BioFactors 23 (2005) 189195 IOS Press

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The inhibitory effects of berry polyphenols on digestive enzymes

Gordon J. McDougall and Derek Stewart
Quality, Health and Nutrition Programme, Genes to Products Theme, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK
Abstract. The evidence for the effect of polyphenol components of berries on digestive enzymes is reviewed. Anthocyanins inhibit -glucosidase activity and can reduce blood glucose levels after starch-rich meals, a proven clinical therapy for controlling type II diabetes. Ellagitannins inhibit -amylase activity and there is potential for synergistic effects on starch degradation after ingestion of berries such as raspberries and strawberries, which contain substantial amounts of ellagitannins and anthocyanins. A range of berry polyphenols (e.g. avonols, anthocyanidins, ellagitannins and proanthocyanidins) can inhibit protease activities at levels which could affect protein digestion in the gastrointestinal tract. In contrast, potential for the inhibition of gastrointestinal lipase activity, a proven therapeutic target for the control of obesity through reduced fat digestion, may be limited to proanthocyanidins. Taking into account the manifold possible synergies for inhibition of starch, protein and/or lipid digestion by the spectrum of polyphenol components present within berry species, the inhibition of digestive enzymes by dietary polyphenols may represent an under-reported mechanism for delivering some of the health benets attributed to a diet rich in fruit and vegetables. Keywords: -Amylase, anthocyanins, -glucosidase, inhibition, lipases, polyphenols, proteases

1. Introduction A consensus has developed over the last few decades that the health benets associated with a diet rich in fruit in vegetables may be derived, in part, from the intake of natural antioxidants [4]. The main antioxidants in fruits are vitamin C and the polyphenols [28]. The polyphenols encompass a range of chemical classes that share the ability to act as chain breaking antioxidants which are proposed to protect against the damage caused by oxygen free radicals to DNA, membrane and cellular components [18]. However, it is becoming clear that different classes of polyphenol compounds differ greatly in their bioavailability [29] and components such as anthocyanins, which are abundant in berries [10], have low bioavailability and/or poor stability in vivo [33] and are therefore unlikely to provide protection at the cellular level. A large proportion of the ingested polyphenol dose from berries will not be taken up into the circulation and passes through the upper gastrointestinal tract (GIT) to the large intestine where they may be biotransformed or broken down by the indigenous microora [3,14]. As a result, the health benets derived from a diet rich in polyphenol antioxidants may be partly delivered through effects carried out within the GIT. In this short review, we discuss the effects that berry polyphenols have during transit through the GIT, particularly their effects on digestive enzymes. In some cases, we include information on inhibition of digestive enzymes by polyphenols from other plant sources which are also present in berries or are structurally similar to berry polyphenols.

Corresponding author. Tel.: +44 1382 562731; Fax: +44 1382 562426; E-mail: gmcdou@scri.sari.ac.uk.

0951-6433/05/$17.00 2005 IOS Press and the authors. All rights reserved

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2. Starch degradation Polyphenolic fractions from plants can cause insulin-like effects in reducing blood glucose levels after food intake (see [5] for background). Certain polyphenols such as anthocyanins can directly induce secretion of insulin from pancreatic cells in ex vivo assays [22] but this effect may be marginal in vivo because of the low serum bioavailability of anthocyanins. The main effect on post-meal blood glucose may be due to inhibition of starch degradation within the GIT. Polyphenolic extracts from a number of plants were found to be effective inhibitors of intestinal -glucosidase/maltase activity [30] with Ki values in the same range as synthetic inhibitors (acarbose and voglibose) that are currently used therapeutically to control non-insulin dependent diabetes mellitus (NIDDM) [47]. The most effective inhibitory agents against -glucosidase activity proved to be diacylated anthocyanins [31], which alone were capable of inducing an anti-hyperglycemic effect in rats [32]. However, -glucosidase activity in vitro was also signicantly inhibited by anthocyanin-rich extracts of blueberry and blackcurrant which contain only a small proportion of acylated anthocyanins [34]. The enhanced inhibition exhibited by acylated anthocyanins over their deacylated forms [31] may reect the enhanced stability of the acylated anthocyanins at intestinal pH but such differences in effectiveness may not be particularly relevant when 200 mg of anthocyanins are available from a single portion of berries [8]. The mechanism of -glucosidase inhibition action by anthocyanins is not fully understood but one can assume that the inhibition, like that of acarbose, is competitive and results from the structural similarity between the normal substrate maltose and the glucosyl groups -linked to the anthocyanin which bind to the active site but are not hydrolysed. Further work is required to dene structure-activity relationships as regard to the aglycone and the attached sugars. The insulin-like action of certain polyphenol-rich plant extracts may also be due to inhibition of amylase activity [30,38]. Polyphenol-rich extracts of raspberry and strawberry were much more effective against -amylase than equivalent extracts from blackcurrant or blueberry [34]. An examination of the polyphenol composition of these berries suggested that ellagitannins may be the inhibitory agents and this was proved by fractionation and purication of the inhibitory ellagitannins from raspberry (Fig. 1). Tannins and proanthocyanidins, especially in forage crops and seeds [41], have long been known as anti-nutritive agents probably due to their ability to bind digestive enzymes. The effect is relatively non-specic but certain proteins, including -amylase [17], are much more susceptible. The possibility that anthocyanins and ellagitannins present in berries could synergistically inuence starch degradation, which could enhance their potential therapeutic effect on post-meal blood glucose levels, requires further attention. Little is known about the effects of polyphenols on the activity of intestinal brush border debranching enzymes, such as glucoamylase and isomaltase, which are required for the complete hydrolysis of the branched amylopectin fraction of starch [16]. As cereal starches can contain up to 50% amylopectin, these enzymes could have a large inuence of glucose load post-meals. These enzymes share similar tertiary structures with -amylase and -glucosidase [23] and may also be susceptible to inhibition by anthocyanins, ellagitannins or other polyphenols. 3. Lipid degradation Inhibition of lipid digestion through inhibition of lipase by compounds such as Orlistat TM has been a well dened clinical target for the treatment of obesity [7,11,36]. Extracts of Salacia reticulata are used in Japan to prevent diabetes and obesity and they exhibit pancreatic lipase inhibition which was partly

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191

3.0 2.5
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Fig. 1. Identication of ellagitannins as -amylase inhibitors in raspberry extracts. Figure redrawn from data contained in reference [34].

attributed to (-)epigallocatechin, (-)epigallocatechin dimers and a tannin fraction [49]. Polyphenolrich extracts of green tea inhibit both gastric and pancreatic lipase activity under GIT conditions [24] but the most active components were not identied. A recent, comprehensive study on the inhibition of pancreatic lipase by polyphenols isolated from oolong tea [35] suggested that epigallocatechin 3O-gallate (EGCG) was an effective inhibitor (IC50 = 0.35 M) but that unesteried avan-3-ols such as epicatechin and epigallocatechin were much less effective (IC50 > 20 M) or ineffective. This may explain the anti-obesity effects of TEAVIGO TM , a commercial source of EGCG, in rodents [48] and the reduction of postprandial hypertriacylglycerolemia by EGCG though delay of fat breakdown in the GIT [20]. However, epigallocaetchin-3, 5-digallate was even more effective (IC50 = 0.1 M) and polymers of avan-3-ols formed during oolong tea preparation were also potent inhibitors. The effectiveness of the avan-3-ol dimers and oligomers suggests that soluble proanthocyanidins found in a range of berries [25] could also inhibit lipase. Indeed, preliminary research showed that polyphenol-rich grape extracts also inhibit pancreatic and gastric lipase activity (personal communication, Professor Dennis Lairon, Universit e de la M editerran ee, Marseille). The requirement for the galloyl group for lipase inhibition by EGCG gives encouragement for studies on ellagitannin-rich strawberry and raspberry extracts on lipase activity [25]. 4. Protein degradation Green tea polyphenols, especially EGCG, have been found to inhibit a range of proteolytic activities ranging from invasive activities such as the matrix metalloproteases (MMPs) involved in tumour survival, expansion and metastatis [21,27] to proteases present in sh muscle [40]. The galloyl group seems be crucial for protease inhibition by EGCG and modelling studies have suggested that EGCG inhibits the serine protease urokinase by binding to the active site [21]. However, it is also possible that EGCG may chelate metal ions important for activity of metalloproteases [12], an ability which it shares with many polyphenolic compounds [6]. However, other polyphenols, such as the avonols, quercetin and myricetin and the anthocyanidin, pelargonidin, can inhibit matrix metalloproteases and serine proteases implicated in tumour metastasis at levels only 210 fold higher than EGCG [42]. Similarly, the avonols, isorhamnetin and rhamnocitrin

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have been reported to inhibit prolyl endopeptidase more effectively than the peptidase inhibitor, bacitracin [45]. In addition, hydrolysable ellagitannins, such as punicafolin, have been shown to be more potent inhibitors of MMPs and in vitro tumour invasiveness than EGCG [46]. Taking the low levels of bioavailability of some polyphenols into account, it is clear that the levels of polyphenol compounds will be much higher in the GIT than circulating in the serum. Therefore, compounds that inhibit proteases with IC50 values substantially higher than (say) EGCG can still cause physiologically-important changes in protein digestion within the GIT. Digestion of proteins in the human GIT is accomplished by the synergistic action of endoproteinases such as pepsin (stomach), trypsin, chymotrypsin and elastase (pancreatic) and exo-proteases such as carboxypeptidase and aminopeptidase. These enzymes share structural features and mechanisms to the proteases discussed above. Inhibition of protein digestion may be benecial to health in that it may reduce calorie intake or slow down food digestion leading to increased satiety due to enhanced gastrointestinal transit of the food bolus. However, high protein content meals may invoke greater satiety than equi-caloric low protein meals [1]. Whether inhibition of protein digestion could be benecial for health is less clear-cut than for carbohydrate or lipid digestion. Very little protein is stored in the liver, in muscles or other cells and reduced protein digestion in the GIT may be compensated by enhanced cellular protein catabolism to maintain homeostasis of amino acid levels [9]. In any case, examples of inhibition of proteases involved in digestive processes by polyphenols are scarce. Trypsin was inhibited by phenolic-rich extracts of pears, cocoa and lentils [38] but the composition of the extracts was not elucidated and probably contained proanthocyanidins or condensed tannins. These compounds achieve their anti-nutritional effect through a rather non-specic binding of proteins, including proteolytic enzymes [41]. Smaller Mr, soluble proanthocyanidins are present in a range of berries [28] and these compounds may inhibit protease activity in the GIT. Initial, unpublished studies have shown that raspberry extracts can inhibit trypsin at levels easily obtainable in the GIT post-meal (Fig. 2). The drastic reduction in trypsin activity noted with increasing amounts of extract suggests that the enzyme is being removed from solution, perhaps by binding to ellagitannins. Further work is required to discover if this inhibition occurs by the same mechanism as the inhibition of MMPs by punicafolin [46]. 5. Other digestive processes The presence of high levels of polyphenolic components in the GIT may have effects, other than on digestive enzymes, that may inuence the digestive system and health in general. Polyphenols may protect other components of the diet such as carotenoids and vitamin C from oxidation due to free radicals generated from other foodstuffs in the stomach [15]. The presence of these effective anti-oxidants may also protect digestive enzymes and the gut epithelial cells from oxidative damage. Polyphenols can interfere with the uptake of glucose [13,43] and thus provide another means to inuence post-meal blood glucose levels. Although polyphenols may protect vitamin C against oxidative damage, they may also inhibit uptake [44]. 6. Conclusions and directions The Food and Agriculture Organisation of the United Nations and the World Health Organization have recognized that the developed world faces an epidemic of chronic life-threatening and debili-

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Fig. 2. Inhibition of trypsin by a polyphenolic-rich extract of raspberry.

tating disease caused or exacerbated by poor diet [2]. The future demographic pattern in the developed world is of an ageing and more obese population increasingly faced by degenerative conditions such as cardiovascular disease, strokes, cancers and diabetes. But this is not a problem conned to the elderly, as increasing numbers of younger people are increasingly being diagnosed with symptoms covered by the umbrella term, Metabolic Syndrome [39] which can increase the likelihood of developing these degenerative diseases [26]. The substantial epidemiological evidence that a diet rich in fruit and vegetables can prevent these conditions, reduce premature death and reduce obesity has lead many governments to promote the benets of eating more fruit and vegetables (e.g. www.dh.gov.uk/PolicyAndGuidance/HealthAndSocialCareTopics/FiveADay). The mechanisms behind this dietary protection are still not clear but fruits and vegetables may offer multiple benets. Increased intake of minerals and natural antioxidants from fruit and vegetables may reduce oxidative damage associated with many disease states [19]. Although the anti-cancer effects of diets rich in fruit and vegetables have not been unambiguously proven [37], it is clear that polyphenols (and other phytochemicals) found in fruit and vegetables may have anti-proliferative activities against cancer cells ([50] and article by Juranic et al. in this edition). The reduced caloric density and higher bre content of fruit and vegetables compared to other foods may lead to increased satiety and reduced hunger. As highlighted in this review, the possibility that polyphenolic compounds found in fruits such as berries can inuence the digestion and subsequent uptake of foods and affect post-meal blood glucose and lipid levels deserves closer scrutiny. The benecial effects may lead to individual polyphenolic compounds being used as non-toxic alternatives to pharmaceutical treatments whereas polyphenolic-rich berry extracts (or the berries themselves) may provide nutraceutical treatments for non-insulin dependent or type II diabetes and obesity. The potential synergy for inhibition of lipid and starch by different polyphenolic components has particular value.

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