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Resveratrol, a grape- and red wine-derived polyphenolic phytoalexin, shows diverse health benefits including cardioprotection. Recent studies implicate that resveratrol displays hormetic action, protecting the cells at a lower dose while killing them at relatively higher doses. Because such hormetic behaviour may have a significant impact on epidemiological and clinical studies, the present study sought to determine dose-response curves for resveratrol action. In parallel, another resveratrol formulation was tested, namely, Longevinex (Resveratrol Partners LLC, USA). A group of rats were force-fed three different doses of resveratrol or Longevinex (2.5 mg/kg, 25 mg/kg and 100 mg/kg) for up to 30 days, while the control group was only given placebo. The results showed hormesis for pure resveratrol, which was cardioprotective at lower doses and detrimental for higher doses, but surprisingly Longevinex did not display any hormetic action. In the concentration range studied, Longevinex remained cardioprotective even at 100 mg/100 g body weight – a dose that killed 100% of the hearts when tested with pure resveratrol. To further test whether Longevinex doses are beneficial for other animal species, Longevinex was gavaged to a group of rabbits for six months, and showed exactly the same degree of cardioprotection. Cardioprotection was examined in isolated working hearts subjected to 30 min of ischemia followed by 2 h of reperfusion; left ventricular performance and infarct size was also examined. It appears that Longevinex does not show any hormetic action, while resveratrol clearly does.
Resveratrol is a unique polyphenolic antioxidant that possesses diverse health benefits from chemoprevention to cardioprotection (1,2). Resveratrol is present in significant amounts in certain fruits and vegetables including grapes, bilberries, blueberries and cranberries, as well as in peanuts and red wine (3). Resveratrol has drawn considerable attention because of its presence in red wine, which became the target of the so-called ‘French Paradox’ (4). It is generally believed that resveratrol is responsible for the cardioprotective effects related to red wine consumption (5).
Numerous studies have conclusively demonstrated the cardioprotective effects of resveratrol, but one significant fact is often neglected – the hormetic action of resveratrol. Resveratrol is a phytoalexin and many plant-derived products display hormesis (6). Hormesis is defined as a dose-response relationship that is stimulatory at low doses, but detrimental at higher doses resulting in a J-shaped or an inverted U-shaped dose-response curve. It is known that cardioprotective effects of alcohol or wine intake follow a J-shaped curve (7). An extensive literature search implicated that resveratrol present in red wine also demonstrated similar health benefits – being highly effective at lower doses and detrimental at higher doses.
The present investigation was undertaken to determine a dose-response curve for resveratrol-mediated cardioprotection and to compare this dose-response curve with another commercially available resveratrol supplement, Longevinex (Resveratrol Partners LLC, USA). The results of the study revealed that while resveratrol displayed hormetic acion, Longevinex did not.
All animals used in the present study received humane care in compliance with the regulations relating to animals and experiments involving animals, and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health [USA], 1996 edition). All the protocols were approved by the Institutional Animal Care Committee of the University of Connecticut Health Center (USA). Male Sprague-Dawley rats weighing between 250 g and 300 g were fed ad libitum regular rat chow with access to water until the start of the experimental procedure. Animals were randomly subdivided in three groups, of which the control group was gavaged with 1 mL of water containing 5% quartering and 5% hydrate, and the other two groups were gavaged with either resveratrol or Longevinex.
After completing the feeding protocol, the animals were anesthetized with sodium pentobarbital (80 mg/kg, intraperitoneally) (Abbott Laboratories, USA); heparin sodium (500 U/kg, intravenously) (Elkins-Sinn Inc, USA) was used as an anticoagulant. After deep anesthesia, the hearts were excised, the aorta was cannulated and the hearts were perfused through the aorta in Langendorff mode at a constant (100 cm of water) perfusion pressure at 37°C with Krebs-Henseleit bicarbonate for a 5 min washout period as described previously (8). The perfusion medium consisted of a modified Krebs-Henseleit bicarbonate buffer (sodium chloride 118 mmol/L, potassium chloride 4.7 mmol/L, calcium chloride 1.7 mmol/L, sodium bicarbonate 25 mmol/L, potassium dihydrogenphosphate 0.36 mmol/L, magnesium sulfate 1.2 mmol/L and glucose 10 mmol/L), and after oxygenization, pH was 7.4 at 37°C. During the washout period, the left atrium was cannulated, and the Langendorff preparation was switched to the working mode for 10 min with a left atrial filling pressure of 17 cm H2O; the aortic afterload pressure was set to 100 cm of water. At the end of 10 min, the baseline cardiac function such as heart rate (beats/min), aortic flow (AF, mL/min), coronary flow (mL/min), left ventricular developed pressure (LVDP, mmHg) and first derivative of developed pressure (LVdP/dt, mmHg/s) were recorded. Later, 30 min of global ischemia was initiated by clamping the left atrial inflow and aortic outflow lines at a point close to their origins. After 30 min, reperfusion was initiated for 120 min by unclamping the atrial inflow and aortic outflow lines. The first 10 min of reperfusion was in Langendorff mode to avoid ventricular fibrillation, and the hearts were switched to an anterograde working mode (8).
After 10 min of the working mode, baseline parameters were recorded. To monitor the recovery of the heart, the left ventricular cardiac function was recorded after 60 min and 120 min of reperfusion. A calibrated flow meter (Gilmont Instrument Inc, USA) was used to measure the AF. Coronary flow was measured by timed collection of the coronary effluent dripping from the heart. During the entire experiment, the aortic pressure was monitored using a Gould P23XL pressure transducer (Gould Instrument Systems Inc, USA) connected to the side arm of the aortic cannula; the signal was amplified using a Gould 6600 (Gould Instrument Systems Inc) series signal conditioner and data were analyzed with the CORDAT II real-time data acquisition and analysis system (Triton Technologies, USA) (10). The heart rate, LVDP and LVdP/dt were all calculated from the continuously generated pressure signal.
The infarct size was measured using the triphenyl tetrazolium chloride (TTC) staining method (9). After 2 h of reperfusion, 40 mL of 1% (weight/volume) solution of TTC in phosphate buffer was infused into the aortic cannula, and the heart samples were stored at −70°C for subsequent analysis. Sections (0.8 mm) of frozen heart were fixed in 2% paraformaldehyde, placed between two cover slips and digitally imaged using a Microtek ScanMaker 600z (Microtek, USA). To quantitate the areas of infarct in pixels, a standard National Institutes of Health image program was used. The infarct size was quantified and expressed in pixels.
Immunochemical detection of apoptotic cells was performed using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) method (9). The sections were incubated with mouse monoclonal antibody recognizing cardiac myosin heavy chain to specifically detect apoptotic cardiomyocytes. The fluorescence staining was viewed with a confocal laser microscope. The number of apoptotic cells was counted and expressed as a percentage of the total myocyte population.
The values for myocardial function parameters, infarct size and apoptosis were expressed as mean ± SEM. A one-way ANOVA was performed to test for any differences in mean values between groups. If differences were established, the values of the drug-treated groups were compared with those of the drug-free group by modified Student’s t test. P<0.05 was considered to be statistically significant.
First, the animals were treated with different doses of resveratrol (2.5 mg/kg, 25 mg/kg and 100 mg/kg) by daily gavaging for 21 days. At the end of the 21 days, the animals were sacrificed, their hearts were excised and isolated, and ischemia was induced for 30 min by terminating the coronary flow (as described in the Methods section). This was then followed by 2 h of reperfusion in the working mode; during the reperfusion, left ventricular function was monitored. As depicted in Figures 1 to to4,4, resveratrol at doses of 2.5 mg/kg and 25 mg/kg conferred cardioprotection as evidenced by improved AF, LVDP and maximum LVdP/dt. Above 25 mg/kg, ventricular function was deteriorated as evidenced by significant reductions of AF, LVDP and maximum LVdP/dt. Above 50 mg/kg (data not shown), especially at 100 mg/kg, there was no AF or developed pressure indicating that the hearts ceased functioning.
At the end of each experiment, the hearts were either subjected to TTC staining to determine infarct size or TUNEL staining to detect apoptosis. The results are shown in Figures 5 and and6.6. Resveratrol significantly reduced the myocardial infarct size and cardiomyocyte apoptosis at doses of 2.5 mg/kg and 25 mg/kg. However, above 50 mg/kg (data not shown at 50 mg/kg), the myocardial infarct size and number of apoptotic cardiomyocytes were significantly increased, indicating cellular injury.
A parallel experiment was conducted with Longevinex by gavaging the rats with three different doses of Longevinex (2.5 mg/kg, 25 mg/kg and 100 mg/kg) for up to one month. The results are shown in Figures 1 to to6.6. Unlike resveratrol, which showed hormesis, Longevinex displayed the same degree of cardioprotection up to a dose of 100 mg/kg. It is interesting to note that even at a dose as low as 25 mg/kg, Longevinex could provide the same degree of cardioprotection as depicted in the results of left ventricular function, LVDP, maximum LVdP/dt as well as infarct size and cardiomyocyte apoptosis (Table 1 and Figure 8). The dose-response curves of resveratrol (J-shaped) and Longevinex (Figure 1) clearly demonstrate that only pure resveratrol displays hormesis and not Longevinex.
Because Longevinex proved to be cardioprotective over a wide range of concentrations, it was further tested on another animal species. A group of New Zealand white rabbits were gavaged with Longevinex (100 mg/kg) for six months, while the control group was only given a placebo. After completion of the feeding protocol, isolated working rabbit hearts were subjected to 30 min of ischemia followed by 2 h of reperfusion. The results of the infarct size are shown in Figure 7. Again, the infarct size remained improved for up to six months of Longevinex feeding, and apoptosis (not shown) remained lowered for the same duration of time.
The results of the present study clearly demonstrate that resveratrol is beneficial to the heart only at low doses and detrimental at higher doses. Also, the action of resveratrol is quickly realized, in most cases within 14 days to 30 days; resveratrol does not provide any additional benefits. However, we did not study whether prolonged use of resveratrol could cause any adverse effects. Such hormetic effects have been known for more than 100 years, and frequently observed among toxins. Resveratrol is a phytoalexin, whose growth is stimulated by environmental stresses such as fungal infection, ultraviolet radiation and water deprivation (11). The cardioprotective effects of resveratrol are exerted through its ability to precondition a heart, which causes the development of intracellular stress leading to the upregulation of intracellular defense systems such as antioxidants and heat shock proteins (12). Preconditioning is another example of hormesis, which is potentiated by subjecting an organ (such as the heart) to cyclic episodes of short durations of ischemia, each followed by another short duration of reperfusion (13). Such small but therapeutic amounts of stress render the heart resistant to subsequent lethal ischemic injury. Such an adaptive response is commonly observed with aging. Consistent with this idea, resveratrol has been found to stimulate longevity genes and, at least in prokaryotic species, extends the life span (14,15). In this respect, resveratrol may fulfill the definition of a hormetin (16). There is no doubt that alcohol, wine and wine-derived resveratrol all display hormesis. It is known that cardioprotective effects of alcohol or wine intake follow a J-shaped curve (6,7), and the present study echoed this finding (Figure 7). At lower doses, resveratrol acts as an antiapoptotic agent providing cardioprotection as evidenced by increased expression in cell survival proteins, improved postischemic ventricular recovery and reduction of myocardial infarct size and cardiomyocyte apoptosis by maintaining a stable redox environment compared with control. At higher doses, however, resveratrol depresses cardiac function, elevates levels of apoptotic protein expressions, results in an unstable redox environment, and increases myocardial infarct size and the number of apoptotic cells. A significant number of reports are available in the literature to show that at a high dose, resveratrol not only hinders tumour growth but also inhibits the synthesis of RNA, DNA and protein; causes structural chromosome aberrations, chromatin breaks, chromatin exchanges, weak aneuploidy and higher S-phase arrest; blocks cell proliferation; decreases wound healing, endothelial cell growth by fibroblast growth factor-2 and vascular endothelial growth factor; and inhibits angiogenesis in healthy tissue cells leading to cell death (17).
We tested another resveratrol formulation, Longevinex, side by side; it is commercially available as a resveratrol supplement. Longevinex did not show any hormetic action up to a dose of 100 mg/kg. It should be noted that any dose of pure resveratrol over 50 mg/100 g body weight stops the heart (17). We also determined the long-term effect of Longevinex on different animal species, eg, rabbits, and found that even after six months of treatment, Longevinex provided cardioprotection. Longevinex consists of resveratrol supplemented with rice bran phytate, ferulic acid and quercetin. Ferulic acid and quercetin are well-known antioxidants found in most fruits and vegetables, while phytate is present in rice bran and wheat and possesses iron-chelating properties. It is micronized to increase bioavailability. The results found in the present study are important for scientists, clinicians and the nonmedical community because it highlights the significance of using pure resveratrol only at lower doses; opposite effects can occur at higher doses, resulting in adverse effects on health. Epidemiological and clinical trials need to be based on the clear understanding of hormetic responses of resveratrol.
This study was supported by a grant from Resveratrol Partners LLC and OTKA 78223.