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Rooibos (Aspalathus linearis) offers cardiac protection against ischaemia/reperfusion in the isolated perfused rat heart.

ABSTRACT

Rooibos, a unique South African herbal tea, is known to be an important source of unique polyphenols compounds. In the present study we have quantified the main polyphenols compounds in both fermented/traditional and unfermented/"green" rooibos (Aspalathus linearis) and evaluated its cardioprotective effects against ischaemia/reperfusion injury. Male Wistar rats consumed aqueous rooibos and green tea (Camellia sinensis) extracts (2%, w/v) for 7 weeks before their hearts were rapidly excised and perfused in a working heart perfusion apparatus. The results showed that the rooibos supplemented hearts significantly improved aortic output recovery after reperfusion when compared to the green tea supplemented hearts. Additionally, we showed that the rooibos extracts, containing the highest amount of flavonols, significantly decreased the level of cleaved caspase-3 and PARP, both pro-apoptotic proteins, during reperfusion when compared to green tea. Green tea supplementation increased phosphorylation of total PKB/Akt, Akt (threonine 308) and Akt (serine 473). The rooibos extracts did not cause significant change in the levels of the pro-survival PKB/Akt (threonine 308 and serinet 473). The GSH/GSSG ratio in the hearts of the green tea supplemented group was significantly (p < 0.05) lower when compared to RF (37.78 [+ or -]28.63), RU (33.20[+ or -]4.13) and C (45.50 [+ or -]14.96). The results clearly demonstrate the cardioprotective properties of aqueous rooibos extracts via the inhibition of apoptosis which can possibly be related to the flavonol content of this unique South African herbal tea.

[c] 2011 Elsevier GmbH. All rights reserved.

ARTICLE INFO

Keywords: Rooibos

Aspalathus linearis

Green tea

Flavonoids

Oxidative stress

Ischemia/reperfusion

Apoptosis

Introduction

Myocardial ischemia/reperfusion (I/R) occurs following partial or complete cessation of blood circulation to the myocardium. The involvement of oxidative stress in the pathogenesis of myocardial I/R injury has been previously reported, with the generation of reactive oxygen species being one of the major mechanisms underlying myocardial reperfusion injury (McCord et al. 1985; Kloner et al. 1989; Dhalla et al. 2000). Observational studies have repeatedly shown that diets high in plant-based foods and beverages are associated with a lower risk of chronic diseases such as cardiovascular disease (Hertog et al. 1993; Hollman et al. 1999; Hu 2003). In addition, it has been reported that antioxidant-rich oils have cardioprotective effects against 1R injury (Esterhuyse et al. 2005; Bester et al. 2006). Flavonoids possess properties that alleviate ischemia/reperfusion injury by helping to re-establish blood flow in post-ischemic hearts. A number of flavonoids and polyphenolic compounds have shown the capacity to dilate vessels (Achike and Kwan 2003; Engler and Engler 2006; Sanchez et al. 2006; Nishioka et al. 2007; Jochmann et al. 2008). Research has shown that flavonols possess antioxidant, antiinflammatory and vasorelaxant activities (Chan et al. 2000; Perez-Vizcaino et al. 2006). Epidemiological studies report an inverse association between dietary flavonol intake and mortality from coronary heart disease (Geleijnse et al. 2002; Lin et al. 2007).

According to the World Health Organization, approximately 80% of the world's population currently relies on indigenous or traditional medicines for their primary health needs. Most of these therapies involve the use of aqueous solutions of plant extracts (Zhang 2002). Rooibos is a herbal tea made from the leaves and stems of the indigenous South African plant, Aspaluthus linearis (Brum./) Dahlg. (family Fabaceae; tribe Crotalarieae) (McKay and Bloemberg 2007; Marnewick 2009). The use of herbal preparations, typically prepared by steeping or heating the crude plant material, has prevailed for centuries and healthcare providers in South Africa and worldwide today often recommend herbal preparations. Rooibos is gaining popularity as a health/functional beverage both locally and worldwide (Joubert et al. 2008; Joubert and Schulz 2006). This is mainly due to the natural absence of caffeine and low tannin content (Blommaert and Steenkamp 1978; Galasko et al. 1989). Rooibos is a good dietary source of antioxidants containing mostly flavonoids such as flavonols, but also the unique C-C linked dihydrochalcone glucoside, aspalathin (Koeppen and Roux 1965) as well as cyclic dihydrochalcone, aspalalinin (Shimamura et al. 2006). Studies have reported on the in vivo and in vitro antioxidant (Yoshikawa et al. 1990; Gadow et al. 1997a, b; Marnewick et al. 2003; Joubert et al. 2004), anti-inflammatory properties (Baba et al. 2009) as well as the modulation of oxidative stress by rooibos (Ulicna et al. 2006; Fukasawa et al. 2009; Marnewick et al. 2011). Beltran-Debon et al. (2011) found rooibos beneficial effects in a hyperlipidemic mouse model when they fed 10g/l of rooibos extract for 14 weeks. They speculated that the possible mechanism may be activation of AMPK. These results motivated to consider the possible use of rooibos as a natural therapeutic substance to manage metabolic disease. No experimental data have been published to demonstrate the chronic intake effect of rooibos on ischaemia/reperfusion injury in an ex vivo rat heart model. Based on results from previous in vitro, animal and human studies, the aim of the present study was to determine whether dietary rooibos supplementation could protect against ischaemia/reperfusion injury in the isolated perfused rat heart. The possible biochemical mechanisms of protection are also addressed.

Materials and methods

Chemicals

Rutin, quercetin and luteolin were purchased from Sigma-Aldrich (Johannesburg, South Africa). Orientin, isoorientin, vitexin, isovitexin, hyperoside and chrysoeriol were purchased from Extrasynthese (Genay, France). Aspalathin was a gift from the South African Medical Research Counsel (PROMEC Prof WCA Gelderblom). Methanol, dimethylsulfoxide (DMSO) and trifluoroacetic acid were purchased from Merck (Johannesburg, South Africa). Standards were dissolved in methanol and DMSO (1 mg/ml) as per the instructions of the manufacturer and stored at -40 [degrees]C. Aliquots of 20 jxg/ml were injected into the HPLC

Phenylmethanesulfonyl fluoride (PMSF), sodium fluoride (NaF) and sodium orthovanidate ([Na sub 3][V0 sub 4]) were all obtained from Sigma-Aldrich, SA. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), polyvinylidene fluoride (PVDF) membranes and tris-buffered saline with 2% tween (TBST) were all obtained from Biorad. Antibodies from Cell Signaling were also used and protein bands on the gels were quantified using the UnscanKit program.

Rooibos and green tea preparation

Aqueous extracts were prepared by the addition of freshly boiled tap water to the tea leaves and stems of unfermented/"green" and fermented/traditional rooibos (Superior grade, generous gift from Mr. Arend Redelinghuys, Rooibos Ltd., Clanwilliam, South Africa) and green tea (commercially available in South Africa) to a final concentration of 2g/100ml, which compares well with the dose and supplementation time of the product used by Beltran-Debon et al. (2011). These concentrations are customary used for tea making purposes. The mixture was allowed to cool at room temperature for 30 min, filtered (Whatman no. 4) and dispensed into water bottles.

Antioxidant content and activity of the rooibos herbal teas and green tea

The soluble solid content of the aqueous green tea and rooibos extracts was determined gravimetrically (sixteen repetitions) during the course of the study after drying aliquots at 110 [degrees] C for 12 h. The flavanol and flavonol contents of each extract were determined spectrophotometrically at 640 nm and 360 nm, respectively (Treutter 1989; Mazza et al. 1999). The total polyphenol content of the aqueous extracts was determined using the Folin Ciocalteu method (Singleton and Rossi 1965), while the oxygen radical absorbance capacity (ORAC) was determined using a fluorometric method (Ou et al. 2001).

High-performance liquid chromatography analysis of the rooibos extracts

The tea samples were chromatographically separated using the method described by Bramati et al. (2002) with modifications. An Agilent Technologies 1200 Series HPLC system with a diode array detector and a 5 [micro] m YMC-PackPro CI 8 (150 mm x 4.6 mm i.d.) column was used for the separation. Acquisition was set at 287 and 360 nm and the mobile phases consisted of water (A) containing 300 [micro] l/l trifluoroaceticacid and methanol (B) containing 300 [micro] 1/1 trifluoroaceticacid. The gradient elution started at 95% A changing to 75% A after 5 min and to 20% A after 25 min. The flow rate was set at 0.8 ml/min, the injection volume was 20 [micro] l and the column temperature was set at 21 [degrees] C. Peaks were identified based on the retention time of the standards and confirmed by comparison of the wavelength scan spectra (set between 210 nm and 400 nm).

Animal experimental treatment

Male Wistar rats were used as experimental animals. They received humane care in accordance with the Principle of Laboratory Animal Care of the National Medical Research and the Guide for the care and use of Laboratory animals of the National Academy of Sciences (National Institutes of Health Publications no. 80-23, revised 1978). The rats had free access to either the various aqueous tea extracts or tap water for 7 weeks as their sole source of drinking fluid. The rats were fed standard rat chow (SRC) ad libitum, while the fluid intake was monitored on a daily basis. The rooibos and green tea extracts were freshly prepared every second day. The rats were housed in an experimental animal facility kept at a constant temperature of 25 [degrees] C and exposed to a twelve-hour artificial day-night cycle. Ethical clearance for this study was granted by the Faculty of Health and Wellness Sciences' Research Ethics Committee of the Cape Peninsula University of Technology (Ref: CPUT/HAS-REC2009/A001).

Male Wistar rats (120-150g) were divided into four groups fed standard rat chow (SRC) and supplemented with either tap water (Control, C), unfermented/"green" rooibos (RU), fermented/traditional rooibos (RF) or green tea (GT) for a period of seven weeks. At the end of the seven week feeding period, animals were sacrificed. Hearts (n = 10 per group) were excised and mounted on the working heart perfusion apparatus and perfused with Krebs-Henseleit buffer. Mechanical function was documented at 2 time points i.e. before ischaemia and 25 min after ischemia into reperfusion. Aortic output recovery (AO) was the functional parameter measured. Hearts were freeze clamped 10 min into reperfusion for biochemical analysis of PKB/Akt, threonine and serine residues, caspase-3 and poly (ADP-ribose) polymerase (PARP), glutathione (GSH), glutathione disulfide (CSSG) and GSH/GSSG ratio as shown in Fig. 1.

Working heart perfusion protocol

After the feeding period, rats were anaesthetized using diethyl ether. Hearts were rapidly excised, then mounted on a working heart perfusion apparatus and were perfused at a constant pressure (75 mm Hg) using a Krebs-Henseleit buffer gassed with a mixture of 5% carbon dioxide and 95% oxygen. After mounting, hearts were subjected to 10 min stabilization, followed by 15 min of normoth-ermic global ischaemia and 25 min of reperfusion. Hearts were freeze-clamped l0 min into reperfusion and stored at -80 [degrees] C for biochemical analysis.

[FIGURE 1 OMITTED]

Western blot

Heart tissue was homogenized by adding homogenization buffer and phenylmethanesulfonyl fluoride (PMSF) to the sample. For phosphoprotein determination sodium fluoride (NaF) and sodium orthovanidate ([Na sub 3][V0 sub 4]) were added to the sample where after samples were sonicated 3 times for 10 s at a time and then centrifuged at 15.000 rpm for 15min. The protein concentrations of the various homogenates were determined using the bicin-choninic acid method. Samples were diluted with Laemmli sample buffer and boiled, after which 40 fxg of protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis the proteins were transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were routinely stained with Ponceau in order to check for equal loading and sufficient transfer. Nonspecific binding was blocked by overnight incubation in 5% fat free milk in Tris-buffered saline with 2% tween (TBST). Membranes were then incubated with primary antibodies that recognize total PKB/Akt, Ser 473, Thr 389, caspase-3 and PARP. Membranes were subsequently washed and incubated with a secondary antibody. After thorough washing with TBST, membranes were covered with a chromogenic substrate (Protein Detector BCIP/NBT Western Blotting Kit. Invitrogen). A photo of the individual protein bands was captured and calculations were expressed in pixels (arbitrary units).

Glutathione analysis

Total glutathione (GSH and GSSG) was measured according to a modified method of Asensi et al. (1999). Heart samples were homogenized (1:25) in 15% (w/v) TCA containing 1 mM EDTA for GSH determination and in 6% (v/v) PCA containing freshly prepared 3 mM M2VP and 1 mM EDTA for GSSG determination on ice. After centrifugation at 1400 rpm in 4 [degrees] C for 30 s, 50 [micro] l of supernatant was added to glutathione reductase (1 U) and 75 [micro] M DTNB. The reaction was initiated by addition of 0.25 mM NADPH to a final volume of 200 [micro] l. The change in absorbance was monitored at 410 nm for 5 min and levels were calculated using pure GSH and GSSG as standards. The ratio of GSH/GSSG was also calculated using the results.

Statistical methods

Functional parameters are presented as percentage of the baseline values. Results are expressed as mean [+ or -] standard error of the mean (SEM). Differences between two groups were determined using an unpaired Students t-test and to compare differences in multiple groups a one-way ANOVA with a Bonferoni Multiple comparison as a post hoc test was used. p < 0.05 was considered to be statistically significant. Pearson correlation coefficients were calculated when two sets of data were tested for a correlation between the results of the different assays.

Results

Soluble solid content and daily intake of selected rooibos and green tea flavonoids

The soluble solids of GT were significantly (p < 0.05) higher when compared to the rooibos herbal teas (RF and RU). Rats consumed significantly (p < 0.05) more fermented/traditional rooibos (RF) (69.00 [+ or -] 14.73 ml/day) and unfermented/"green" rooibos (RU) (55.10 [+ or -] 13.06ml/day) when compared to green tea (35.10 [+ or -] 4.33ml/day). Rats that consumed the RF (67.70 [+ or -] 8.12mg/day) ingested the lowest (p < 0.05) amount of total polyphenols compared to those consuming RU (74.62 [+ or -] 3.41 mg/day) and GT (95.58 [+ or -] 7.16 mg/day). When considering the flavanol and flavonol intake, the rats receiving GT (31.47 [+ or -] 0.89 mg/day) had a significantly higher flavanol intake compared to rats consuming the RU (5.07 [+ or -] 0.14mg/day) and RF (2.67 [+ or -] 0.56 mg/day). However, the flavonol intake of the rats receiving the GT (3.81 [+ or -] 0.56 mg/day) was significantly lower compared to RU (13.61 [+ or -] 1.07 mg/day) and RF (20.65 [+ or -] 3.41 mg/day). The daily ORAC (oxygen radical absorbance capacity) unit intake were significantly higher (p < 0.05) in the GT groups when compared to the RF and RU groups as shown in Table 1.
Table 1 Daily intake profile of the rooibos and green
tea extracts.

Treatment           C         RF         RU        GT

Tea intake          74.50 [+  69.00 [+   55.10 [+   35.10 [+
(ml/day)            or           or -]      or -]      or -]
                    -]12.93    14.73-1    13.06 b     4.33 c

Soluble solids      None       0.22 [+    0.35 [+    0.63 [+
(mg/ml)                          or -]      or -]      or -]
                                0.08a       0.03b      0.05c

Polyphenol          None      67.70 [+   74.62 [+   95.58 [+
(mg/day)                            or      or -]      or -]
                               -]8.12a      3.41b      7.16C

Flavanol(mg/day)    None       2.67 [+    5.07 [+   31.47 [+
                                 or -]      or -]         or
                                 0.56a      0.14b    -]0.89c

Flavonol (mg/day)   None      20.65 [+   13.61 [+    3.81 [+
                                 or -]      or -]      or -]
                                 3.41a      1.07b      0.56c

ORAC(|xmoleTE/day)  None     210.96 [+     379.11  950.16 [+
                                    or      [+ or         or
                              -]61.41a         -]   -]72.00c

Values in the rows represent the means [+ or -] SEM
of 10 rats per group. Abbreviations: ORAC: oxygen
radical absorbance capacity (ORAC), C: control, RF:
fermented rooibos herbal tea, RU: unfermented rooibos
herbal tea and GT: green tea and. Values followed by
the different letters differ significantly (p < 0.05).


HPLC quantification of major flavonoids in the rooibos and green tea extracts

Only the major constituents of rooibos were quantified, with aspalathin being the major flavonoid in the unfermented/"green" rooibos extracts, and still one of the major flavonoids in the fermented rooibos, together with orientin and isoorientin. Trace quantities of quercetin, luteolin and chrysoeriol are also present in the fermented and unfermented rooibos extract (Table 2 and Figs. 2 and 3). Epigallocatechin gallate (EGCG) was the major flavonoid in the green tea extract while EC and ECG were also present (Table 2 and Fig. 4).
Table 2 Major rooibos and green tea flavonoids
(mg/l) in the aqueous extracts quantified by HPLC.

Tea                      RF         RU     GT

Aspalathin             31.72[+    361.84  N.D.
                            or     [+ or
                        -]3.78   -]13.62

Orientin              24.43 [+  28.65 [+  N.D.
                         or -]     or -]
                          0.33      4.63

Isoorientin           35.35 [+  44.93 [+  N.D.
                         or -]        or
                          0.49    -]1.14

Vitexin                7.30 [+   7.03 [+  N.D.
                            or     or -]
                        -]0.18      0.08

Isovitexin             7.99 [+   8.97 [+  N.D.
                            or     or -]
                        -]0.36      0.23

Hyperoside/Rutin      13.38 [+  42.34 [+  N.D.
                            or        or
                        -]0.27    -]0.28

Quercetin              2.16 [+      N.D.  N.D.
                            or
                        -]0.04

Luteolin                  0.88      N.D.  N.D.
                  [+ or -]0.01

Chrysoeriol               0.41      N.D.  N.D.
                  [+ or -]0.01

Epigallocatechin          N.D.      N.D.  156.11
gallate                                   [+ or -] 9.88

Epicatechin               N.D.      N.D.  95.69 [+ or -]
                                          5.29

Epicatechin               N.D.      N.D.  24.56
gallate                                   [+ or -] 3.01

N.D.: none detected.


[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

Function: aortic output (AO) recovery

AO recovery of hearts from the RU (61.58 [+ or -] 1.31%) and RF (61.06 [+ or -] 2.26%) supplemented groups was significantly (p < 0.05) increased when compared with the control (49.77 [+ or -] 1.83%) and GT (54.03 [+ or -] 1.50%) groups, respectively (Fig. 5).

Signaling pathway markers: protein kinase B (PKB/Akt), serine473 (Ser 473) and threonine308 (Thr 308)

There was a significant (p < 0.05) increase in phosphorylation of total PKB/Akt in GT (75.52 [+ or -] 3.07 pixels) supplemented hearts compared to all other groups, i.e. C (55.91 [+ or -] 4.01 pixels), RF (62.58 [+ or -] 2.097 pixels), and RU (55.51 [+ or -] 2.669 pixels) during repeinfusion (Fig. 6A). Fig. 6B shows that phosphorylation of Thr 308 was significantly increased in the hearts of GT (36.78 [+ or -] 3.045 pixels) supplemented group compared to the control (33.18 [+ or -] 2.045 pixels) and RF (32.36 [+ or -] 1.47pixels) groups. Phosphorylation of Ser 473 was significantly increased in the GT (43.31 [+ or -] 2.77 pixels) supplemented hearts compared to the control (36.33 [+ or -] 4.01 pixels) and RF (37.32 [+ or -] 4.68 pixels) but not the RU (41.27 [+ or -] 4.92) group (Fig. 6C).

Apoptotic proteins: poly(ADP-ribose) polymerase (PARP) and caspase-3

PARP cleavage in the RF (45.36 [+ or -] 2.67 pixels) and RU (47.82 [+ or -] 3.82 pixels) supplemented hearts is significantly (p < 0.05) lower when compared to the control (54.37 [+ or -] 3.47 pixels) and GT (54.39 [+ or -] 1.33 pixels) supplemented groups (Fig. 7A). Hearts of RF (32.56 [+ or -] 0.78 pixels) and GT (30.87 [+ or -] 1.19 pixels) supplemented groups showed a significant (p < 0.05) decrease in caspase-3 activation during reperfusion when compared with caspase-3 activation of hearts of the control group (43.80 [+ or -] 2.23 pixels). Caspase-3 activation of hearts from the RU (53.41 [+ or -] 1.32 pixels) group showed a significant (p < 0.05) increase when compared to both RF and GT supplemented groups, with the control hearts (43.80 [+ or -] 2.23 pixels) showing a significant decrease when compared to the RU supplemented hearts (Fig. 7B).

[FIGURE 6 OMITTED]

Reduced glutathione (GSH) and oxidized glutathione (GSSG) levels

The tGSH level in the GT (3.22 [+ or -] 1.08 [micro] M/g) supplemented group was significantly (p < 0.05) lower when compared to the control (7.45 [+ or -] 0.74 [micro] M/g), RF (6.82 [+ or -]2.05 [micro] M/g) and RU (8.74[+ or -]1.09 [micro] M/g) groups. Furthermore, the tGSH level in the RU supplemented group was significantly (p<0.05) higher than in the RF supplemented group (Fig. 8A). The GSSG level in the RF (0.48 [+ or -] 0.37 [micro] M/g) and GT (0.62[+ or -]0.17 [micro] M/g) supplemented groups was significantly (p < 0.05) higher when compared to the control (0.13 [+ or -] 0.08 [micro] M/g) and RU (0.373 [+ or -] 0.06 [micro] M/g) groups (Fig. 8B). The GSH/GSSG ratio in the hearts of the GT (10.58 [+ or -] 7.48) supplemented group was significantly (p < 0.05) lower when compared to that of the RF (37.78 [+ or -] 28.63), RU (33.20 [+ or -] 4.13) and control (45.50 [+ or -] 14.96) groups (Fig. 8C).

[FIGURE 7 OMITTED]

Discussion

Rooibos is a rich source of unique antioxidants due to its flavonoid content (Joubert et al. 2005). Flavonoids are known to protect against myocardial ischemia/reperfusion injury by their multifaceted properties, such as anti-inflammatory, antioxidant, vasodilatory, and antiplatelet aggregation (Akhlaghi and Band 2009). The flavonoid constituents of unfermented/"green" rooibos (RU) and fermented/"traditional" rooibos (RF) differ from those of green tea (GT). Although, the cardioprotective effect of green tea was demonstrated in other animal studies (Babu et al. 2006; Pasini et al. 2009). The data obtained in this study show, for the first time, that both RF and RU supplemented hearts are able to improve aortic output (AO) recovery when compared to the control groups with GT supplemented hearts not showing any improved AO recovery when compared to the control. The antioxidant quantitative data from the present study show that the flavonol content of RU and RF are higher when compared to that of GT, with flavanol content of GT higher than that of RU and RF. A good correlation (r= 0.9276) was also shown between the aortic output recovery and flavonol content but not the flavanol content of the extracts. It could therefore be concluded that rats consuming the RF and RU showed improved AO recovery associated with the higher intake of flavonols. This argues that in our model, rooibos flavonols are more effective as protectors against ischaemia/reperfusion injury compared to green tea flavanols. Animal studies that have investigated the cardioprotective effects of natural or synthetic flavonoids have focused mainly on the acute pharmacological activity of these compounds. For example, in vivo studies using animal models have reported on acute cardioprotection obtained from intravenous injections of natural or synthetic flavonoids (Ji et al. 2004; Wang et al. 2004). More recently, results from human studies provide evidence that rooibos can offer protection against oxidative stress conditions such as cardiovascular disease (Villano et al. 2010; Marnewick et al. 2011). To our knowledge, no investigation has been done regarding the effect of rooibos flavonols on the isolated perfused heart after an episode of ischaemia/reperfusion injury. The increased phosphorylation of protein kinase B (PKB/Akt) on serine 473 (Ser473) and threonine 308 (Thr 308) by GT, could be attributed to the fact that GT has more flavanols and a higher polyphenol content compared to RU and RF. Correlation analysis also showed a negative correlation (r= -0.814) between the flavonol content of the rooibos extracts and total PKB/Akt, This finding may suggest that the flavonol content of RF and RU had little effect on phosphorylation of PKB/Akt. Evidence is accumulating suggesting that the cellular effects of flavonoids may be mediated by their interactions with the PKB/Akt signaling pathway cascade (Schroeter et al. 2002). In addition, EGCG, the main catechin found in GT, could have an effect on phosphorylation of PKB/Akt.

[FIGURE 8 OMITTED]

Apoptosis has been consistently observed in cardiomyocytes after reperfusion and may represent a mechanism by which cardiac cells are damaged (Abe et al. 2000). Our results show that both rooibos extract (RF and RU) supplemented rat hearts significantly decreased PARP cleavage compared to the control and green tea supplemented hearts, showing a negative correlation (r= -0.9979) between the flavonol content of the aqueous extracts and the decreased PARP cleavage. This correlation could not be shown for the flavanol content of the extracts. This finding strongly suggests that the protection offered by RU and RF may be as a result of inhibition of apoptosis. It would appear under the current conditions, that the herbal tea extracts with the highest flavonol content are likely to attenuate PARP cleavage. However, our results show that GT, with the highest polyphenol content than both RU and RF, had no effect on PARP cleavage. Rat hearts supplemented with RU and RF showed a significant decrease in cleaved PARP after 10 min into reperfusion, compared to other groups. This provides circumstantial evidence to show that rooibos flavonols are highly effective in protecting against ischaemia/reperfusion injury. These results are also supported by the improved AO recovery at 25 min into reperfusion in the RU and RF supplemented groups. This finding may therefore argue that decreased PARP cleavage may be a possible mechanism of protection offered by RF and RU.

Caspase-3 is one of the key executioners of apoptosis, capable of cleaving or degrading many key proteins including nuclear lamins, fodrin as well as PARP (Lawen 2003). Recent studies have shown that quercetin, a flavonol, can prevent apoptosis by altering the expression of Bax, Bcl-2 and caspase-3 (Chao et al. 2009). The present study showed that caspase-3 cleavage in the RF group was significantly decreased when compared to the control group. This was also associated with a decrease on PARP cleavage, strongly arguing for an anti-apoptotic role for RF. Our results are also in agreement with those obtained for quercetin, which suppresses the expression of cleaved caspase-3 in the liver (Liu et al. 2010). Furthermore, our results suggested that rooibos flavonoids could alleviate ischaemia/reperfusion injury by suppressing apoptosis. In addition, the higher flavonol content of RF may contribute to the attenuation of PARP cleavage. However, in the RU group, caspase-3 cleavage was significantly increased. Since PARP cleavage in the RU group was decreased, this result needs to be further investigated. It is known that caspase-3 has a direct effect on PARP cleavage and that these two proteins should change in the same direction. Although this is not the case in the current study, it may be argued that one or more of the many components of RU could have influenced cleavage of caspase-3 differently, but further investigation is needed.

While beneficial effects of dietary rooibos flavonoids were observed ex vivo in isolated perfused rat hearts, it is unlikely that the protection might be due to a direct antioxidant effect of the circulating rooibos flavonoids or their respective metabolites. It has been suggested that flavonoids might affect endogenous antioxidant defenses of cells by modulating the glutathione and glutathione related enzyme systems (Masella et al. 2005). In the present study, the tGSH level was significantly higher in the RU and RF group compared to the GT. This may suggest that, in the present study, fiavonols have an effect on the tGSH levels. Despite the higher polyphenol, flavanol and ORAC values in the GT fed rats, tGSH levels in this group were lower when compared to RU and RF supplemented groups. In the GT supplemented group, the GSSG levels were increased in the heart, resulting in a decrease of the GSH/GSSG ratio. It is known that the GSH/GSSG ratio decreases under stress conditions due to increased GSSG levels or as a result of decreased GSH levels (Dickson and Forman 2002). Changes in the GSH and GSSG levels resulted in a significantly reduced GSH/GSSG ratio in GT fed rats compared to the control, RF and RU fed rats. Both rooibos extracts caused a significant recovery of this ratio (similar to that of the negative control) when compared to the rats consuming the green tea. The significant decrease in GSH/GSSG ratio could be associated with the poor AO recovery in GT fed rats. Green tea in the current study did not improve aortic output recovery despite the higher flavanol, polyphenol content and ORAC. Even though not significantly, RU and RF showed a higher GSH/GSSG ratio when compared to GT and comparable to that of the control group. This was also associated with improved AO recovery in the RU and RF fed hearts.

Most significant of the current findings, is that it confirms results by Beltran-Debon et al. (2011) in a hyperlipidemic mouse model. In the Beltron-Debon model it was concluded that cellular signaling pathways could be involved in the beneficial effects of rooibos. Also it is interesting to note that the daily dosage and the feeding period were similar if you closely compare the two models used. Beltran-Debon et al. (2011) used a daily dose of 10g/l in a mouse model for 14weeks and in the current study 2g/100ml/day for 7weeks. These findings may suggest that rooibos extract can be used as a therapeutic substance in oxidative stress related diseases.

In conclusion, rooibos has been shown to favorably modulate oxidative stress, the plasma lipid profile and antioxidant status (Villano et al. 2010; Marnewick et al. 2011), metabolic complications (Beltran-Debon et al. 2011) as well as inhibit the activity of angiotensin-converting enzyme with no effect on nitric oxide in humans (Persson et al. 2010). Results from the present study showed rooibos to protect against myocardial ischaemia/reperfusion injury in vivo and. taking into account previous results, it is suggested that the unique combination of rooibos flavonoid compounds have beneficial effects on vascular function. The protective effect could partially be due to the antioxidant effects of the rooibos polyphenols, in particular flavonols, and targeting specific cellular pathways such as the apoptotic pathway. However, further studies are needed to determine the vascular effects of specific rooibos compounds and/or their respective metabolites as well as to confirm that rooibos could be a potential therapeutic substance.

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W.G. Pantsi (a), J.L Marnewick (b), A.J. Esterhuyse (a), F Rautenbach (b), J. van Rooyen (a), (*)

(a.) Department of Biomedical Sciences, Faculty of Health and Wellness Sciences, Cape Peninsula University of Technology, Bellville, South Africa

(b.) Oxidative Stress Research Centre, Faculty of Health and Wellness Sciences, Cape Peninsula University of Technology, Bellville, South Africa

* Corresponding author. Tel.: + 27 21 9596523; fax: +27 21 9596874.

E-mail address: vanrooyenj@cput.ac.za (J van Rooyen).

doi:10.0l6/j.phymed.20l1.09.069
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Author:Pantsi, W.G.; Marnewick, J.L.; Esterhuyse, A.J.; Rautenbach, F.; van Rooyen, J.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:Nov 15, 2011
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