Accepted Manuscript

Cardioprotective properties of grape seed proanthocyanidins: an update

M. Antónia Nunes, Filipa Pimentel, Anabela S.G. Costa, Rita C. Alves, Maria Beatriz
P.P. Oliveira

PII: S0924-2244(15)30095-9
DOI: 10.1016/j.tifs.2016.08.017
Reference: TIFS 1872

To appear in: Trends in Food Science & Technology

Received Date: 2 September 2015

Revised Date: 26 July 2016
Accepted Date: 24 August 2016

Please cite this article as: Antónia Nunes, M., Pimentel, F., Costa, A.S.G., Alves, R.C., Oliveira,
M.B.P.P., Cardioprotective properties of grape seed proanthocyanidins: an update, Trends in Food
Science & Technology (2016), doi: 10.1016/j.tifs.2016.08.017.

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1 Cardioprotective properties of grape seed proanthocyanidins: an update

3 M. Antónia Nunesa, Filipa Pimentela, Anabela S. G. Costaa, Rita C. Alvesa*, Maria Beatriz P. P.

4 Oliveiraa
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5 REQUIMTE, LAQV/ Departamento de Ciências Químicas, Faculdade de Farmácia da

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6 Universidade do Porto, R. Jorge Viterbo Ferreira 228, 4050-313, Porto, Portugal.

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9 Corresponding author:

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10 Rita C. Alves

11 REQUIMTE, LAQV/ Departamento de Ciências Químicas, Faculdade de Farmácia da

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Universidade do Porto, R. Jorge Viterbo Ferreira 228, 4050-313, Porto, Portugal.
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13 E-mail: rita.c.alves@gmail.com

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27 Abstract

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29 Background

30 The nutritional properties of grapes (Vitis vinifera L.) and derived-grape products are known for

31 a long time. To produce those food products, the agro-industry generates great amounts of by-

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32 products. The re-use of these undervalued materials is challenging because it represents an

33 attractive opportunity to convert them into value-added sources of natural ingredients with

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34 potential human health benefits.

35 Proanthocyanidins, naturally occurring polyphenolic compounds in fruits, nuts, seeds, or bark,

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36 are an important group of compounds present in grape skin and seeds.

37 Several studies have demonstrated the beneficial effects of grape seeds, a grape by-product,

38 regarding cardiovascular disease.

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40 Scope and Approach

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41 This review aims to provide an overview on the major impact of grape seed proanthocyanidins
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42 on modulating cardiovascular disease risk markers in humans, namely blood pressure, blood
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43 lipids, endothelium and anti-inflammatory function. Therefore, current and relevant data

44 regarding the functional properties of grape seed proanthocyanidins in cardiovascular disease

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45 was explored.

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47 Key Findings and Conclusions

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48 Grape seed proanthocyanidins reveal a promising potential in human health, particularly in

49 cardiovascular disease. Nevertheless, additional long-term studies, and larger sample sizes are

50 necessary to better understand the individual effect of those compounds and/or their synergism

51 with other individual bioactive substances.

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53 Keywords

54 Cardiovascular; grape seeds; bioactive; protective.

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55 Introduction

56 Grape seeds contain a high concentration of polyphenols, mostly monomeric flavan-3-ols and

57 oligomeric proanthocyanidins (Carlson, Peng, Prasain, & Wyss, 2008; Naziri, Nenadis,

58 Mantzouridou, & Tsimidou, 2014). Grape seed proanthocyanidins (GSP) are mostly dimers,

59 trimers and highly polymerized oligomers of monomeric catechins (Nandakumar, Singh, &

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60 Katiyar, 2008). Several clinical trials, in vitro and in animal studies, show that these compounds

61 have a positive effect in modulating different factors associated to cardiovascular diseases

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62 (CVD), such as hypertension and dyslipidemia (Graf, Raskin, Cefalu, & Ribnicky, 2010).

63 Epidemiological studies also strongly suggest that proanthocyanidins could protect against CVD

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64 (Blade, Arola, & Salvado, 2010).

65 Among non-communicable diseases, CVD are one of the major causes of morbidity and

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mortality and continue to rise worldwide. They occur not only in developing countries but also
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67 in low and middle-income ones, as a consequence of globalization (WHO, 2011). It is estimated

68 that, in 2030, nearly 23.3 million people will die from cardiovascular disorders (7.1 million in
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69 2002) (Mathers & Loncar, 2006). There are life style factors that can predispose to CVD such as
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70 a sedentary life and an unhealthy diet (WHO, 2011). Nevertheless, CVD seem to be also
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71 consequence of a complex interaction between those factors and genetics (Sarajlic & Przulj,

72 2014).
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73 It is known that an adequate consumption of fruit and vegetables reduces the risk of CVD and

74 other chronic diseases (Eilat-Adar, Sinai, Yosefy, & Henkin, 2013; Schroeter, et al., 2010).
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75 Indeed, there are several products that have been pointed out as being of interest in preventing
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76 CVD, namely cocoa, tea, red wine, pine bark or grape seeds (Habauzit & Morand, 2012; Khan,

77 et al., 2014; Nandakumar, et al., 2008; O'Keefe, et al., 2013).

78 A great number of products derived from the grape processing can be found in the market (e.g.

79 wine, juice, jam, raisins, and others), entailing a huge amount of agro-industrial by-products.

80 Simultaneously, there is a huge interest in identifying and characterizing natural compounds in

81 those products in order to provide food industry natural ingredients, ameliorate human health

82 and increase the sustainability of the process (Georgiev, Ananga, & Tsolova, 2014).

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83 This review aims to enlighten the possible health value of GSP associated to CVD prevention or

84 treatment. Blood pressure, blood lipids, endothelial and anti-inflammatory function were

85 considered are the risk markers for CVD considered in this review.

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87 Review strategy

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88 Extraction of current and relevant data was performed using electronic database Pubmed.

89 Reviews and primary articles were searched using advanced search terms in “all fields” and

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90 with the publication type “reviews” or none, using the keywords “grape seeds” and

91 “cardiovascular disease”, “hypertension”, “blood pressure”, “hyperlipidemia”, “blood lipids”,

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92 “endothelial function”, “platelet function”, “coronary artery disease”, “coronary heart disease”,

93 and “myocardial function” in individual searches. The same keywords were searched using in

94 “all fields”, the keyword “grape seed”.

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95 The search was limited to English language articles. The titles and abstracts were scanned to

96 exclude any studies that were clearly irrelevant. Studies that evaluated concomitant drugs
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97 administration and/or polyherbal formulations were excluded.

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98
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99 Bioactive compounds of grape seeds

100 Although there are many species of grapevines, wine is usually produced from Vitis vinifera L..
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101 Among fruits, grape is one of the richest sources of phytochemicals. Flavonoids are the most

102 abundant biologically active compounds present in this fruit (Georgiev, et al., 2014). In general,
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103 a grape is constituted by about 2-6% stems, 5-12% skin, 80-90% juice and 0-5% seeds.
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104 During the processing of grapes into juice, only small amounts of anthocyanins are extracted.

105 Throughout the fermentation/maceration process other bioactive compounds, such as

106 proanthocyanidins and oligostilbenes, are obtained (Georgiev, et al., 2014). This step of the

107 process originates grape pomace, a by-product that includes skin, pulp, stems and grape seeds,

108 retaining more than 70% of grape polyphenols (Ratnasooriya & Rupasinghe, 2012).

109 Grape products (such as wine and grape juice) are also known to contain high amounts of

110 polyphenols, particularly phenolic acids, anthocyanins and simple and complex flavonoids, as

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111 proanthocyanidins (Leifert & Abeywardena, 2008). The highest concentrations of grape

112 polyphenols are found in stems, skin and seeds. In red grape berries, phenolic compounds are

113 mainly present in skin and seeds (Dohadwala & Vita, 2009).

114 Grape seeds can be separated from the pomace, being a potential source of bioactive compounds

115 (Kar, Laight, Shaw, & Cummings, 2006; Teixeira, et al., 2014). The seeds contain fibre (40%),

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116 essential oils (16%), protein (11%) and phenolic compounds (7%), like tannins, and other

117 substances, such as sugars and minerals (de Campos, Leimann, Pedrosa, & Ferreira, 2008).

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118 Grape seed extract is an outstanding source of polyphenols, mostly proanthocyanidins

119 (approximately 90%) that can be found mostly in red wine (rather than in white wine) but also

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120 commercially available as capsules or tablets at different concentrations (Feringa, Laskey,

121 Dickson, & Coleman, 2011). The health benefits of seeds or its extracts and grape-derived

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products, such as wine, have been attributed to its polyphenolic compounds (Graf, et al., 2010;
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123 Peng, et al., 2005).

124 Proanthocyanidins are structurally a complex subclass of polyphenolic compounds and may
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125 occur as monomers, dimmers, trimers and oligomers of 20 or more units, although higher
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126 molecular weight polymers are regularly found (Wang, Chung, Song, & Chun, 2011). Grape
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127 skin and seeds contain proanthocyanidins, most of which are water soluble, thus allowing the

128 use of clean extraction methods for their recovery (Koyama, Goto-Yamamoto, & Hashizume,
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129 2007; Nandakumar, et al., 2008; White, 2012).

130 The grape skin contains anthocyanins and flavonols; hydroxycinnamates are present in the flesh
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131 and skin; seeds contain flavan-3-ol monomers and the majority of gallic acid derivatives. The
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132 flavan-3-ol subclass covers the simple monomers (+)-catechin and its isomer (-)-epicatechin, as

133 well as oligomeric (from two to five units) and polymeric (more than five units) molecules, also

134 known as proanthocyanidins or condensed tannins. Procyanidins, exclusively composed by

135 catechin and/or epicatechin subunits are the most abundant type of proanthocyanidins in plants.

136 On the other hand, the less common prodelphinidins are composed by gallocatechin or

137 epigallocatechin subunits, and have been identified in grape skin (Tsang, et al., 2005).

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138 In the U.S.A., the estimated dietary intake of polyphenols is about 100 mg/day, distributed into

139 22% monomers, 16% dimmers, 5% trimmers, and 30% polymers (Choy, Jaggers, Oteiza, &

140 Waterhouse, 2013).

141 Polyphenols can be naturally conjugated with sugars. Glucose is most often found due to its

142 prevalence in plant physiology (Manach, Scalbert, Morand, Remesy, & Jimenez, 2004). These

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143 conjugates can also be linked to other phenol groups, organic acids, proteins and even lipids,

144 that may affect their absorption (Parada & Aguilera, 2007). The type of sugar bounded to a

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145 flavonoid skeleton determines the site and the extent of absorption of the glycosylated

146 flavonoids. Once free from the sugar, they are further metabolized and a large number of

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147 modified molecules are produced (D'Archivio, Filesi, Vari, Scazzocchio, & Masella, 2010).

148 To understand the effectiveness of grape seeds polyphenolic compounds in preventing CVD it is

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important to determine the nature and distribution of these compounds in an organic system.
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150 The bioactive composition of grapes and its seeds is strongly determined by external factors

151 such as the variety or stage of ripeness, geographic location, climatic conditions or cultivars
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152 (Nandakumar, et al., 2008; Rathel, Samtleben, Vollmar, & Dirsch, 2007). Other aspects may
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153 influence polyphenols bioavailability, for instance, individual differences that can lead to a wide
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154 rate variation and extent of the compounds absorption. This inter-individual variability can be

155 due to several factors, such as genetic profile, composition/activity of the gut microbiota, and
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156 diet (Rein, et al., 2013).

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158 Proanthocyanidins metabolism

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159 Bioavailability can be defined as the proportion of a given compound that is ingested, digested,

160 absorbed and reaches the systemic circulation (Carbonell-Capella, Buniowska, Barba, Esteve, &

161 Frígola, 2014). In turn, bioaccessibility refers to digestive transformations, the absorption into

162 the intestinal epithelium cells, presystemic intestinal and hepatic metabolism, tissue distribution

163 and bioactivity (Courraud, Berger, Cristol, & Avallone, 2013). Therefore, the bioavailability

164 strictly depends on the bioaccessibility (Palafox-Carlos, Ayala-Zavala, & Gonzalez-Aguilar,

165 2011). Bioaccessibility is usually determined by in vitro studies, accessed by simulated gastric

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166 and small intestine digestion. The bioavailability of a compound should ideally be determined in

167 vivo, in animals or humans, as the area under the plasma-concentration curve, obtained after the

168 administration of a chronic or acute dose, through food or in isolated forms (Rein, et al., 2013).

169 Many recent reviews have been focusing on the study of bioavailability of polyphenolic

170 compounds. The consensus is that bioavailability of most polyphenols could be affected by

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171 several factors that will reduce their effectiveness. Factors like low absorption, instability,

172 excessive metabolism or gut microbial transformation can influence it. Some bioactive

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173 compounds exhibit rates of absorption ranging from 0.3% to 43%, as well as low concentrations

174 of circulating plasma metabolites (Abourashed, 2013; Manach, Williamson, Morand, Scalbert,

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175 & Remesy, 2005). Nevertheless, there are some evidences that, although present in low

176 concentration in plasma, polyphenols and their metabolites show activity in their targets.

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Indeed, some efforts have been performed to enhance the bioavailability of polyphenols, namely
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178 chemical derivation, modified formulation and processing (Abourashed, 2013; Nandakumar, et

179 al., 2008; Nifli, Kampa, Alexaki, Notas, & Castanas, 2005).
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180 To access nutrients bioavailability, blood plasma concentrations are usually used (in vivo
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181 assays). The presence of other meal components and the dose used should be taken into account,
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182 since these can affect the evaluation (Faulks & Southon, 2005; Parada & Aguilera, 2007). One

183 of the major factors that interfere on bioavailability is the chemical structure of the compounds.
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184 Yet, host related factors, like intestinal and systemic factors, can also influence the

185 bioavailability of bioactive compounds, as already referred. All the digestive process may
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186 extensively modify the biological activity of polyphenols. Unavoidably, those that reach cells
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187 and tissues can be chemically, biologically and functionally different from the original dietary

188 form (D'Archivio, et al., 2010).

189 High molecular weight compounds, as complex lipids and oligomeric proanthocyanidins for

190 example, cannot pass through intestinal cells unless they are previously broken down

191 (Appeldoorn, Vincken, Aura, Hollman, & Gruppen, 2009). The bioavailability of these

192 molecules is largely influenced by their degree of polymerization, as polymeric forms can pass

193 intact through the gastrointestinal tract and reach the colon, where they can be transformed by

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194 microbiota before absorption. Microbial metabolites are then absorbed, undergo phase II

195 metabolism, enter in the circulation, being finally eliminated in urine (Monagas, et al., 2010).

196 The absorption and bioavailability of oligomeric forms of procyanidins is not as clear as for the

197 monomeric ones (Serra, et al., 2010). The degree of polymerization settles on the polymer

198 length. After oral administration, due to oligomers polymerization structure, cellular absorption

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199 is restricted to those with a lower degree of polymerization, being the larger degree polymerized

200 molecules absorbed only at the gut lumen (Cheah, Howarth, Bindon, Kennedy, & Bastian,

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201 2014; Choy, et al., 2013). The absorption rate of dimmers is around 5-10%, while trimers and

202 tetramers have lower absorption rates. In fact, the majority of proanthocyanidins reaches the

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203 colon intact. There, they are metabolized by the microbiota into smaller molecules and the

204 resultant metabolites are those really associated with health benefits (Ou & Gu, 2014; Rein, et

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al., 2013). The small intestine is the primary site for glucuronidation of phenolic compounds
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206 including proanthocyanidins. This process occurs in the luminal part of the endoplasmatic

207 reticulum and in the liver (Monagas, et al., 2010).

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208 Another parameter taken into account, in these studies, is the applied doses that should reflect
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209 what is normally ingested by humans. High doses in in vitro studies can lead to outcomes that
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210 necessarily do not reflect the real antioxidants consumption in a standard dietary pattern.

211 Therefore, the results should be cautiously extrapolated (D'Archivio, et al., 2010).
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212 So far, there is no consensus about the absorption and metabolism of proanthocyanidins,

213 although it is recognized that gut is an important place where the biotransformation of these
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214 compounds occur (Choy, et al., 2013). In vivo studies demonstrated the presence of microbial
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215 derived metabolites in urine after the intake of rich proanthocyanidins diets. In urine, the main

216 derivatives from flavan-3-ol microbial phenolics acids are phenylacetic acids and mono- and di-

217 hydroxylated phenyl propionic acids along with hydroxyhippuric acids (Monagas, et al., 2010).

218 The diverse structure and high molecular size of proanthocyanidins make their identification

219 and quantification a challenging task. Furthermore, the proanthocyanidins metabolism is

220 partially understood mainly due to their amphiphilic physical properties, the natural complex

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221 food matrices where they exist, combined with the high metabolization extent that occurs at

222 colon (Choy & Waterhouse, 2014).

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224 Grape seeds and cardiovascular protection

225 It is known that CVD are caused by multiple factors. However, the majority of CVD events are

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226 related to atherosclerosis complications (Habauzit & Morand, 2012). Atherosclerosis is a

227 multistage disease of the intima, the innermost layer of arterial wall, originated by a

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228 pathophysiological process (Sessa, Pietro, Filardo, & Turriziani, 2014).

229 Endothelium is more than a simple physical barrier, since endothelium cells maintain vascular

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230 homeostasis by secreting local acting mediators (Shih & Cherng, 2014). This thin layer of cells

231 is directly related to the development of atherosclerosis. In fact, it plays an important role in the

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regulation of vascular tone, leucocytes adhesion, platelet activity and thrombosis. Evidence
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233 suggests that alterations in the functional properties of the vascular endothelium are involved in

234 the initiation, progression, and clinical complications of atherosclerotic diseases (Habauzit &
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235 Morand, 2012).

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236 Recently, the PREDIMED study showed an inverse association between cardiovascular
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237 disorders risk and polyphenols intake, which is independent of other dietary and non-dietary

238 CVD risk factors. Furthermore, similar significant associations were established for lignans,
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239 hydroxybenzoic acids and flavanols (Tresserra-Rimbau, et al., 2014). Classical biological

240 actions of polyphenols have been associated to antioxidant capacity but other recent evidence
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241 suggests other abilities that also contribute to CVD risk reduction, namely their
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242 immunomodulatory and vasodilatory properties (Tangney & Rasmussen, 2013).

243 Food products with antioxidant properties, mainly flavonoids that have radical scavenging

244 action, can play a protective role in CVD pathogenesis related to reactive oxygen species

245 (Wang, Mehendale, & Yuan, 2007).

246 Grape seed properties have been evaluated in many animal studies and in human trials,

247 suggesting a potential beneficial effect on the cardiovascular system (Feringa, et al., 2011;

248 Fernández & Labra, 2013; Rasmussen, Frederiksen, Struntze Krogholm, & Poulsen, 2005;

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249 Yang, Kim, Yang, Kim, & Kwon, 2012). Grape seed extracts contain a high concentration of

250 proanthocyanidins, which can be responsible for that effect in several cardiovascular markers.

251 The seed extracts may be protective against CVD through various mechanisms, which comprise

252 antioxidant, anti-platelet and anti-inflammatory effects, blood pressure lowering capacity, and

253 endothelial function improvement. The main in vitro, in vivo studies and human trials are

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254 summarized in Table 1.

255

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256 Blood pressure

257 Procyanidins indirectly increase the nitrogen oxide lifetime by inhibiting the production of

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258 superoxide, its major physiological scavenger, and directly inhibit angiotensin-converting

259 enzyme (Belcaro, et al., 2013).

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In a double-blind, placebo-controlled, randomized, parallel-group intervention study, the effect
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261 of a specific grape seed extract, rich in polyphenolic compounds (300 mg/day) versus placebo,

262 was evaluated in subjects with pre- and stage I hypertension. Data showed that the consumption
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263 of the polyphenol extract did not significantly decrease ambulatory blood pressure (Ras, et al.,
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264 2013).
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265 During 4 weeks, 36 adults were followed in a study conducted by Clifton (2004) who did not

266 found differences in systolic and diastolic blood pressure, serum lipids and oxidized low-density
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267 lipoprotein (LDL) after a daily intake of a grape seed extract. Interestingly, in the subjects that

268 presented increased cardiovascular risk factors, including smoking habits, high cholesterol
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269 levels, or high blood pressure, there was a significant difference in flow mediated dilation
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270 (FMD) (Clifton, 2004). FMD corresponds to a physiological response, measured as the vascular

271 diameter increase subsequent to resumed blood flow after transient ischemia, and has been

272 validated as a prognostic parameter of cardiovascular events (Thijssen, et al., 2011).

273 Grape seed extract has also shown not to influence blood pressure, and even other

274 cardiovascular risk markers, in healthy people (van Mierlo, Zock, van der Knaap, & Draijer,

275 2010). However, in some studies, using a standardized grape seed extract in subjects with

276 mildly hypertensive or metabolic syndrome, a decrease of the systolic blood pressure was

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277 observed (Belcaro, et al., 2013; Sivaprakasapillai, Edirisinghe, Randolph, Steinberg, &

278 Kappagoda, 2009).

279 Fernández and Labra (2013) investigated the effect of in vitro gastrointestinal digestion on the

280 stability and composition of a raw and purified skin and seed grape extract, respectively.

281 Simultaneously, they evaluated the effects of digestion on the inhibitory activities of those

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282 extracts in angiotensin I-converting enzyme (ACE). The inhibition of ACE is a therapeutic

283 intervention to control CVD-associated hypertension. After simulated gastric digestion the mean

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284 degrees of polymerization of both extracts were stable. In simulated intestinal digestion, the

285 proanthocyanidins in the grape skin extracts were degraded, whereas the grape seed extract,

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286 containing proanthocyanidins, was more resistant. After digestion, more than 90% of the

287 inhibition of ACE by the raw extracts was preserved and, at the end of intestinal digestion, these

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extracts were capable to inhibit ACE for more than 80%. Purified extracts showed low ACE
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289 inhibitory activities. It was verified that the level of inhibition of ACE is influenced by the

290 percentage of low-molecular-weight compounds in the extracts (Fernández & Labra, 2013).
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291 Some animal studies have shown an improvement in blood pressure. In ouabain-induced
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292 hypertensive rats treated with grape seed proanthocyanidins, it has been reported a significant
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293 decrease in systolic blood pressure, an increase in oxide nitric production and ameliorate

294 endothelium aspect (Liu, et al., 2012). Moreover, grape seed proanthocyanidins significantly
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295 blocked the ouabain-induced increase of blood pressure (Cui, Liu, Feng, Zhao, & Gao, 2012).

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297 Blood lipids

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298 The progression of atherosclerosis and CVD is related to an alteration in plasma cholesterol

299 levels (Lichtenstein, et al., 2006). The accumulation of cholesterol deposits in macrophages in

300 the intima of arteries is the initial phase of atherosclerosis. The foam cells can aggregate and

301 progressively form fibrous plaques covered by a connective tissue cap with embedded smooth

302 muscle cells, increasing as well the secretion of inflammation mediators, such as interleukins

303 (IL) (IL-1 and IL-6) and tumor necrosis factor (TNF)-α (Sessa, et al., 2014). Activated

304 macrophages express myeloperoxidase (MPO) and NADPH oxidase, which generate a range of

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305 oxidants (e.g. superoxide, hydrogen peroxide and hypochlorous acid) (Ronald, et al., 2009;

306 Sessa, et al., 2014). Moreover, the inflammatory state can induce oxidative stress by increasing

307 reactive oxygen species (ROS) production in vascular wall, leading to the progression and

308 destabilization of atherosclerotic plaque and, consequently, to CVD (Hulsmans & Holvoet,

309 2010).

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310 In that way, this pathologic condition is associated with a chronic low-intensity inflammation

311 that involves the participation of a wide range of cells, namely endothelial and smooth muscular

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312 cells, monocytes, lymphocytes and platelets, adhesion molecules, cytokines, chemokines,

313 growth factors and some enzymes (Khan, et al., 2014). All of these inflammatory mediators

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314 seem to be linked to all of the different steps of atherogenesis (Libby, 2012).

315 Grape seeds are considered to have an anti-hyperlipidemic beneficial effect related with

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polyphenolic compounds, specifically gallic acid, catechins and epicatechin, which have been
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317 demonstrated to inhibit pancreatic cholesterol esterase, pancreatic and lipoprotein lipases, bind

318 bile acids capacity, reduce the solubility of cholesterol in micelles, and protect LDL against
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319 oxidation (Moreno, et al., 2003; Ngamukote, Makynen, Thilawech, & Adisakwattana, 2011;
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320 Shafiee, Carbonneau, Urban, Descomps, & Leger, 2003).

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321 Vinson et al. (2002) assessed the efficacy of grape seed proanthocyanidins extract using a

322 hamster model of atherosclerosis. These authors found that these compounds induced a large
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323 reduction up to 34% in triglyceride level, and 25% in plasma cholesterol levels, showing also a

324 significant decrease in plasma lipid peroxidation levels (Vinson, Mandarano, Shuta, Bagchi, &
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325 Bagchi, 2002). Additionally, in rats fed with a high fat diet specifically planned to induce
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326 metabolic syndrome, results indicate that a grape seed proanthocyanidins extract decreased

327 triglycerides synthesis in these obese rats (Baiges, Palmfeldt, Blade, Gregersen, & Arola, 2010).

328 Grape seeds proanthocyanidins may also improve lipid homeostasis by reducing plasmatic

329 levels of apoliprotein B-triglyceride-rich lipoproteins and low-density lipoprotein cholesterol

330 (LDL-c) and increasing high-density lipoprotein cholesterol (HDL-c), despite the outcomes in

331 humans are less clear (Blade, et al., 2010). Moreover, these compounds also reduced cholesterol

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332 accumulation, preventing the development of foam cells, which partially inhibit the

333 atherosclerosis process as previously mentioned (Terra, et al., 2009).

334 Thiruchenduran et al. (2011), using Wistar rats fed with a high-cholesterol diet, found that grape

335 seed proanthocyanidins acted against CVD promoters, in particular the ones induced by the diet,

336 partially restoring those changes (Thiruchenduran, Vijayan, Sawaminathan, & Devaraj, 2011).

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337 In another study, using animals as well, the authors observed an improvement on the blood

338 pressure and in hypertriglyceridemia, what is probably related with the oxidative stress (Pons, et

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339 al., 2014).

340 In a randomized, double-blind, placebo controlled study, analogous extracts were given to 40

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341 hypercholesteraemic subjects, during 8 weeks. The results showed a significant reduction in

342 LDL-c and in total cholesterol (T-c) levels for the grape seed proanthocyanidins group.

343
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However, this group was additionally supplemented with niacin-bound chromium, what
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344 suggests that other factors acting in synergy may enhance the nutritional benefits (Bagchi, et al.,

345 2003; Leifert & Abeywardena, 2008). In another randomized double-blind placebo-controlled
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346 clinical trial, the effect of red grape seed proanthocyanidins extract consumption by 52 mildly
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347 hyperlipidemic subjects was evaluated. During 2 months, two different groups received either
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348 200 mg/day of the seed extract or placebo. The results showed a reduction of T-c, LDL-c and

349 oxidized low-density lipoprotein particles, decreasing the risk of atherosclerosis and
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350 cardiovascular disorders in those individuals (Razavi, et al., 2013).

351 Sano and colleagues (2007) performed a similar study with 61 healthy subjects with LDL-c
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352 levels of 100 to 180 mg/dl, following them during 12 weeks of administration of 0 (placebo),
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353 200 or 400 mg of grape seed extract. Reducing effects on oxidized LDL were observed,

354 compared to patients who took the placebo tablets (Sano, et al., 2007).

355 High doses of grape seeds, often higher than the normally amounts consumed by humans, may

356 be necessary to proficuously decrease blood lipid levels. Nevertheless, the major benefit effects

357 of grape seeds are more likely to be related with its antioxidant capacity, such as scavenging of

358 hydroxyl and peroxyl radicals and inhibition of LDL, rather than lipid reduction (Feringa, et al.,

359 2011; Leifert & Abeywardena, 2008).

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360

361 Endothelial function

362 Vascular endothelium has the capacity to produce substances that modify vascular tone and

363 protect the wall from thrombus formation, inflammatory cell infiltration and smooth muscle cell

364 proliferation (Dzau, Gnecchi, Pachori, Morello, & Melo, 2005). Endothelial damage can be

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365 implicated in atherosclerosis development, so the balance between injury and recover is

366 important to prevent cardiovascular events (Shantsila, Watson, & Lip, 2007).

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367 In a randomized, double-blind, placebo-controlled crossover trial to assess the muscadine grape

368 seed effect on vascular function and cardiovascular risk factors, 50 adults with coronary disease,

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369 or having more than one cardiac risk factor were supplemented with 1300 mg daily and placebo,

370 for 4 weeks. Results showed no evidence on the FMD improvement or significant changes in

371
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other biomarkers of inflammation, lipid peroxidation, or antioxidant capacity (Mellen, Daniel,
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372 Brosnihan, Hansen, & Herrington, 2010). In turn, in in vitro studies have demonstrated

373 endothelium-dependent relaxation as the key mechanism of action of grape seeds extracts
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374 (Aldini, Carini, Piccoli, Rossoni, & Facino, 2003; Fitzpatrick, Bing, Maggi, Fleming, &
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375 O'Malley, 2002; Graf, et al., 2010; Mendes, Desgranges, Cheze, Vercauteren, & Freslon, 2003).
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376 In rabbit’s aorta, grape seed extract has demonstrated to cause a dose-dependent endothelium

377 relaxation (Edirisinghe, Burton-Freeman, & Tissa Kappagoda, 2008). An oxidative stress
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378 reduction could enhance the bioactivity of oxide nitric, therefore, causing the observed

379 reduction on blood pressure (Feringa, et al., 2011).

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380 Notwithstanding, other study failed to demonstrate the effect of grape seeds on heart rate,
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381 although a significant decrease in blood pressure was verified (Peng, et al., 2005).

382 Polyphenols stimulate antioxidant activities of enzymes, such as catalase, and contribute to the

383 antioxidant defence of endothelial cells by reducing NADPH oxidase expression and its activity.

384 Also, antioxidants inhibit free oxygen species with pro-inflammatory activity generated by

385 enzymes like cyclooxygenase, lipoxygenase and inducible nitric oxide synthase. These

386 antioxidant properties are largely based on in vitro models. Nevertheless, few trials show

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387 undoubtedly the beneficial effect of polyphenol exposure, similar to in vitro studies (Tangney &

388 Rasmussen, 2013).

389

390 Anti-inflammatory function

391 Regarding anti-inflammatory function, it has been suggested that grape seed extracts could have

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392 interesting properties, demonstrated in in vitro studies. Inflammation, as described above, has

393 been found to play a key role in the development of atherosclerotic changes (Kar, et al., 2009)

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394 in every step of atherosclerotic plaque formation (Wolf, Stachon, Bode, & Zirlik, 2014). Also,

395 c-reactive protein (CRP) has been considered as an important biomarker of inflammation linked

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396 to CVD. CRP is produced by the liver in response to inflammation or tissue damage (Libby,

397 2012).

398
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In a meta-analysis of randomized controlled trials, Feringa et al. (2011) did not found an effect
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399 of grape seed on CRP, neither in the diastolic blood pressure, nor in lipid levels. Yet, a

400 significant effect of grape seed extract in lowering the systolic blood pressure and heart rate was
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401 verified (Feringa, et al., 2011). Moreover, this extract has been shown to have anti-
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402 inflammatory effect on experimental inflammation (Li, Zhang, Wu, & Tian, 2001), besides
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403 reducing body weight and plasmatic systemic markers of inflammation, tumour necrosis factor

404 alpha and CRP in diet-induced rats (Terra, et al., 2011). Other researchers have demonstrated
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405 positive effects on inflammatory-related diseases as well, such as in obesity and in type 2

406 diabetes, by using human and animal adipocytes and macrophage-like cell lines (Chacon, et al.,
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407 2009; Sakurai, et al., 2010).

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408

409 Conclusion

410 Grape seeds are an agro-industrial by-product with huge potential to be a functional ingredient

411 and/or a dietary supplement. The seeds bioactive compounds, namely, proanthocyanidins have

412 been demonstrating to have positive effects on human health. Several trials report positive

413 evidences, but often performed with grape seed extract doses much higher than those normally

414 consumed through a regular diet. Furthermore, grape seeds proanthocyanidins metabolism is

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415 only partially understood. Based on that it is important to study how these compounds influence

416 gut microbiota and their bioavailability.

417 Taking into account the promissory value of grape seeds, it is mandatory to conduct larger

418 randomized, double-blinded trials, considering variables such as grape seed extracts dosages

419 and larger follow-up periods, in order to support the potential positive benefits for human

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420 health, specifically regarding CVD prevention.

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442 Conflict of interests

443 The authors declare that there is no conflict of interests regarding the publication of this paper.

444

445 Acknowledgments

446 The authors thank the financial support to the project Operação NORTE-01-0145-FEDER-

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447 000011–denominada Qualidade e Segurança Alimentar-uma abordagem (nano)tecnológica. This

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448 work was also supported by the project UID/QUI/50006/2013-POCI/01/0145/FEDER/007265

449 with financial support from FCT/MEC through national funds and co-financed by FEDER.

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450 Filipa B. Pimentel is grateful to FCT for a PhD research grant (SFRH/BD/109042/2015),

451 financed by National Funds (Ministério da Educação e da Ciência) and by the European Social

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452 Fund through POCH-Programa Operacional Capital Humano.
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713 Terra, X., Fernández-Larrea, J., Pujadas, G., Ardevol, A., Blade, C., Salvado, J., Arola, L., &

714 Blay, M. (2009). Inhibitory effects of grape seed procyanidins on foam cell formation in

715 vitro. Journal of Agricultural and Food Chemistry, 57, 2588-2594.

716 Terra, X., Pallares, V., Ardevol, A., Blade, C., Fernández-Larrea, J., Pujadas, G., Salvado, J.,

717 Arola, L., & Blay, M. (2011). Modulatory effect of grape-seed procyanidins on local

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718 and systemic inflammation in diet-induced obesity rats. The Journal of Nutritional

719 Biochemistry, 22, 380-387.

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720 Thijssen, D. H., Black, M. A., Pyke, K. E., Padilla, J., Atkinson, G., Harris, R. A., Parker, B.,

721 Widlansky, M. E., Tschakovsky, M. E., & Green, D. J. (2011). Assessment of flow-

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723 Journal of Physiology: Heart and Circulatory Physiology, 300, H2-12.

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725 effect of grape seed proanthocyanidins against cholesterol cholic acid diet-induced

726 hypercholesterolemia in rats. Cardiovasculat Pathology, 20, 361-368.

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727 Tresserra-Rimbau, A., Rimm, E. B., Medina-Remon, A., Martinez-Gonzalez, M. A., de la

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729 M., Ros, E., Serra-Majem, L., Pinto, X., Saez, G. T., Basora, J., Sorli, J. V., Martinez, J.

730 A., Vinyoles, E., Ruiz-Gutierrez, V., Estruch, R., Lamuela-Raventos, R. M., &
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735 (2005). The absorption, metabolism and excretion of flavan-3-ols and procyanidins

736 following the ingestion of a grape seed extract by rats. British Journal of Nutrition, 94,

737 170-181.

738 van Mierlo, L. A., Zock, P. L., van der Knaap, H. C., & Draijer, R. (2010). Grape polyphenols

739 do not affect vascular function in healthy men. Journal of Nutrition, 140, 1769-1773.

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740 Vigna, G. B., Costantini, F., Aldini, G., Carini, M., Catapano, A., Schena, F., Tangerini, A.,

741 Zanca, R., Bombardelli, E., Morazzoni, P., Mezzetti, A., Fellin, R., & Maffei Facino, R.

742 (2003). Effect of a standardized grape seed extract on low-density lipoprotein

743 susceptibility to oxidation in heavy smokers. Metabolism, 52, 1250-1257.

744 Vinson, J. A., Mandarano, M. A., Shuta, D. L., Bagchi, M., & Bagchi, D. (2002). Beneficial

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745 effects of a novel IH636 grape seed proanthocyanidin extract and a niacin-bound

746 chromium in a hamster atherosclerosis model. Molecular and Cellular Biochemistry,

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748 Vinson, J. A., Proch, J., & Bose, P. (2001). MegaNatural® Gold grapeseed extract: in vitro

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749 antioxidant and in vivo human supplementation studies. Journal of Medicinal Food, 4,

750 17-26.

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752 and skin extracts inhibit platelet function and release of reactive oxygen intermediates.

753 Journal of Cardiovascular Pharmacology, 46, 445-451.

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754 Wang, C. Z., Mehendale, S. R., & Yuan, C. S. (2007). Commonly used antioxidant botanicals:
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755 active constituents and their potential role in cardiovascular illness. American Journal
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758 proanthocyanidin intake and major food sources in the U.S. diet. Journal of Nutrition,

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760 Weseler, A. R., Ruijters, E. J., Drittij-Reijnders, M. J., Reesink, K. D., Haenen, G. R., & Bast,
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761 A. (2011). Pleiotropic benefit of monomeric and oligomeric flavanols on vascular

762 health-a randomized controlled clinical pilot study. PLoS One, 6, e28460.

763 White, S. X. L. E. (2012). Extraction and characterization of proanthocyanidins from grape

764 seeds. The Open Food Science Journal, 6, 5-11.

765 WHO (2011). Global status report on noncommunicable diseases 2010 [online]

766 http://www.who.int/nmh/publications/ncd_report2010/en/.

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768 atherosclerosis. Hamostaseologie, 34, 63-71.

769 Yang, Y. J., Kim, Y. J., Yang, Y. K., Kim, J. Y., & Kwon, O. (2012). Dietary flavan-3-ols

770 intake and metabolic syndrome risk in Korean adults. Nutrition Research and Practice,

771 6, 68-77.

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Table 1 – Effects of grape seeds bioactive compounds in human intervention studies, animal models and in vitro.
Dose,
Model Product Main endpoints Reference
duration, sample size

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Mildly hyperlipidemic subjects Red grape seed extract 200 mg/day Reduce T-c Razavi, et al. (2013)
8 weeks Reduce LDL-c

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n=52 Reduce Ox-LDL

Healthy, pre and Grape seed procyanidins extract 150 and 300 mg/day Improvement in BP, heart rate, Belcaro, et al. (2013)

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mildly hypertensive subjects 16 weeks plasma oxidative and
n=119 microcirculatory status

Healthy subjects with systolic Grape seed extract 300 mg/day No significant decrease in Ras, et al. (2013)

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blood pressure between 120 and 8 weeks ambulatory BP
159 mmHg n=70

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Male smokers Grape seeds 200 mg/day Decrease in T-c and LDL-c Weseler, et al. (2011)
(monomeric and oligomeric 8 weeks Improvement of overall vascular
flavanols) n=28 health

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Healthy men Wine grape and grape seed 800 mg/day No major impact on FMD van Mierlo, et al. (2010)
polyphenols 2 weeks

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n=35

Adults with cardiovascular risk
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Muscadine grape seed 1300 mg/day No evidence of improved FMD Mellen, Daniel, Brosnihan,
4 weeks No significant change in Hansen, & Herrington (2010)
n=50 biomarkers of inflammation,
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lipid peroxidation or antioxidant
capacity
Significant increase in resting
brachial diameter
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Adults with type 2 diabetes on Grape seed extract 600 mg/day Improvement of markers of Kar, et al. (2009)
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diet or oral glucose-lowering 4 weeks inflammation and glycaemia

medications, high cardiovascular n=32
risk
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Table 1 – Effects of grape seeds bioactive compounds in human intervention studies, animal models and in vitro (cont.).
Dose,
Model Product Main endpoints Reference
duration, sample size

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Adults with metabolic Grape seed extract 150 or 300 mg/day Decrease in SBP and DBP Sivaprakasapillai, Edirisinghe,
syndrome 4 weeks No significant changes in serum Randolph, Steinberg, &

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n=27 lipids Kappagoda (2009)
No significant changes in blood
glucose

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Adults with LDL-c levels Grape seed extract 200 or 400 mg/day Reducing effects on Ox-LDL Sano, et al. (2007)
between 12 weeks
100-180 mg/dl n=61

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Adults with above-average Grape seed extract 2000 mg/day Improved FMD Clifton (2004)

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vascular risk due to smoking, 4 weeks
high cholesterol, or hypertension n=36

Healthy male heavy smokers Grape seed extract 150 mg/day No significant modification of Vigna, et al. (2003)

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4 weeks T-c, triglycerides, HDL-c and
n=24 LDL-c
TBARS concentration reduced

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Hypercholesterolemic subjects Grape seed proanthocyanidins 200 mg/day Reduce Ox-LDL Bagchi, et al. (2003)

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(210 – 300 mg/dl) extract 8 weeks
n=40
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Normal and Grape seed extract 600 mg/day Improvement of plasma Vinson, Proch, and Bose (2001)
hypercholesterolemic subjects n=17 antioxidant capacity and lipid
profile in hypercholesterolemic
subjects
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Hypercholesterolemic subjects Grape seed proanthocyanidins 200 mg/day No significant modifications on Preuss, et al. (2000)
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extract 8 weeks BP
(combined administration of n=40 Decrease in T-c and LDL-c
niacin-bound chromium)
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Table 1 – Effects of grape seeds bioactive compounds in human intervention studies, animal models and in vitro (cont.).
Dose,
Model Product Main endpoints Reference
duration, sample size

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Rats Low molecular grape seed 375 mg/kg body weight Reduced oxidative stress Pons, et al. (2014)
(high-fat diet-fed rats) proanthocyanidins extract 6 h post-administration Decrease of BP

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Improvement of
hypertriglyceridaemia
No alterations in hepatic-reduced

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glutathione and plasma ACE

Hamsters Grape seed procyanidins extract 25 mg/kg body weight Improvement in the lipid profile Caimari, del Bas, Crescenti, and
(standard diet or a high-fat diet) 15 days Arola (2013)

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Rats Grape seed proanthocyanidins 250 mg/kg body weight Increase of NO production Cui, Liu, Feng, Zhao,

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extracts 5 weeks Regulates ouabain induced & Gao (2012)
hypertension

Rats Grape seed proanthocyanidins 250 mg/kg body weight Reduced SBP Liu, et al. (2012)

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(ouabain-induced hypertensive extract 5 weeks Improvement of vascular
rats) endothelial function
Reduced TGF-β1 expression in

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the thoracic aorta

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Rats Grape seed proanthocyanidins 100 mg/kg body weight Improvement of antioxidant Thiruchenduran, Vijayan,
(male Wistar rats induced with a 30 days status Sawaminathan,
hypercholesterolemic diet) Reverse of high lipids levels & Devaraj (2011)
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Rats Grape seed proanthocyanidins 25 mg /kg body weight Decreased lipid synthesis in liver Baiges, Palmfeldt, Blade,
(fed a high fat diet) extract 10 days Gregersen, & Arola
(2010)
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Rats Grape seed extract Up to 0.5% Decrease arterial pressure Peng, et al. (2005)
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(estrogen-depleted female 10 weeks

spontaneously hypertensive rats)
Rats Grape seed procyanidins extract 250 mg/kg body weight Improved plasma lipidic profile Del Bas, et al. (2005)

Hamsters Grape seed proanthocyanidins Up to 100 mg/kg body weight Reduce foam cells Vinson, Mandarano, Shuta,
(atherosclerotic hamsters) extract 10 weeks Reduce plasma cholesterol Bagchi, & Bagchi (2002)
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Table 1 – Effects of grape seeds bioactive compounds in human intervention studies, animal models and in vitro (cont.).
Dose,
Model Product Main endpoints Reference
duration, sample size

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Murine macrophage cell Grape seed procyanidins 45 µg/ml Attenuate the development of Terra, et al. (2009)
foam cell formation

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Modulate the expression of key
genes in cholesterol flux and
inflammation

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Rabbit aortic rings Grape seed extract 100 µmol/l Dose-dependent relaxation of the Edirisinghe, Burton-Freeman
aortic ring & Tissa Kappagoda (2008)

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Rat cardiac H9C2 cells Grape seed extract 25 – 100 µM Increased resistance to cardiac Du, Guo, and Lou (2007)
(catechin and proanthocyanidins) 24 hours cell apoptosis elicited by ROS

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Platelets Grape seed extract 50 to 100 mg/l Inhibits platelet function Vitseva, Varghese, Chakrabarti,
Inhibits release of ROS Folts, and Freedman (2005)

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Hamster aortic rings Grape seed extract 18.4 mg/kg up to 70 µg/ml Reduce plasma cholesterol Auger, et al. (2004)
(in bath) Reduce atherosclerosis
12 weeks

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Human umbilical endothelial Procyanidins 1 µM – 20 µM Protect endothelial cells from Aldini, Carini, Piccoli, Rossoni,

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cells peroxynitrile damage & Facino (2003)

Rat isolated aortic rings Grape seeds procyanidins 10-7 to 1 g/l Synthesis and release of NO Mendes, Desgranges,
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Cheze, Vercauteren, &
Freslon (2003)

Rat aortic rings Concord grape seed extract Up to 0.25 µg/ml (bath) Increase endothelium –dependent Fitzpatrick, Fleming, Bing,
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Minutes relaxation Maggi, and O'Malley (2000)

Ox-LDL - Oxidized low-density lipoprotein; T-c – Total-cholesterol; LDL-c – Low-density lipoprotein cholesterol; HDL-c - High-density lipoprotein cholesterol; FMD - Flow-mediated dilation;
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TBARS - Thiobarbituric acid reactive substances; ROS - Reactive oxygen species; TGF-β1 - Transforming growth factor-β 1; NO – Nitric oxide; ACE - Angiotensin converting enzyme.
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Highlights

- Grape pomace is an abundant by-product of winery agro-industry;

- Grape seeds, separated from the pomace, are a rich source of proanthocyanidins;

- Proanthocyanidins can have a positive effect on cardiovascular disease prevention.

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