such as GSH. In a diseased state such as diabetes, cardiomyocytes become more vulnerable to cell damage and apoptosis due to an imbalance in the antioxidant-oxidative stress status. Our results showed that rooibos was able to preserve ATP and GSH levels under stressful conditions. It therefore makes sense that dietary supplementation with plant derived antioxidants such as that found in rooibos could be of benefit to protect against these increased levels of oxidative stress.
Abbreviations: ATP, adenosine triphosphate; Ca[Cl.sub.2], calcium chloride; CVD, cardiovascular disease; DCFH-DA, 2',7'-dichlorfluorescein-diacetate; DCM, diabetic cardiomyopathy; DM, diabetes mellitus; DMEM, Dulbecco's modified Eagle's medium; FBS, foetal bovine serum; FFAs, free fatty acids; FRE, fermented rooibos extract; GSH, glutathione; HBSS, Hank's balanced salt solution; HFABP, heart fatty acid binding protein; HPLC-DAD, high performance liquid chromatography with diode-array detection; [H.sub.2][O.sub.2], hydrogen peroxide; KCl, potassium chloride; Mg[Cl.sub.2] x 6[H.sub.2]O, magnesium chloride hexahydrate; NAC, n-acetyl cysteine; NaCl, sodium chloride; NaHC[O.sub.3], sodium hydrogen carbonate; Na[H.sub.2]P[O.sub.4] x 2[H.sub.2]O, sodium dihydrogen phosphate; PBS, phosphate buffered saline; PI, propidium iodide; PPAG, phenylpyruvic acid-2-O-[beta]-D-glucoside; ROS, reactive oxygen species; SGLT, sodium-dependent glucose transporters; STZ, streptozotocin; T2D, type 2 diabetes mellitus.
Received 2 September 2013
Received in revised form 26 September 2013
Accepted 27 October 2013
Appendix A. Supplementary data
Conflict of interest
Authors report no conflict of interest.
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.Org/10.1016/j.phymed.2013.10.029.
The study was funded by the Diabetes Discovery Platform of the South African Medical Research Council.
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P.V. Dludla (a,b), C.J.F. Muller (a), J. Louw (a), E. Joubert (c,d), R. Salie (a,e), A.R. Opoku (b), R. Johnson (a), *
(a) Diabetes Discovery Platform, South African Medical Research Council, Tygerberg 7505, South Africa
(b) Department of Biochemistry and Microbiology, University of Zululand, KwaDlangezwa 3886, South Africa
(c) Post-Harvest and Wine Technology Division, Agricultural Research Council (ARC) Infruitec-Nietvoorbij, Stellenbosch 7599, South Africa
(d) Department of Food Science, Stellenbosch University, Stellenbosch 7602, South Africa
(e) Division of Medical Physiology, Faculty of Health Sciences, Stellenbosch University, Tygerberg 7505, South Africa
* Corresponding author at: South African Medical Research Council, Diabetes Discovery Platform, P.O. Box 19070,Tygerberg 7505, South Africa. Tel.: +27 219380866; fax: +27 219380456.
E-mail address: email@example.com (R. Johnson).
Table 1 Content of PPAG and the major flavonoids in FRE. Compound Content (g/100 g) of FRE PPAG (a) 0.713 Aspalathin 0.364 Nothofagin 0.070 Isoorientin 0.924 Orientin 0.721 Quercetin-3-O-robinobioside 0.446 Vitexin 0.152 Hyperoside 0.087 Rutin 0.185 Isovitexin 0.140 Isoquercitrin 0.063 Luteolin-7-O-glucoside 0.069 (a) Phenylpyruvic acid-2-O-p-D-glucoside.
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|Author:||Dludla, P.V.; Muller, C.J.F.; Louw, J.; Joubert, E.; Salie, R.; Opoku, A.R.; Johnson, R.|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||Apr 15, 2014|
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