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DOI 10.1002/mnfr.201300774
Mol. Nutr. Food Res. 2014, 58, 1374–1378
FOOD & FUNCTION
Hibiscus sabdariffa extract lowers blood pressure and improves endothelial function 1 ´ Jorge Joven1 , Isabel March2 , Eugenia Espinel3 , Salvador Fernandez-Arroyo , 1 1 1 ` , Raul ´ Beltran-Deb ´ ´ , on Esther Rodr´ıguez-Gallego , Gerard Aragones Carlos Alonso-Villaverde1 , Lidia Rios2 , Vicente Martin-Paredero1 , Javier A. Menendez4 , Vicente Micol5 , Antonio Segura-Carretero6 and Jordi Camps1 1
` ` Unitat de Recerca Biomedica, Hospital Universitari Sant Joan, Institut d’Investigacio´ Sanitaria Pere Virgili, Universitat Rovira i Virgili, Campus of International Excellence Southern Catalonia, Reus, Spain 2 Hospital lleuger de Cambrils, Tarragona, Spain 3 Department of nephrology, Hospital Universitari de la vall d’Hebron 4 Catalan Institute of Oncology and Girona Biomedical Research Institute, Girona, Spain 5 ´ Instituto de Biolog´ıa Molecular y Celular, Universidad Miguel Hernandez, Alicante, Spain 6 Department of Analytical Chemistry, Faculty of Sciences, University of Granada, Granada, Spain Polyphenols from Hibiscus sabdariffa calices were administered to patients with metabolic syndrome (125 mg/kg/day for 4 wk, n = 31) and spontaneously hypertensive rats (125 or 60 mg/kg in a single dose or daily for 1 wk, n = 8 for each experimental group). The H. sabdariffa extract improved metabolism, displayed potent anti-inflammatory and antioxidant activities, and significantly reduced blood pressure in both humans and rats. Diuresis and inhibition of the angiotensin I-converting enzyme were found to be less important mechanisms than those related to the antioxidant, anti-inflammatory, and endothelium-dependent effects to explain the beneficial actions. Notably, polyphenols induced a favorable endothelial response that should be considered in the management of metabolic cardiovascular risks.
Received: October 17, 2013 Revised: November 29, 2013 Accepted: January 13, 2014
Keywords: Diuresis / Hypertension / Inflammation / Oxidative stress / Phytochemicals
Additional supporting information may be found in the online version of this article at the publisher’s web-site
The effects of different nutrients on metabolic syndrome are not completely understood [1, 2]. Dietary management of hypertension, a key component, may prevent the onset of hypertensive events (systolic blood pressure (SBP) ≥ 135–140 mm Hg; diastolic blood pressure (DBP) ≥ 85–90 mm Hg) and improve or eradicate mild hypertension [3, 4]. However, adherence to diets is usually not sustained, mainly because to receive high amounts of bioactive components from foods,
` Correspondence: Dr. Jorge Joven, Unitat de Recerca Biomedica, ` Hospital Universitari Sant Joan, Institut d’Investigacio´ Sanitaria Pere Virgili, Universitat Rovira i Virgili, Reus, Spain, carrer Sant Llorenc¸ 21, 43201 Reus, Spain E-mail: [email protected]; [email protected] Abbreviations: ACE, angiotensin converting enzyme; BP, blood pressure; DBP, diastolic blood pressure; eNOS, endothelial nitric oxide synthase; HS, Hibiscus sabdariffa; SBP, systolic blood pressure; SHR, spontaneously hypertensive rats; XO, xanthine oxidase; WKY, Wistar Kyoto
a large quantity must be ingested, which can be unpleasant, harmful, obesogenic, or stimulatory [5–7]. An alternative to consumption is providing botanical extracts. Based on knowledge acquired for polyphenols from Hibiscus sabdariffa (HS) calices [8–12], we present here a proof of concept demonstrating the effectiveness of these components in hypertension management in patients with metabolic syndrome and in spontaneously hypertensive rats (SHR). Detailed material and methods and content in polyphenols of HS extract (Monteloeder S.L., Spain) may be obtained in Supporting Information Material and Methods and Supporting Information Table 1. We recruited patients with metabolic syndrome [13] and data were collected from clinical records or obtained using standardized guidelines and routine laboratory methods [14] as assessed by the Ethics Committee at the Hospital Universitari de Sant Joan de Reus (EPINOLS, 1203-29/3proj6). Participants provided full informed consent. HS extract (125 mg/kg/day) was fractionated and given twice (morning and evening) during a 4-wk intervention period
C 2014 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.mnf-journal.com This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
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Table 1. Differences in selected variables at baseline and after the intervention period
Baseline Metabolic variables Glucose (mmol/L) Insulin (pmol/L) Triglycerides (mmol/L) Total-cholesterol (mmol/L) HDL-cholesterol (mmol/L) Apolipoprotein B (g/L) Apolipoprotein A-I (g/L) AST (kat/L) ALT (kat/L) ␥−GT (kat/L) Inflammatory variables 2-microglobulin (mg/L) hsCRP (mg/L) TNF-␣ (pg/mL) IL-6 (pg/mL) IL-1 (pg/mL) IL-8 (pg/mL) CCL2 (pg/mL) Other selected variables Endothelin-1 (pg/mL) Adiponectin (ng/mL)a) Leptin (pmol/L)a) 8-Isoprostane-F2␣ (pg/mL)a) Paraoxonase activity (U/L)a) Total peroxides (mol/L)a) Renin (ng/mL h)a) Aldosterone (pmol/L) ACE (U/L)
Intervention
p value
5.2 (3.9−7.3) 71.5 (29.5−248.8) 1.1 (0.5−4.6) 5.9 (4.4−7.8) 1.07 (0.7−1.4) 1.4 (0.9−2.0) 1.5 (1.2−2.0) 0.37 (0.26−0.81) 0.38 (0.23−1.4) 0.39 (0.14−2.9)
5.2 (4.0−7.1) 72.1 (11.9−205.6) 1.2 (0.5−5.3) 5.5 (4.5−6.6) 1.10 (0.7−1.7) 1.4 (0.9−1.9) 1.5 (1.1−2.1) 0.37 (0.25−0.63) 0.37 (0.15−2.1) 0.37 (0.15−2.2)
0.647 0.443 0.372 0.020 0.040 0.210 0.327 0.857 0.279 0.142
2.0 (1.5−2.8) 2.6 (0.23−13.1) 15.8 (0.1−46.5) 5.1 (1.1−11.6) 3.1 (0.1−16.2) 36.7 (0.1−126.4) 185.4 (60.2−232.5)
1.9 (1.7−3.1) 2.4 (0.31−11.2) 12.7 (0.1−32.1) 2.6 (1.8−5.2) 1.7 (0.1−8.1) 10.2 (0.1−19.7) 96.5 (65.1−143.7)
0.532 0.864 0.050 0.049 0.043 0.031 0.042
3.7 (0.3−6.2) 7221 (4100) 1250 (1114) 64.5 (40.2) 290.5 (145.6) 102.4 (36.5) 3.9 (1.7−5.2) 542 (340−900) 40.1 (18−63)
2.6 (0.3−4.1) 10 624 (5567) 298 (312) 21.8 (16.5) 362.9 (125.2) 85.7 (34.2) 3.5 (1.2−4.6) 390 (270−625) 32.5 (15−48)
0.125 0.039 0.041 0.019 0.049 0.057 0.513 0.297 0.112
ACE, angiotensin converting enzyme; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CCL2, C-C-chemokine ligand 2; GGT, gamma-glutamyltransferase; hsCRP, high-sensitivity C-reactive protein. a) Values are the geometric mean (95% CI) or median (interquartile range) for n = 31 participants.
without interfering with the patient’s lifestyle and medication [10–12]. We limited treatment to a 4-wk period to assess safety and conditions of use. We used 24-h ambulatory blood pressure oscillometric monitoring for blood pressure (BP) measurements [15]. The baseline characteristics of included patients displayed the expected heterogeneity (Supporting Information Table 2). As expected [16], most of the inflammatory variables were decreased during the intervention period. We also found a significant increase in serum adiponectin and a significant decrease in serum leptin, likely indicating improvement in overweight-induced metabolic alterations (Table 1). The HS extract significantly reduced BP after the intervention period in the form of 24-h SBP (a change of −11.0 ± 6.3 mmHg; p < 0.001 versus baseline) and 24-h DBP (−4.2 ± 1.9 mmHg; p < 0.001 versus baseline) Supporting Information Table 3. The SBP and DBP reductions only occurred during the daytime (−12.7 ± 4.3 and −3.7 ± 1.1 mmHg, respectively; p < 0.001 for both measurements). Similarly, the percentage of patients with relatively high SBP and/or DBP decreased over the course of the HS treatment. It is therefore possible that this treatment may provide BP control throughout the night and may cover the early morning period when
the BP surges. Of note, the heart rate was significantly (p = 0.012) decreased after the intervention period (70.5 ± 11.5 beats/min) with respect to the baseline value (76.3 ± 11.8 beats/min). We found that DBP decreased significantly in 18 of 31 patients (58%) and SBP in 20 of 31 patients (64.5%). The condition of responder was associated with a higher baseline SBP (p = 0.0005), a higher baseline DBP (p = 0.0002), a greater decrease in heart rate (p = 0.001), a higher decrease in circulating 8-isoprostane-F2␣ (p = 0.042) and higher baseline values for some inflammatory biomarkers (CCL2, p = 0.031; IL-6, p = 0.022; IL-1, p = 0.0011; IL-8, p = 0.011). The antioxidant effect of the extract in humans is clearly indicated by the decreased levels of circulating 8-isoprostaneF2␣ (p = 0.019) and increased serum paraoxonase activity (p = 0.049) [17] (Table 1). In vitro assays indicated a significant effect as peroxyl radical scavenger (Supporting Information Table 4) and inhibitor of xanthine oxidase and 15lipoxygenase (IC50 ± SD in g/mL, 3.1 ± 0.1 and 35.2 ± 0.5, respectively). Serum sodium, potassium, and chloride concentrations did not significantly vary between the beginning and end of the intervention period and there were no significant changes
C 2014 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Figure 1. The effects of a single oral administration or a daily treatment for 1 wk of saline or a HS extract (60 mg/kg/day, H60, or 125 mg/kg/day, H125) on BP in SHR and their corresponding normotensive controls (WKY). *p < 0.05 when compared to the control (saline) group (n = 8 for each group).
in plasma aldosterone, renin, and angiotensin converting enzyme (ACE) concentrations (Table 1). Some values may indicate that aldosterone per se has a role in hypertension but patients with primary aldosteronism, resistant hypertension, or hypokalemia were not invited to participate to avoid inaccuracies in the diagnosis of metabolic syndrome. The aqueous HS extract slightly inhibited ACE activity in vitro, IC50 of 34.9 g/mL. Therefore, this mechanism and an effect on diuresis are unlikely. Our primary and most consistent finding is that the consumption of polyphenol-rich HS by humans with metabolic syndrome decreased daytime BP and heart rate, reduced lipid disturbances, and improved oxidative and inflammatory stresses. All procedures in SHR and experimental protocols [18,19] were examined and approved by the Ethics Review Committee for Animal Experimentation of the Universitat Rovira i Virgili (URV, 235/6780). The SBPs, DBPs, and the heart rate were monitored by the tail-cuff plethysmographic method. The extract was tested after a single administration and af-
ter daily administration for 1 wk (Fig. 1). The initial SBPs and DBPs among the individual SHR were similar (162 ± 3 and 91 ± 7 mmHg, respectively). The same uniformity was found among the Wistar Kyoto (WKY) rats (130 ± 2 and 72 ± 3 mmHg, respectively). The extract’s effects were only observed in SHR. After a single oral administration, the hypertensive rats displayed significantly lower DBP and SBP at the 4- and 6-h time points, but at the end of the day, those values were restored almost to baseline (Fig. 1). During the 7-day administration, the HS extract significantly decreased the BP; the effects were more intense in the DBP, reaching a peak 25% decrease (22–23 mmHg) between days 4 and 5. In a separate experiment, we included groups treated with saline (control), furosemide (6 mg/kg) and two different doses of the HS extract to explore whether the HS extract is diuretic in rats. In a 24-h experiment, the urinary volume of rats treated with the HS extract was significantly increased with respect to the control. Curiously, values were similar in both
Figure 2. In endothelial cells cultured without TNF-␣, incubation with 40 g/mL HS extract (HS40) increased the production of the eNOS protein (A), eNOS’s mRNA expression (B), and the production of NO (C). The addition of L-NAME, an eNOS inhibitor, nearly eliminated NO production, and the effects of indomethacin, a cyclooxygenase inhibitor, were not significant (C). *p < 0.05 compared to the control. Values represent results obtained in triplicate from at least four experiments. C 2014 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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the normotensive and hypertensive rats. The urinary electrolytes did not change in the 24-h experiment. However, in a 6-h experiment performed in hypertensive rats, the electrolyte levels did vary (Supporting Information Table 5). In endothelial cells, tumor necrosis factor ␣ (TNF-␣) significantly induced the production and/or secretion of cytokines (Supporting Information Fig. 1A). Pretreating the cells with different concentrations of the HS extract (20– 40 g/mL) significantly reduced (by 40–80%) but did not abolish the TNF-␣-induced cytokine production (Supporting Information Fig. 1B). This effect was most likely exerted transcriptionally because the relative mRNA levels (normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as constitutive gen) also displayed a similar decreased expression (Supporting Information Fig. 1C). TNF-␣ also promoted the expression of E-selectin at the transcription level (132 ± 25fold increase). This expression increase was partially but significantly (p = 0.047) inhibited by HS (40 g/mL) pretreatment (to only 73 ± 10-fold). The HS extract significantly reduced the amount of TNF␣-induced nuclear factor kappa light chain enhancer of activated B cells [NF-B (p65)] (Supporting Information Fig. 1D), and the reactive oxygen species production (Supporting Information Fig. 1E), indicating that HS treatment plays a role in reducing inflammatory and oxidative conditions. In the absence of TNF-␣, the HS extract (40 g/mL) significantly induced in these cells the production of endothelial nitric oxide synthase (eNOS) at the protein and mRNA levels and these effects were accompanied by a significant increase in nitric oxide (NO) production (Fig. 2). To further ascertain the mechanism leading to such an increase in NO production, we performed the test in the presence of a NO synthase inhibitor (L-NAME, 80 M), which almost entirely suppressed NO production. Conversely, the effect of cyclooxygenase inhibitor indomethacin (10 M) was not significant. Therefore, endothelial dysfunction may be nutritionally alleviated. This study provides a research framework for assessing the effects of HS polyphenols on conditions associated with hypertension and the multifaceted metabolic syndrome [20], combining human and animal studies with ex vivo and in vitro experiments. There are several mechanisms involved. Diuresis, or inhibition of ACE activity are probably not important mechanisms of HS’ hypotensive effect. Rather, we interpret our findings as proof of the beneficial effects of polyphenols on free radicals and immune modulation. It is likely that these actions could improve cardiovascular health. Our ex vivo data also contribute to this conclusion and add a beneficial endothelial response, probably vasodilation, to a better understanding of the possible mechanisms of action. Taken together, our results suggest that a multifaceted and likely synergistic mechanism accounts for the hypotensive action of polyphenol-rich HS, which acts through improved endothelium-dependent response. The Unitat de Recerca Biom`edica is currently being supported by the program of consolidated groups from the Universitat Rovira
i Virgili and grants from the Fondo de Investigaci´on Sanitaria (FIS PI08/1032, PI08/1381 and PI11/00130). This and other laboratories involved in this study performed experiments in network under the bioactive food components platform, which share know-how, equipment and financial support (IDI-20120741, CIBER CB12/03/30038 y PROMETEO/2012/007). The authors have declared no conflict of interest.
References [1] Savica, V., Bellinghieri, G., Kopple, J. D., The effect of nutrition on blood pressure. Annu. Rev. Nutr. 2010, 30, 365–401. [2] Joven, J., Micol, V., Segura-Carretero, A., Alonso-Villaverde, C. et al., Polyphenols and the modulation of gene expresssion pathways: can we eat our way out of the danger of chronic disease? Crit. Rev. Food Sci. Nutr. 2014, 54, 985–1001. [3] Vollmer, W. M., Sacks, F. M., Ard, J., Appel, L. J. et al., Effects of diet and sodium intake on blood pressure: subgroup analysis of the DASH-Sodium Trial. Ann. Intern. Med. 2001, 135, 1019–1028. [4] Lien, L. F., Brown, A. J., Ard, J. D., Loria, C. et al., Effects of PREMIER lifestyle modifications on participants with and without the metabolic syndrome. Hypertension 2007, 50, 609–616. [5] Ried, K., Sullivan, T. R., Fakler, P., Frank O. R., Stocks N. P., Effect of cocoa on blood pressure. Cochrane Database Syst. Rev. 8, 2012, CD008893. [6] Hodgson, J. M., Puddey, I. B., Woodman, R. J., Mulder, T. P. et al., Effects of black tea on blood pressure: a randomized controlled trial. Arch. Intern. Med. 2012, 172, 186–188. ˜ [7] Rubio-Ruiz, M. E., El Hafidi, M., Perez-Torres, I., Banos, G. et al., Medicinal agents and metabolic syndrome. Curr. Med. Chem. 2013, 20, 2626–2640. ´ ´ ´ ´ R., [8] Fernandez-Arroyo, S., Herranz-Lopez, M., Beltran-Deb on, ´ Borras-Linares, I. et al., Bioavailability study of a polyphenolenriched extract from Hibiscus Sabdariffa in rats and associated antioxidant status. Mol. Nutr. Food Res. 2012, 56, 1590-1595. ` G., Rodr´ıguez[9] Joven, J., Espinel, E., Rull, A., Aragones, Gallego, E. et al., Plant-derived polyphenols regulate expression of miRNA paralogs miR-103/107 and miR-122 and prevent diet-induced fatty liver disease in hyperlipidemic mice. Biochim. Biophys. Acta 2012, 1820, 894–899. ´ ´ ´ [10] Herranz-Lopez, M., Fernandez-Arroyo, S., Perez-Sanchez, ´ ´ E. et al., Synergism of plant-derived A., Barrajon-Catal an, polyphenols in adipogenesis: perspectives and implications. Phytomedicine 2012, 19, 253–261. [11] Segura-Carretero, A., Puertas-Mej´ıa, M. A., Cortacero´ R. et al., Selective extraction, separaRam´ırez, S., Beltran, tion and identification of anthocyanins from Hibiscus sabdariffa L. Using solid phase extraction-capillary electrophoresismass spectrometry (time-of-flight/ion trap). Electrophoresis 2008, 29, 2852–2861.
C 2014 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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´ ´ ´ [12] Fernandez-Arroyo, S., Rodr´ıguez-Medina, I., Beltran-Deb on, R., Pasini, F. et al., Quantification of the polyphenolic fraction and in vitro antioxidant and in vivo anti-hyperlipemic activities of Hibiscus sabdariffa aqueous extract. Food Res. Int. 2011, 44, 1490–1495. [13] Grundy, S. M., Brewer, H. B., Jr., Cleeman, J. I., Smith, S. C., Jr. et al., Definition of metabolic syndrome: report of the National Heart, Lung, and Blood Institute/American Heart Association conference on scientific issues related to definition. Circulation 2004, 109, 433–438. ´ J. M., Castellano, I., Ferre, ´ N., Joven, J. et al., Evalu[14] Simo, ation of a homogeneous assay for high-density lipoprotein cholesterol: limitations in patients with cardiovascular, renal, and hepatic disorders. Clin. Chem. 1998, 10, 1233–1241. [15] Mancia, G., Omboni, S., Parati, G., Ravogli, A. et al., Lack of placebo effect on ambulatory blood pressure. Am. J. Hypertens. 1995, 8, 311–315. ´ ´ ` [16] Beltran-Deb on, R., Alonso-Villaverde, C., Aragones, G.,
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Rodr´ıguez-Medina, I. et al., The aqueous extract of Hibiscus sabdariffa calices modulates the production of monocyte chemoattractant protein-1 in humans. Phytomedicine 2010, 17, 186–191. ´ N., Camps, J., Prats, E., Vilella, E. et al., Serum paraox[17] Ferre, onase activity: a new additional test for the improved evaluation of chronic liver damage. Clin. Chem. 2002, 48, 261–268. ´ N., Escola-Gil, ` [18] Joven, J., Rull, A., Ferre, J. C. et al., The results in rodent models of atherosclerosis are not interchangeable: the influence of diet and strain. Atherosclerosis 2007, 195, e85–e92. ´ [19] Cadiz-Gurrea, M., Fernandez-Arroyo, S., Joven, J., SeguraCarretero, A., Comprehensive characterization by UHPLCESI-Q-TOF-MS from an Eryngium bourgatii extract and their antioxidant and anti-inflammatory activities. Food Res. Int. 2013, 50, 197–204. [20] Das, U. N., Is metabolic syndrome X an inflammatory condition? Exp. Biol. Med. 2002, 227, 989–997.
C 2014 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.mnf-journal.com
DOI 10.1002/mnfr.201300774
Mol. Nutr. Food Res. 2014, 58, 1374–1378
FOOD & FUNCTION
Hibiscus sabdariffa extract lowers blood pressure and improves endothelial function 1 ´ Jorge Joven1 , Isabel March2 , Eugenia Espinel3 , Salvador Fernandez-Arroyo , 1 1 1 ` , Raul ´ Beltran-Deb ´ ´ , on Esther Rodr´ıguez-Gallego , Gerard Aragones Carlos Alonso-Villaverde1 , Lidia Rios2 , Vicente Martin-Paredero1 , Javier A. Menendez4 , Vicente Micol5 , Antonio Segura-Carretero6 and Jordi Camps1 1
` ` Unitat de Recerca Biomedica, Hospital Universitari Sant Joan, Institut d’Investigacio´ Sanitaria Pere Virgili, Universitat Rovira i Virgili, Campus of International Excellence Southern Catalonia, Reus, Spain 2 Hospital lleuger de Cambrils, Tarragona, Spain 3 Department of nephrology, Hospital Universitari de la vall d’Hebron 4 Catalan Institute of Oncology and Girona Biomedical Research Institute, Girona, Spain 5 ´ Instituto de Biolog´ıa Molecular y Celular, Universidad Miguel Hernandez, Alicante, Spain 6 Department of Analytical Chemistry, Faculty of Sciences, University of Granada, Granada, Spain Polyphenols from Hibiscus sabdariffa calices were administered to patients with metabolic syndrome (125 mg/kg/day for 4 wk, n = 31) and spontaneously hypertensive rats (125 or 60 mg/kg in a single dose or daily for 1 wk, n = 8 for each experimental group). The H. sabdariffa extract improved metabolism, displayed potent anti-inflammatory and antioxidant activities, and significantly reduced blood pressure in both humans and rats. Diuresis and inhibition of the angiotensin I-converting enzyme were found to be less important mechanisms than those related to the antioxidant, anti-inflammatory, and endothelium-dependent effects to explain the beneficial actions. Notably, polyphenols induced a favorable endothelial response that should be considered in the management of metabolic cardiovascular risks.
Received: October 17, 2013 Revised: November 29, 2013 Accepted: January 13, 2014
Keywords: Diuresis / Hypertension / Inflammation / Oxidative stress / Phytochemicals
Additional supporting information may be found in the online version of this article at the publisher’s web-site
The effects of different nutrients on metabolic syndrome are not completely understood [1, 2]. Dietary management of hypertension, a key component, may prevent the onset of hypertensive events (systolic blood pressure (SBP) ≥ 135–140 mm Hg; diastolic blood pressure (DBP) ≥ 85–90 mm Hg) and improve or eradicate mild hypertension [3, 4]. However, adherence to diets is usually not sustained, mainly because to receive high amounts of bioactive components from foods,
` Correspondence: Dr. Jorge Joven, Unitat de Recerca Biomedica, ` Hospital Universitari Sant Joan, Institut d’Investigacio´ Sanitaria Pere Virgili, Universitat Rovira i Virgili, Reus, Spain, carrer Sant Llorenc¸ 21, 43201 Reus, Spain E-mail: [email protected]; [email protected] Abbreviations: ACE, angiotensin converting enzyme; BP, blood pressure; DBP, diastolic blood pressure; eNOS, endothelial nitric oxide synthase; HS, Hibiscus sabdariffa; SBP, systolic blood pressure; SHR, spontaneously hypertensive rats; XO, xanthine oxidase; WKY, Wistar Kyoto
a large quantity must be ingested, which can be unpleasant, harmful, obesogenic, or stimulatory [5–7]. An alternative to consumption is providing botanical extracts. Based on knowledge acquired for polyphenols from Hibiscus sabdariffa (HS) calices [8–12], we present here a proof of concept demonstrating the effectiveness of these components in hypertension management in patients with metabolic syndrome and in spontaneously hypertensive rats (SHR). Detailed material and methods and content in polyphenols of HS extract (Monteloeder S.L., Spain) may be obtained in Supporting Information Material and Methods and Supporting Information Table 1. We recruited patients with metabolic syndrome [13] and data were collected from clinical records or obtained using standardized guidelines and routine laboratory methods [14] as assessed by the Ethics Committee at the Hospital Universitari de Sant Joan de Reus (EPINOLS, 1203-29/3proj6). Participants provided full informed consent. HS extract (125 mg/kg/day) was fractionated and given twice (morning and evening) during a 4-wk intervention period
C 2014 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.mnf-journal.com This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
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Mol. Nutr. Food Res. 2014, 58, 1374–1378
Table 1. Differences in selected variables at baseline and after the intervention period
Baseline Metabolic variables Glucose (mmol/L) Insulin (pmol/L) Triglycerides (mmol/L) Total-cholesterol (mmol/L) HDL-cholesterol (mmol/L) Apolipoprotein B (g/L) Apolipoprotein A-I (g/L) AST (kat/L) ALT (kat/L) ␥−GT (kat/L) Inflammatory variables 2-microglobulin (mg/L) hsCRP (mg/L) TNF-␣ (pg/mL) IL-6 (pg/mL) IL-1 (pg/mL) IL-8 (pg/mL) CCL2 (pg/mL) Other selected variables Endothelin-1 (pg/mL) Adiponectin (ng/mL)a) Leptin (pmol/L)a) 8-Isoprostane-F2␣ (pg/mL)a) Paraoxonase activity (U/L)a) Total peroxides (mol/L)a) Renin (ng/mL h)a) Aldosterone (pmol/L) ACE (U/L)
Intervention
p value
5.2 (3.9−7.3) 71.5 (29.5−248.8) 1.1 (0.5−4.6) 5.9 (4.4−7.8) 1.07 (0.7−1.4) 1.4 (0.9−2.0) 1.5 (1.2−2.0) 0.37 (0.26−0.81) 0.38 (0.23−1.4) 0.39 (0.14−2.9)
5.2 (4.0−7.1) 72.1 (11.9−205.6) 1.2 (0.5−5.3) 5.5 (4.5−6.6) 1.10 (0.7−1.7) 1.4 (0.9−1.9) 1.5 (1.1−2.1) 0.37 (0.25−0.63) 0.37 (0.15−2.1) 0.37 (0.15−2.2)
0.647 0.443 0.372 0.020 0.040 0.210 0.327 0.857 0.279 0.142
2.0 (1.5−2.8) 2.6 (0.23−13.1) 15.8 (0.1−46.5) 5.1 (1.1−11.6) 3.1 (0.1−16.2) 36.7 (0.1−126.4) 185.4 (60.2−232.5)
1.9 (1.7−3.1) 2.4 (0.31−11.2) 12.7 (0.1−32.1) 2.6 (1.8−5.2) 1.7 (0.1−8.1) 10.2 (0.1−19.7) 96.5 (65.1−143.7)
0.532 0.864 0.050 0.049 0.043 0.031 0.042
3.7 (0.3−6.2) 7221 (4100) 1250 (1114) 64.5 (40.2) 290.5 (145.6) 102.4 (36.5) 3.9 (1.7−5.2) 542 (340−900) 40.1 (18−63)
2.6 (0.3−4.1) 10 624 (5567) 298 (312) 21.8 (16.5) 362.9 (125.2) 85.7 (34.2) 3.5 (1.2−4.6) 390 (270−625) 32.5 (15−48)
0.125 0.039 0.041 0.019 0.049 0.057 0.513 0.297 0.112
ACE, angiotensin converting enzyme; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CCL2, C-C-chemokine ligand 2; GGT, gamma-glutamyltransferase; hsCRP, high-sensitivity C-reactive protein. a) Values are the geometric mean (95% CI) or median (interquartile range) for n = 31 participants.
without interfering with the patient’s lifestyle and medication [10–12]. We limited treatment to a 4-wk period to assess safety and conditions of use. We used 24-h ambulatory blood pressure oscillometric monitoring for blood pressure (BP) measurements [15]. The baseline characteristics of included patients displayed the expected heterogeneity (Supporting Information Table 2). As expected [16], most of the inflammatory variables were decreased during the intervention period. We also found a significant increase in serum adiponectin and a significant decrease in serum leptin, likely indicating improvement in overweight-induced metabolic alterations (Table 1). The HS extract significantly reduced BP after the intervention period in the form of 24-h SBP (a change of −11.0 ± 6.3 mmHg; p < 0.001 versus baseline) and 24-h DBP (−4.2 ± 1.9 mmHg; p < 0.001 versus baseline) Supporting Information Table 3. The SBP and DBP reductions only occurred during the daytime (−12.7 ± 4.3 and −3.7 ± 1.1 mmHg, respectively; p < 0.001 for both measurements). Similarly, the percentage of patients with relatively high SBP and/or DBP decreased over the course of the HS treatment. It is therefore possible that this treatment may provide BP control throughout the night and may cover the early morning period when
the BP surges. Of note, the heart rate was significantly (p = 0.012) decreased after the intervention period (70.5 ± 11.5 beats/min) with respect to the baseline value (76.3 ± 11.8 beats/min). We found that DBP decreased significantly in 18 of 31 patients (58%) and SBP in 20 of 31 patients (64.5%). The condition of responder was associated with a higher baseline SBP (p = 0.0005), a higher baseline DBP (p = 0.0002), a greater decrease in heart rate (p = 0.001), a higher decrease in circulating 8-isoprostane-F2␣ (p = 0.042) and higher baseline values for some inflammatory biomarkers (CCL2, p = 0.031; IL-6, p = 0.022; IL-1, p = 0.0011; IL-8, p = 0.011). The antioxidant effect of the extract in humans is clearly indicated by the decreased levels of circulating 8-isoprostaneF2␣ (p = 0.019) and increased serum paraoxonase activity (p = 0.049) [17] (Table 1). In vitro assays indicated a significant effect as peroxyl radical scavenger (Supporting Information Table 4) and inhibitor of xanthine oxidase and 15lipoxygenase (IC50 ± SD in g/mL, 3.1 ± 0.1 and 35.2 ± 0.5, respectively). Serum sodium, potassium, and chloride concentrations did not significantly vary between the beginning and end of the intervention period and there were no significant changes
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Figure 1. The effects of a single oral administration or a daily treatment for 1 wk of saline or a HS extract (60 mg/kg/day, H60, or 125 mg/kg/day, H125) on BP in SHR and their corresponding normotensive controls (WKY). *p < 0.05 when compared to the control (saline) group (n = 8 for each group).
in plasma aldosterone, renin, and angiotensin converting enzyme (ACE) concentrations (Table 1). Some values may indicate that aldosterone per se has a role in hypertension but patients with primary aldosteronism, resistant hypertension, or hypokalemia were not invited to participate to avoid inaccuracies in the diagnosis of metabolic syndrome. The aqueous HS extract slightly inhibited ACE activity in vitro, IC50 of 34.9 g/mL. Therefore, this mechanism and an effect on diuresis are unlikely. Our primary and most consistent finding is that the consumption of polyphenol-rich HS by humans with metabolic syndrome decreased daytime BP and heart rate, reduced lipid disturbances, and improved oxidative and inflammatory stresses. All procedures in SHR and experimental protocols [18,19] were examined and approved by the Ethics Review Committee for Animal Experimentation of the Universitat Rovira i Virgili (URV, 235/6780). The SBPs, DBPs, and the heart rate were monitored by the tail-cuff plethysmographic method. The extract was tested after a single administration and af-
ter daily administration for 1 wk (Fig. 1). The initial SBPs and DBPs among the individual SHR were similar (162 ± 3 and 91 ± 7 mmHg, respectively). The same uniformity was found among the Wistar Kyoto (WKY) rats (130 ± 2 and 72 ± 3 mmHg, respectively). The extract’s effects were only observed in SHR. After a single oral administration, the hypertensive rats displayed significantly lower DBP and SBP at the 4- and 6-h time points, but at the end of the day, those values were restored almost to baseline (Fig. 1). During the 7-day administration, the HS extract significantly decreased the BP; the effects were more intense in the DBP, reaching a peak 25% decrease (22–23 mmHg) between days 4 and 5. In a separate experiment, we included groups treated with saline (control), furosemide (6 mg/kg) and two different doses of the HS extract to explore whether the HS extract is diuretic in rats. In a 24-h experiment, the urinary volume of rats treated with the HS extract was significantly increased with respect to the control. Curiously, values were similar in both
Figure 2. In endothelial cells cultured without TNF-␣, incubation with 40 g/mL HS extract (HS40) increased the production of the eNOS protein (A), eNOS’s mRNA expression (B), and the production of NO (C). The addition of L-NAME, an eNOS inhibitor, nearly eliminated NO production, and the effects of indomethacin, a cyclooxygenase inhibitor, were not significant (C). *p < 0.05 compared to the control. Values represent results obtained in triplicate from at least four experiments. C 2014 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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the normotensive and hypertensive rats. The urinary electrolytes did not change in the 24-h experiment. However, in a 6-h experiment performed in hypertensive rats, the electrolyte levels did vary (Supporting Information Table 5). In endothelial cells, tumor necrosis factor ␣ (TNF-␣) significantly induced the production and/or secretion of cytokines (Supporting Information Fig. 1A). Pretreating the cells with different concentrations of the HS extract (20– 40 g/mL) significantly reduced (by 40–80%) but did not abolish the TNF-␣-induced cytokine production (Supporting Information Fig. 1B). This effect was most likely exerted transcriptionally because the relative mRNA levels (normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as constitutive gen) also displayed a similar decreased expression (Supporting Information Fig. 1C). TNF-␣ also promoted the expression of E-selectin at the transcription level (132 ± 25fold increase). This expression increase was partially but significantly (p = 0.047) inhibited by HS (40 g/mL) pretreatment (to only 73 ± 10-fold). The HS extract significantly reduced the amount of TNF␣-induced nuclear factor kappa light chain enhancer of activated B cells [NF-B (p65)] (Supporting Information Fig. 1D), and the reactive oxygen species production (Supporting Information Fig. 1E), indicating that HS treatment plays a role in reducing inflammatory and oxidative conditions. In the absence of TNF-␣, the HS extract (40 g/mL) significantly induced in these cells the production of endothelial nitric oxide synthase (eNOS) at the protein and mRNA levels and these effects were accompanied by a significant increase in nitric oxide (NO) production (Fig. 2). To further ascertain the mechanism leading to such an increase in NO production, we performed the test in the presence of a NO synthase inhibitor (L-NAME, 80 M), which almost entirely suppressed NO production. Conversely, the effect of cyclooxygenase inhibitor indomethacin (10 M) was not significant. Therefore, endothelial dysfunction may be nutritionally alleviated. This study provides a research framework for assessing the effects of HS polyphenols on conditions associated with hypertension and the multifaceted metabolic syndrome [20], combining human and animal studies with ex vivo and in vitro experiments. There are several mechanisms involved. Diuresis, or inhibition of ACE activity are probably not important mechanisms of HS’ hypotensive effect. Rather, we interpret our findings as proof of the beneficial effects of polyphenols on free radicals and immune modulation. It is likely that these actions could improve cardiovascular health. Our ex vivo data also contribute to this conclusion and add a beneficial endothelial response, probably vasodilation, to a better understanding of the possible mechanisms of action. Taken together, our results suggest that a multifaceted and likely synergistic mechanism accounts for the hypotensive action of polyphenol-rich HS, which acts through improved endothelium-dependent response. The Unitat de Recerca Biom`edica is currently being supported by the program of consolidated groups from the Universitat Rovira
i Virgili and grants from the Fondo de Investigaci´on Sanitaria (FIS PI08/1032, PI08/1381 and PI11/00130). This and other laboratories involved in this study performed experiments in network under the bioactive food components platform, which share know-how, equipment and financial support (IDI-20120741, CIBER CB12/03/30038 y PROMETEO/2012/007). The authors have declared no conflict of interest.
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