Analytical Methods for Determining Bioavailability and Bioaccessibility of Bioactive Compounds from Fruits and Vegetables: a Review Juana M

Analytical Methods for Determining Bioavailability and Bioaccessibility of Bioactive Compounds from Fruits and Vegetables: A Review Juana M. Carbonell-Capella, Magdalena Buniowska, Francisco J. Barba, Mar´ıa J. Esteve, and Ana. Fr´ıgola

Abstract: Determination of bioactive compounds content directly from foodstuff is not enough for the prediction of potential in vivo effects, as metabolites reaching the blood system may be different from the original compounds found in food, as a result of an intensive metabolism that takes place during absorption. Nutritional efficacy of food products may be ensured by the determination of bioaccessibility, which provides valuable information in order to select the appropriate dosage and source of food matrices. However, between all the methods available, there is a need to establish the best approach for the assessment of specific compounds. Comparison between in vivo and in vitro procedures used to determine bioaccessibility and bioavailability is carried out, taking into account the strengths and limitations of each experimental technique, along with an intensive description of actual approaches applied to assess bioaccessibility of bioactive compounds. Applications of these methods for specific bioactive compound’s bioaccessibility or bioavailability are also discussed, considering studies regarding the bioavailability of carotenoids, polyphenolic compounds, glucosinolates, vitamin E, and phytosterols. Keywords: bioaccessibility, bioactive compounds, bioavailability, in vitro methods, in vivo methods

Introduction ever, comparison of results between different studies is difficult to Nowadays, consumers are more and more aware of the benefits accomplish, as there is no defined experimental model for study- beyond basic nutrition provided by food and food compounds. ing bioaccessibility and bioavailability. Analysis of the procedures Between these, plant foods including fruits and vegetables have for measuring or predicting bioactive compounds bioavailability is been demonstrated to exhibit multiple health benefits, closely re- therefore required, particularly as a result of continuous develop- lated to their high contents in vitamins and other bioactive com- ments of new products by food industries considered “functional” pounds (vitamin C, carotenoids, phenolic compounds, vitamin because of their specific antioxidant or phytochemical contents. E, glucosinolates) with antioxidant properties (Nehir and Sim- The aim of the present article is to critically review different ap- sek 2012; Barba and others 2013; Carbonell-Capella and others proaches used in the estimation of bioaccessibility and bioavailabil- 2013a). However, when studying the role of bioactive compounds ity of food compounds, focusing on bioactive compounds, as these in human health, their bioavailability is not always well known. are of major interest in current functional food development. Fur- Before becoming bioavailable, they must be released from the food thermore, results of studies in which bioaccessibility and bioavail- matrix and modified in the gastrointestinal (GI) tract. Therefore, ability of bioactive compounds were investigated are also discussed. it is important before concluding on any potential health effect, to analyze whether the digestion process affects bioactive compounds Bioaccessibility, Bioavailability, and Bioactivity and their stability, as this, in turn, will affect their bioavailability The concept of bioaccessibility can be defined as the quan- and their possible beneficial effects. tity or fraction which is released from the food matrix in the GI Different digestion models have been developed by the scientific tract and becomes available for absorption (Heaney 2001). This community that accurately mimic the complex physicochemical includes digestive transformations of food into material ready for and physiological conditions of the human GI tract, along with assimilation, the absorption/assimilation into intestinal epithelium in vivo models in living organisms (Hur and others 2011). How- cells, and lastly, the presystemic metabolism (both intestinal and hepatic). For some nutrients, beneficial effects of unabsorbed nutrients (such as binding of bile salts by calcium in the tract) would MS 20131126 Submitted 08/9/2013, Accepted 10/17/2013. Authors are with Dept. of Nutrition and Food Chemistry, Univ. de Valencia,` Avda, Vicent Andres´ be missed by absorption-based definitions. Bioaccessibility is usu- Estelles,´ s/n. 46100 Burjassot, Spain. Direct inquiries to author Fr´ıgola (E-mail: ally evaluated by in vitro digestion procedures, generally simulating [email protected]). gastric and small intestinal digestion, sometimes followed by Caco- 2 cells uptake (Courraud and others 2013).

C 2014 Institute of Food Technologists® r doi: 10.1111/1541-4337.12049 Vol.13,2014 ComprehensiveReviewsinFoodScienceandFoodSafety 155 Bioavailability of bioactive compounds . . .

(hydrophilic/lipophilic), distribution in nature (specific to vegetable species/ubiquitous), range of concentrations both in foods In vivo and in the human body, possible site of action, effectiveness against methodologies oxidative species, and specificity and biological action (Porrini and Riso 2008). Among them, polyphenolic compounds, carotenoids, tocopherols, phytosterols, and organosulfur compounds constitute important groups in the human diet. In vitro Indeed, bioavailability of bioactive compounds may be modified methodologies because of interactions with other macronutrients such as fiber in low-processed foods and beverages or proteins and polysaccharides in processed food products (Dupas and others 2006). Furthermore, when different foods come in contact in the mouth or digestive In vitro , ex vivo, and in tract, various interactions may take place affecting phytochemical vivo methodologies bioavailability (for example, fat enhances quercetine bioavailabilty in meals) (Lesser and others 2006). On that basis, significant research effort has recently focused on achieving optimal uptake of phytochemicals to maintain body functions and health and, con- Figure 1–Definition of bioavailability, bioaccessibility, and bioactivity and their potential assessment methodologies. sequently, carefully controlled studies are necessary in order to determine phytochemical bioavailability. As shown in Figure 2, different approaches to study bioacces- Differently, the term bioavailability includes also in its defini- sibility and bioavailability of bioactive compounds include in vitro tion the utilization of a nutrient and therefore can be defined methods, ex vivo techniques, in situ assays, and in vivo models. Ad- as the fraction of ingested nutrient or compound that reaches the vantages of each procedure are summarized in Table 1.However, systemic circulation and is utilized (Wood 2005). Overall, bioavail- comparisons between different approaches are difficult as condi- ability includes GI digestion, absorption, metabolism, tissue distri- tions differ between them and only in vivo studies provide accurate bution, and bioactivity. Consequently, in terms of bioavailability, values (Oomen and others 2002). when a claim is made, it must be demonstrated that the com- ponent analyzed is efficiently digested and assimilated and then, Approaches in the Assessment of Bioaccessibility once absorbed, exerts a positive effect in human health. How- and Bioavailability of Bioactive Compounds ever, practical and ethical difficulties are found when measuring Carotenoids bioactivity, so the term “bioavailability” is usually defined as the Carotenoids are found in fruits and vegetables as carotenes fraction of a given compound or its metabolite that reaches the (unsatured hydrocarbons) and xanthophylls (oxygenated deriva- systemic circulation (Holst and Williamson 2008), without con- tives). Generally, the main carotenoids in vegetables are lutein, sidering bioactivity. According to this definition, bioavailability of β-carotene, violaxanthin, and neoxanthin, whereas in fruits xan- a compound is determined in vivo in animals or humans as the thophylls are usually found in a greater proportion. They are prone area under the curve (plasma-concentration) of the compound to isomerization and/or oxidation due to their unsaturation (Hill obtained after administration of an acute or chronic dose of an and Johnson 2012). isolated compound or a compound-containing food (Rein and Only a very low proportion of carotenoids has been re- others 2013). ported to become bioaccesible (Courraud and others 2013). In Bioactivity is the specific effect upon exposure to a substance. It some fruits (such as mango, papaya) carotenoids are found in oil includes tissue uptake and the consequent physiological response droplets in chromoplast and hydroxycarotenoids are mostly esteri- (such as antioxidant, anti-inflammatory). It can be evaluated in fied with fatty acids, being more easily extracted during digestion. vivo, ex vivo,andin vitro (Figure 1)(Fernandez-Garc´ ´ıa and others Carotenoids bioavailability from foods varies greatly depending 2009). on endogenous (product-related) and exogenous (process-related) Meanwhile, digestibility applies specifically to the fraction of factors. Amount and type of fat present in the vicinity is a key food components that is transformed by digestion into potentially factor that affects bioaccessibility. A minimum amount of fat is accessible matter through all physical–chemical processes that take necessary for absorption (Fernandez-Garc´ ´ıa and others 2012), so place in the lumen. Assimilation, meanwhile, refers to the uptake formulation of carotenoids in an oily matrix may enhance higher of bioaccessible material through the epithelium by some mecha- bioaccessibility. Important steps in carotenoid absorption are re- nism of transepithelial absorption (Etcheverry and others 2012). lease from the food matrix, micelle formation, uptake into mucosal cells, packing into chylomicrons, and transport within the lym- Bioavailability of Bioactive Compounds phatic system. Moreover, carotenoids content might be affected Bioactive compounds are phytochemicals that are present in by oxidative reactions during analytical procedures, so incubation foods and are capable of modulating metabolic processes, resulting time should be kept to a minimum without affecting sensitivity. in the promotion of better health. In general, these compounds Garret and others (2000) added α-tocopherol in order to ensure are mainly found in plant foods such as fruit, vegetables, and protection against oxidation and thus improve carotenoids stability. whole grains (Carbonell-Capella and others 2013b; Gil-Chavez´ Different in vitro methods used in the assessment of carotenoid and others 2013) and typically occur in small amounts. These bioaccessibility comprise simulated GI digestion, intestinal seg- compounds exhibit beneficial effects such as antioxidant action, ments, brush-border and basolateral membrane vesicles, entero- inhibition or induction of enzymes, inhibition of receptor activ- cytes, and transformed intestinal cell lines, mainly Caco-2 human ities, and induction and inhibition of gene expression (Correia cells (Table 2). Garret and others (1999) may be considered the and others 2012). They can be considered an extremely hetero- pioneers in the development of the Caco-2 procedure for the geneous class of compounds with different chemical structures assessment of carotenoid bioaccessibility. The method consists of r 156 Comprehensive Reviews in Food Science and Food Safety Vol. 13, 2014 C 2014 Institute of Food Technologists® Bioavailability of bioactive compounds . . .

Methods used in the assesment of bioaccesibility and bioavailability of bioactive compounds

In vitro models Ex vivo models In situ models In vivo models

-Simulated -Gastrointestinal -Intestinal -Animal studies gastrointestinal organs in laboratory perfusion in -Human studies digestion conditions animals -Artificial membranes -Caco-2 cell cultures -Isolated/Reconstituted cell membranes - Ussing chambers

Figure 2–Methodologies used in the assessment of bioavailability and bioaccessibility of bioactive compounds.

Table 1–Strengths and drawbacks of in vivo and in vitro procedures used to assess bioaccessibility and bioavailability of bioactive compounds.

Advantages Disadvantages In vitro digestion Simulated gastrointestina Relatively inexpensive and technically simple Extrapolation to in vivo digestion Screening of numerous samples is possible Homeostatic mechanisms are not present Focus on small number of components Dynamic conditions of gastrointestinal tract are not fully reproduced with biochemical and cell culture models Specific mechanisms of action can be tested Intestinal bacteria and hepatic metabolism is not always considered Validation with reference material Oral and large intestinal phases are often not included although can readily be added Efficiency of each digestion, absorption or transport Closed system not responsive to composition and quantity of foods mechanism can be studied Exocrine pancreas secretions not only contains pancreatin Caco-2 cells Phenotype is similar to normal absorptive epithelial cells Original from human colonic adencarcinoma Grow on dish surface and on membrane inserts Mucin, biofilms, and other epithelial cell types are not present Secretion of chylomicrons is possible In vivo digestion In vivo conditions Lower throughput Selection of specific subjects Extremely complex functional systems Pharmacokinetic studies can be performed Influence of different factors Extrapolation from animal studies to human Certified reference standards lack High cost of equipment and labor Ethical constraints an in vitro digestion including a gastric and small intestinal step Hedren´ and others (2002) also developed an in vitro digestion based on that described by Miller and others (1981) to estimate method for the estimation of carotenoid bioaccessibility (called in iron availability from foods. Subsequently, the digestate is filtered vitro accessibility) in raw and cooked carrots, which was further (which would be representative of micellarized carotenoids) and used in several different studies assessing carotenoid bioaccessibility added to Caco-2 cells. To ensure that carotenoids were found in (Lemmens and others 2009; Colle and others 2013). The groups micelles, these authors filtered the aqueus fraction. They ascer- carefully examined critical steps in the digestion procedure, such tained that lycopene was poorly micellarized and thus its quantity as the impact of added pancreatic enzymes and different bile salts decreased after filtration, but lutein, α-carotene, and β-carotene amounts, along with shaking conditions used in the micellariza- did not change in their quantities. They also observed that hy- tion step, so as to validate the method. When bile salts were not drophobic species were efficiently micellarized when bile salts and added, β-carotene bioaccessibility decreased by about 80%, but pancreatic enzymes were combined. Furthermore, these authors duplicating the amount of bile salts (from 25 to 50 g/L) resulted found out that differentiated Caco-2 cells were able to accumu- in no additional increment of carotenoid bioaccessibility. More- late carotenoids from mixed micelles. Further modifications were over, orbital shaking gave more reproducible results in comparison made to this method by Thakkar and others (2007)whoin- with reciprocal shaking. In contrast to data by Garret and others cluded an oral digestion phase because of high starch content, (1999), they estimated not only the micellarized fraction, but total and by Chitchumroonchokchai and Failla (2006) who added li- carotenoids released, as the intestinal phase was not achieved by pase and carboxyl ester lipase. These latter authors observed that centrifugation and filtration. Courraud and others (2013)intro- xanthophyll esters were hydrolyzed by carboxyl ester lipase be- duced an oral phase to Hedren’s´ method, without α-amylase as fore xanthophylls are transported into enterocytes, resulting in an most of the matrices were nonstarchy. They obtained a significant enhanced cellular accumulation of zeaxanthin. loss of β-carotene only during the gastric phase and of retinyl

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g palmitate in the oral and in the gastric phases, conﬁrming that

× sensitivity of carotenoids to acidic conditions is higher than to ) alkaline conditions.

2012 Reboul and others (2006) made some modiﬁcations to the C. ◦

C method established by Garret and others (1999). BHT used as ◦

C antioxidant was replaced by pyrogallol, more water soluble. Gas- ◦ tric pH was set at 4 instead of 2, simulating the pH in the human 37 porcine bile, pH 6.5, 2h,37 1h,4 Porcine pepsin, pH 2, 2h, Porcine pancreatin, Centrifugation 3300 stomach after vegetable-rich meals ingestion. Moreover, duodenal pH was adjusted to 6 instead of 7.5 as this is the pH measured in human duodenum during digestion. Instead of 2 h of incu-

× bation time, duodenal conditions were adjusted to 30 min to ) others ( approach the digestive transit time, and amount of bile salts were pH 2, 30 C, C. ◦

◦ increased. They observed that carotenoid bioaccessibility was de- 2010 +

C C pendent of the different food matrix, being more bioaccessible ◦ ◦ in carrot juice and processed tomato in comparison with crude tomato and watermelon sources, which had very low accessibility. ,65min,4 min, 37 min, 37 porcine bile salt, pH 6.9, 2 h, 37 ﬁltration g Wer ner and Bohm¨ (2011) employed this procedure in the assess- Porcine pepsin, pH 4, 30 Porcine pancreatin, Homogenization in saline Homogenization Centrifugation 16500 ment of carotenoid bioaccessibility in durum wheat and egg pasta. Durum wheat pasta exhibited higher carotenoid bioaccessibility. The authors also observed that results were highly dependent on

) others ( bile extract concentration and to a lesser extent on gastric pH and C ◦ incubation time with digestive enzymes. 2007 As previous methods were found unsuitable for xanthophyll ester hydrolysis, Granado-Lorencio and others (2007) adapted a C C ◦ ◦

, taurocholate salts, method originally applied to the evaluation of soil contaminants. 2 pancreatin, human pancreatic lipase, colipase, cholesterol esterase, phospolipase A albumin, porcine pepsin, pH 1.1, 1 h, 37 kitchen blender, 15 s, to simulate mastication pH 7.8, 2 h, 37 sedimentation/ Centrifugation 5000 rpm, 20 min 37 Compared to previous in vitro models, these authors included the -Amylase, pH 6.5, 5 min, Bovine bile, porcine Mucin, bovine serum Homogenization with α Overnight use of human pancreatic lipase, phospholipase A2, cholesterol esterase, and taurocholate salts. They obtained a remainder of over

Method 70% of carotenoids in the ﬁnal digesta and observed that cholesterol esterase hydrolyzed xanthophyll esters, and human pancreatic ) others ( C, C lipase did not. ◦ ◦

2006 The in vitro digestion procedure for carotenoids followed by C

◦ Wright and others (2008), as adapted from Garret and others (1999) and Hedren´ and others (2002) consisted of dissolving the pyrogallol carotenoids in the oily phase, considering thus exclusively the min, 37 + porcine pancreatin, pH 6, 30 min, 37 ﬁltration rpm, 18 h, 10 Porcine pepsin, pH 4, 30 Homogenization in saline Porcine bile extract, Centrifugation 20000 intestinal digestion phase, as previous research had not shown sig- niﬁcant changes when eliminating the gastric step (Garret and β

, others 1999). They observed that -carotene transfer increased as g did bile (from 0 up to 20 mg/mL) and pancreatin concentration × ) others ( C.

◦ (from 0 up to 4.8 mg/mL) and with pH from 3.5 to 9. Colle and others (2010) also introduced several modiﬁcations ´ en Reboul Granado-Lorencio Colle Cilla 2002 to the method established by Hedren´ and others (2002). Both the pH and transit times were adapted to closely simulate human C ◦ conditions. A certain amount of lipid (0% to 10%) was added small pieces, with nitrogen blown 37 20 min porcine bile salt, pH 7.5, 30 min, 37 to tomatoes prior to the in vitro digestion. A signiﬁcant increase Porcine pepsin, pH 2, 1 h, Centrifugation 5000 Porcine pancreatin, of lycopene bioaccessibility was observed when 5% of lipid was additioned. , )Hedr

g Cilla and others (2012) adapted a method used for iron bioacces- ) others ( × C

◦ sibility to determine carotenoid bioaccessibility of fruit juice-milk 2008 beverages, along with other bioactive compounds. Bioaccessibil- 1999 C, ﬁltration

◦ ity of carotenoids was dependent of the type of milk used. Whole C C ◦ ◦ milk-fruit beverage led to a higher carotenoid extraction (11%) in methods for carotenoid bioaccessibility determination. C

◦ comparison to the skimmed milk-fruit beverage. carboxyl ester lipase, porcine pancreatin, lipase, pH 6.5, 2 h, 37 45 min, 4 1h,37 min, 37 As reported in the review by Rodriguez-Amaya (2010), al- (adapted from Garret and and and and and -Amylase, pH 6.8, 10 Failla and others ( in vitro α Porcine bile extract, Centrifugation 5000 Homogenization Finely ground or cut into though these models simulate human digestion closely, a better description of the food sample preparation should be carried out. Furthermore, carotenoid extraction efﬁciency from food and micelles should be similar, so no overestimation or underestimation of micellarization is done. Results obtained in the different studies are shown in Table 3. The dynamic digestion TIMR system that more closely mim- phase micellar fraction preparation Small intestine Step and others Isolation of Cell uptake Caco-2 cells, 4h, 37 Gastric phase Porcine pepsin, pH 2.5, Table 2–Comparison of Food sample Oral phase ics in vivo conditions was also employed in the assessment of

r 158 Comprehensive Reviews in Food Science and Food Safety Vol. 13, 2014 C 2014 Institute of Food Technologists® Bioavailability of bioactive compounds . . .

Table 3–Carotenoid bioaccessibility and bioavailability (%) of plant-derived products.

Carotenoids Method employed Sample bioavailability (%) in the determination Reference Baby food meal Lutein (24); β-carotene (13.8); Simulated gastric and small Garret and others (1999) α-carotene (10.4) intestinal digestion coupled with Caco-2 cells Spinach purée β-Carotene (29); lutein (27) Simulated gastric and small Ferruzzi and others (2001) intestinal digestion (Garret and others 1999) Raw pulped carrot β-Carotene (21) Simulated gastric and small Hedrén and others (2002) intestinal digestion Carrot puree β-Carotene (8.9); α-carotene Simulated gastric and small Reboul and others (2006) (4.4) intestinal digestion Spinach Lutein (37.6); α-carotene (2.4) Simulated gastric and small Reboul and others (2006) intestinal digestion Orange β-Cryptoxanthin (45); zeaxanthin Simulated oral, gastric, and small Granado-Lorencio and others (43); lutein (26) intestinal digestion (2007) Salad (tomato, spinach, carrot, Lutein (+zeaxanthin) (45.6); Simulated gastric and small Huo and others (2007) romaine lettuce, and orange β-carotene (2.8); α-carotene intestinal digestion pepper) (2.0); lycopene (1.1) Boiled cassava β-Carotene (30) Simulated oral, gastric, and small Failla and others (2008) intestinal digestion coupled with Caco-2 (Garret and others 1999). Lycopene from tomato extract Lycopene In vivo single dose design Riso and others (2010) Orange fleshed melons β-Carotene (3.2) Simulated gastric and small Fleshman and others (2011) intestinal digestion Tomato pulp Lycopene (2) Simulated gastric and small Colle and others (2012) intestinal digestion (Colle and others 2012) Butternut squash α-Carotene (17.9); β-carotene Simulated gastric and small Jeffery and others (2012a) (16.5); lutein (15.9); intestinal digestion (Garret and violaxanthin (4.3) others 1999) Carrot Lycopene (38.9); α-carotene Simulated gastric and small Jeffery and others (2012a) (20.2); β-carotene (21.6); intestinal digestion (Garret and lutein (40.5); phytoene (64.2) others 1999) Grapefruit Lycopene (4.5); β-carotene (7.9); Simulated gastric and small Jeffery and others (2012a) lutein (8.7); violaxanthin (8.4); intestinal digestion (Garret and phytoene (47.1) others 1999) Mango β-Carotene (31.8); lutein (13.5); Simulated gastric and small Jeffery and others (2012a) violaxanthin (19.4) intestinal digestion (Garret and others 1999) Papaya β-Carotene (48.5); lutein (37.3); Simulated gastric and small Jeffery and others (2012a) violaxanthin (21.6); phytoene intestinal digestion (Garret and (67.8) others 1999) Tomato Lycopene (1.4); β-carotene Simulated gastric and small Jeffery and others (2012a) (15.5); lutein (58.6); phytoene intestinal digestion (Garret and (96.2) others 1999) Whole milk-fruit beverage Neoxanthin + 9-cis-violaxanthin Simulated gastric and small Cilla and others (2012) (47.3); zeaxanthin (14.7); intestinal digestion lutein (13.9) Soy milk-fruit beverage Neoxanthin + 9-cis-violaxanthin Simulated gastric and small Cilla and others (2012) (18.5); zeaxanthin (4.4); lutein intestinal digestion (3.7) Carrot juice Lutein (22); α-carotene (1.5), Simulated gastric and small Courraud and others (2013) β-carotene (1.5) intestinal digestion (Hedrén and others 2002) lycopene bioaccessibility by Deat´ and others (2009), followed Tyssandier and others 2003). The most frequently used in vivo ap- with Caco-2 cells. At the end of the dynamic experiment (300 proach to study bioavailability of carotenoids involves the single- min), lycopene decreased by 25%, in accordance with static in vitro dose design. An increase in β-cryptoxanthin, β-carotene, and models. zeaxanthin plasma concentrations was observed after supplemen- Animal studies have also been designed in the assessment of tation of blood orange juice in a long-term human study (Riso carotenoid bioavailability (Zuniga and Erdman 2011; Sy and others and others 2005), although this did not exert significant effects on 2012). They obtained more accurate results than with in vitro meth- several markers of oxidative stress. Meanwhile, Riso and others ods. Despite this, human carotenoid absorption and metabolism (2010) found a low increase of lycopene, along with interindivid- is not accurately mimicked by any animal model (Lee and oth- ual variability. Interestingly, Ross and others (2011) demonstrated ers 1999). For instance, in enterocytes, β-carotene is converted the fate of oral lycopene in humans in plasma, with the detec- to vitamin A in rodents much more efficiently than in humans. tion in skin for up to 42 d of lycopene and its metabolites. Goltz Likewise, Failla and others (2008) observed that gerbils and preru- and others (2013) observed that carotenoid absorption increased minant calves, but not ferrets, hydrolyzed the ingested β-carotene when vegetables were consumed in a single meal rather than over to vitamin A with an efficiency similar to humans. multiple meals. Many studies have examined carotenoid bioavailability in hu- Borel and others (1998) demonstrated a high correlation be- mans (Micozzi and others 1992; Castenmiller and others 1999; tween in vitro carotenoid bioaccessibility, in vivo observations

r C 2014 Institute of Food Technologists® Vol. 13, 2014 Comprehensive Reviews in Food Science and Food Safety 159 Bioavailability of bioactive compounds . . . and with results from bioavailability trials with human subjects. Contrary to expectation, several authors (Parada and Aguilera Therefore, in vitro models may constitute a less tedious and less 2007; Courraud and others 2013) have demonstrated that techno- costly alternative to in vivo studies in the assessment of carotenoid logical processes such as cooking of vegetables increase carotenoid bioaccessibility. bioavailability by disruption of the natural food matrix during food Studies show that percentages of bioaccessibility and bioavail- processing. However, severe thermal treatment or inadequate stor- ability of the different carotenoids vary widely. Lutein was more age may cause isomerization during the formation of by-products readily solubilized than α-carotene, β-carotene, and lycopene that can, in turn, reduce the absorption of desirable bioactive (Garret and others 1999), probably because oxycarotenoids are compounds. more hydrophilic than hydrocarbon carotenoids and to different subcellular location and molecular interactions in plant foods. Polyphenolic compounds Sy and others (2012) also obtained a high recovery of lutein Phenolic compounds or polyphenols form a large group of and astaxanthin, whereas lycopene was the least abundantly re- chemical substances considered as secondary metabolites of plants. covered. However, Jeffery and others (2012a) reported for the They have an aromatic ring and a benzene ring with one or more 1st time a high phytoene bioaccessibility, several times that of hydroxide groups, including phenolic acids (hydroxy-benzoic other carotenoids, followed by lutein in carrot and tomato and acids and hydroxy-trans-cinnamic acids), coumarins, flavonoids β-carotene in papaya and mango. In human studies, Tyssiander (flavones, flavonols, flavanones, flavanolols, flavanols, and antho- and others (2003) reported greater bioavailability of lutein and cyanidins), isoflavonoids, lignans, stilbenes, and phenolic poly- β-carotene compared to lycopene. mers (proanthocyanidins and hydrolyzable tanins) (Craft and oth- Furthermore, dietary fat appears to be necessary for the effi- ers 2012). Among the various phenolic compounds, bioavailability cient solubilization of lipophilic compounds. In this line, Failla appears to differ greatly and the most abundant ones in our diet and others (2008) demonstrated an increase in carotenes when do not necessarily correspond to those with best bioavailability triglycerides were added to a carotenoid-rich salad, in accordance profile. Absorption and metabolism of polyphenolic compounds with Hedren´ and others (2002), who observed a significant in- are determined primarily by their physicochemical characteristics. crease in β-carotene bioaccessibility after oil addition. Qian and For example, molecular size, their basic structure, degree of poly- others (2012) found the lowest bioaccessibility of β-carotene (0%) merization or glycosylation, solubility, and conjugation with other when orange oil was used as the carrier lipid, probably because phenolics can be considered critical factors. Phenolic acids with flavored oils do not contain triacylglycerol components and thus small-molecular weight such as gallic acid and isoflavones are eas- cannot be digested into free fatty acids. Moreover, Borel and others ily absorbed through the tract, as well as flavones, catechins, and (1998) demonstrated that β-carotene incorporated into chylomi- quercetin glucosides (Martin and Apple 2010). On the contrary, crons higher in meals with long-chain rather than medium-chain large polyphenols such as proanthocyanidins are poorly absorbed. triglycerides. For this reason, Jeffery and others (2012b) used yo- Most proanthocyanidins are degraded into monomer or dimer gurt as a lipid source with long-chain triglycerides. Human stud- units before being absorbed (Hackman and others 2008). ies have also proved the importance of lipid in the absorption In plant products, most of the phenolic compounds are found of dietary lutein. On this subject, Mamatha and Baskaran (2011) as glycosylated forms or as esters or polymers that must be hy- obtained a higher plasma lutein level in rats when lutein was sol- drolyzed by intestinal enzymes or microflora before the released ubilized in mixed micelles with fat. Brown and others (2004) also aglycones can be absorbed. However, anthocyanins can be ab- observed that consumption in humans of full-fat salad dressing en- sorbed as glycosides and appear as such in blood (D’archivio and hanced a higher carotenoid bioavailability than reduced-fat salad others 2007). Metabolism is another factor, strongly affecting their dressing. bioavailability. Generally, after absorption, polyphenols undergo Interestingly, not only lipid amount, but also qualitative lipid biotransformations of phase I and II into 3 main O-sulfated, O- profile has its influence in carotenoid bioavailability (Goltz glucuronidated, and O-methylated forms. Despite this, antho- and Ferruzi 2013). Monounsatured fatty acids promote a cyanins do not appear to undergo extensive metabolism. Neither higher carotenoid bioavailability than polyunsatured fatty acids, do galloylated monomeric flavonols such as epigallocatechin and as demonstrated by Clark and others (2000) in mesenteric epicatechin gallate, which may appear unconjugated, at least to lymph duct cannulated rats. This was further observed by a large extent, in the systemic circulation (Cermak and others Gleize and others (2013), who found that bioaccessibility of 2009). Thus, the structure of the resulting metabolites could be the xanthophylls lutein and zeaxanthin was higher with sat- totally different from the parent compounds, and they may or not urated fatty acids than with monounsaturated and polyunsat- exert their biological action (Denev and others 2012). Results ured fatty acids both in an in vitro digestion model followed published by Vitaglione and others (2007) suggested that proto- by Caco-2 cell study and in vivo in orally administered rats. catechuic acid, which can be absorbed both from the small and Furthermore, long-chain triglyceride increased the β-carotene large intestine, may be the metabolite involved in the activity ob- bioaccessibility in comparison with medium-chain triglyceride served after the intake of cyanidin-3-glucoside, whose absorption in a simulated intestinal digestion (Salvia-Trujillo and others and excretion are reported to be below 1% of intake. Therefore, 2013). evaluation of polyphenol bioavailability should include the analysis Effect of pH on the transfer efficiency of carotenoids is also not only of native compounds, but also their metabolic products. of importance, as suggested by in vitro results. Wright and others Technological processes may also affect bioavailability of phenolic (2008) demonstrated an increase in the β-carotene transfer to the compounds, showing a significant increase of chlorogenic acid and aqueous phase under higher pH conditions, while Jeffery and naringenin in plasma levels when consuming cooked tomato in others (2012b) obtained a positive correlation of β-carotene and comparison with the fresh product (Bugianesi and others 2004). phytoene with food pH. However, this hypothesis can be rejected Despite the great variability of this group of substances, along in in vivo methods, because there is no significant meal effect on with their occurrence in plant materials as a complex mixture, ex- stomach pH (Tyssandier and others 2003). periments reported in the literature have analyzed bioavailability

r 160 Comprehensive Reviews in Food Science and Food Safety Vol. 13, 2014 C 2014 Institute of Food Technologists® Bioavailability of bioactive compounds . . . of polyphenolic compounds with different chemical structures

and solubility through in vitro and in vivo assays, as shown in ) Table 4. The most widely used procedure for screening polyphe- C 2013 nolic compound bioaccessibility is the in vitro static GI method. ◦ C Gil-Izquierdo and others (2001) may be considered the pioneers ◦ in adapting the method established by Miller and others (1981) porcine pancreatin, taurodeoxycholate, taurocholate, pH 7.4, 2.5 h, 37 to simulate human digestion and absorption of dietary iron in the 1h,37 Porcine bile extract, Porcine pepsin, pH 2, study of phenolic compound release. During the intestinal phase, Homogenization a cellulose dialysis tubing is used to simulate intestinal absorption.

The main modiﬁcation introduced by Gil-Izquierdo and others C. (2002) was the placement of the food and cellulose dialysis tubing ◦ in a polyethylene tube to assure close contact between food and C membrane, reaching faster equilibration of pH values and thus a ◦ C C faster liquid exchange. They observed that phenolic composition ◦ ◦ 37 porcine pancreatin, lipase, pH 7, 2 h, 37 1h,37 rpm, 30 min, 4 was not affected by pepsin digestion in any of the assayed food saline Amylase, pH 6.9, 5 min, Porcine bile extract, Porcine pepsin, pH 2, Centrifugation at 3000 products. This method has been employed in the screening of Homogenization in multiple foods, including orange juice (Gil-Izquierdo and others 2002), pomegranate juice (Perez-Vicente´ and others 2002), broc- C ◦

coli (Vallejo and others 2004), soymilk (Rodriguez-Roque and ) others (2012) others ( C ◦ others 2013) and gooseberry (Chiang and others 2013) among C C ◦ ◦ 2012 other foods. These authors found that gastric digestion increased -amylase, 10 α

polyphenolic concentration, whereas the duodenal fraction signif- 2h, 37 1h,37 porcine pancreatin, pH 7, 2 h, 37 semipermeable cellulose membrane 2 × centrifugation icantly diminished polyphenolic content and even more so in the min, 37 Mucin, Porcine pepsin, pH 1.2, dialyzed fraction. Results are shown in Table 5. Porcine bile extract, Further modiﬁcations (Villanueva-Carvajal and others 2013)

included the use of crushed ice after each digestion phase to ensure )( C the end of enzymatic activity. These authors also studied particle ◦ size and concluded that this was inversely proportional to phenolic 2007 C ◦

release, so the enlargement of the contact area could improve Method ´ digestion efﬁciency with an absorption increase of polyphenols. udez-Soto and Gawlik-Dziki Shim and Chen and porcine pancreatin, pH 7, 2 h, 37 Bermudez-Soto´ and others (2007) removed the employment 2h,37 Porcine bile extract, Filtration Dialysis in a of the dialysis membrane during intestinal digestion as substantial Porcine pepsin, pH 2, losses were observed of some of the phenolic compounds. They

simply determined polyphenolic compounds after separation by ) others ( C

filtration. During gastric digestion no significant changes were ◦ observed in the stability of polyphenols in chokeberry, but antho- 2006 C cyanins were increased, due to the low pH after the gastric step. ◦ During intestinal digestion a significant decrease in anthocyanins porcine pancreatin, pH 6, 2 h, 37 (43%) and flavonols (26%) was observed, whereas chlorogenic acid 1h,37 Porcine bile extract, increased (24%). This method was further employed by Tagliazuc- Porcine pepsin, pH 2, chi and others (2010) in the assessment of grape polyphenols, C ◦ C. Cooling in ice for 10 min pH 6 pH 5.3 pH 5.3 ◦

with the addition of an oral phase. Furthermore, once the pan- ) others (

creatic digestion was ﬁnalized, samples were taken to pH 2 to C ◦ ensure the stability of phenolic compounds. Differently, these au- 2011 thors observed an increase in the bioaccessibility of total polyphenols, ﬂavonoids, and anthocyanins during the gastric digestion in semipermeable cellulose membrane simultaneously with intestinal phase porcine pancreatin, pH 7 to 7.5, 2 h, 37 grape, while intestinal digestion caused a decrease in all classes of 2.5, 1 h, 37 Dialysis in a Porcine bile extract, polyphenols. Porcine pepsin, pH 2 to Bouayed and others (2011) also developed a method to assess

free soluble polyphenols potentially available for further uptake ) others ( than from Miller’s method. They found out that after simulated

GI digestion of apples, polyphenols release was mainly achieved 2001 C methods for polyphenolic bioaccessibility determination. during the gastric phase. Subsequently, a further increase (<10%) ◦ C in total phenolics and ﬂavonoids was obtained after the intestinal ◦ Gil-Izquierdo and Bouayed and Dupas and Berm in vitro porcine pancreatin, lipase, pH 7, 2 h, 37 semipermeable cellulose membrane simultaneously with intestinal phase phase. This increase may be due to the additional time of extrac- 2h,37 Porcine bile extract, tion along with the effect of intestinal enzyme on the complex Homogenization Homogenization Homogenization and food matrix, which facilitates the release of phenolics bound to the matrix. Results showed a dialyzability of 40% for ﬂavonoids and 55% for free soluble phenolics, respectively, in comparison with their undigested counterparts in apples. Regarding anthocyanins, these authors could not measure them after gastric and intestinal phase preparation Separation Dialysis in a Gastric phase Porcine pepsin, pH 2, Cell uptake Caco-2 cell Small intestine Transition step pH 6.5, 45 min, 37 Table 4–Comparison of Food sample Oral phase digestion, probably because they degradate in alkaline intestinal Step others (

r C 2014 Institute of Food Technologists® Vol. 13, 2014 Comprehensive Reviews in Food Science and Food Safety 161 Bioavailability of bioactive compounds . . .

Table 5–Polyphenolic bioaccessibility and bioavailability (%) of plant-derived products.

Polyphenolic Method employed Sample bioavailability (%) in the determination Reference Orange juice (soluble fraction) Narirutin (10.5); hesperidin (16.2); Simulated gastric and small intestinal Gil-Izquierdo and others (2001) total flavanones (12.0), vicenin-2 digestion with cellulose dialysis (18.6) tubing Orange juice (soluble fraction) Narirutin (23.4); hesperidin (24.0); Simulated gastric and small intestinal Gil-Izquierdo and others (2002) hesperetin (21.1); total flavanones digestion with cellulose dialysis (23.5), vicenin-2 (24.5) tubing Strawberry Cyanidin-3-glucoside (6.6), Simulated gastric and small intestinal Gil-Izquierdo and others (2002) pelargonidin-3-glucoside (12.6); digestion with cellulose dialysis pelargonidin-rutinoside (11.7); tubing ellagic acid-arabinoside (20.6); ellagic acid (172.8); quercetin-3-glucoside (28.3); kaempferol-3-glucoside (27.4) Strawberry jam Cyanidin-3-glucoside (2.3), Simulated gastric and small intestinal Gil-Izquierdo and others (2002) pelargonidin-3-glucoside (3.7); digestion with cellulose dialysis pelargonidin-rutinoside (3.8); tubing ellagic acid-arabinoside (6.1); ellagic acid (9.7); quercetin-3-glucoside (6.1); kaempferol-3-glucoside (12.0) Coffee Chlorogenic acid (traces); benzoic In vivo digestion in rats Dupas and others (2006) acid (traces) Chokeberry Cyanidin-3-glucoside (56.7), cyanidin Simulated gastric and small intestinal Bermudez-Soto´ and others (2007) (0), quercetin 3-glucoside (81.2), digestion followed by filtration quercetin (0), neochlorogenic acid (71.6), chlorogenic acid (123.6). Tixia gooseberry Caffeic acid (44.1), epigallocatechin Simulated gastric and small intestinal Chiang and others (2013) gallate (28.1), kaempferol (100.2), digestion according to ρ-coumaric (61.0), pelargonidin Gil-Izquierdo and others (2001) chloride (96.6), quercetin hydrate (516.3), resveratrol (58.1), rutin (95.3). Invicta gooseberry Caffeic acid (59.5), kaempferol Simulated gastric and small intestinal Chiang and others (2013) (82.8), ρ-coumaric (73.4), digestion according to quercetin hydrate (154.7), Gil-Izquierdo and others (2001) resveratrol (94.6), rutin (101.0). environment. Further studies carried out by Bouayed and others cessibility of dietary polyphenols with the isolated indigestible frac- (2012) found that phenolic compounds in the gastric or intesti- tion (small intestine bioaccessibility) and a colonic fermentation nal medium were approximately similar (chlorogenic acid), higher of this fraction (large intestine bioaccessibility). Bioaccessibility of (phloridzin and quercetin 3-О-glucoside) or lower (ρ-coumaric polyphenols in the large intestine was calculated by the difference acid) compared to those found in fresh apples, in accord with of polyphenol contents between the total indigestile fraction and results obtained by Chiang and others (2013). Polyphenol con- the residue after the fermentation; and 48% of dietary polyphe- centration decreased during dialysis through the semipermeable nols were estimated bioaccessible in the small intestine, while 42% cellulose membrane, although all polyphenols in the intestinal became bioaccesible in the large intestine. Only 10% was not medium were dialyzable, which could be indicative of passive dif- accessible and remained in the food matrix after the entire diges- fusion, an important mechanisms for cellular polyphenol uptake, tion process. Furthermore, Nordlund and others (2012) used an at least for several aglycones. in vitro colon model to study the formation of phenolic microbial Gawlik-Dziki (2012) carried out an in vitro digestion includ- metabolites from rye, wheat, and oat bran. The major metabo- ing an oral phase. Dialysis sacks were added after 2 h of intestinal lites found were hydroxylated phenylpropionic acid metabolites, digestion, for a total time of 4 h. They also obtained a decrease closely related to the ferulic acid content in the cereal samples. of phenolic compounds in the dialysate. Shim (2012) also in- The dynamic GI model (TIMR ) has also been extensively used cluded an oral phase. However, they did not use dialysis sacks but to measure phenolic bioaccessibility. Colonic fermentation ex- centrifugation. They used the in vitro method to compare pheno- periments may be incorporated in this model, so the assessment lic bioaccessibility in different parts of Smilax china and obtained of polyphenol bioaccessibility may be more reliable. This model, 36.4%, 17.8%, and 9.9% of the remaining total polyphenols after which mimics the biological environment through the duode- digestion of leaf, root, and stem, respectively. num, jejunum, and ileum, was employed in the monitorization Chen and others (2013) carried out an in vitro digestion model of anthocyanins stability and bioaccessibility in maqui berry and according to the method established for carotenoid bioavailability wild blueberry (Lila and others 2012). These authors observed in the assessment of 9 commercially available tea juices. After the that after intake, most anthocyanins were bioaccessible between gastric phase there was a significant decrease in total polyphenol the 2nd and 3rd hours. Lopez´ de Lacey and others (2012) also content of 5 of the juices. After the duodenal phase, a further used a dynamic GI model to study the bioaccessibility of green tea increase in the total polyphenol content was obtained in 4 of the polyphenols incorporated into agar. Their results revealed that the juices, possibly due to structural transformation of polyphenols. polyphenols incorporated in the agar were bioaccessible, and con- However, these methods did not include a colonic phase and sequently available for absorption. Furthermore, the gelatin used polyphenols may be metabolized by the colonic microflora. For to simulate the presence of protein during the digestion partly this reason, Saura-Calixto and others (2007) estimated the bioac- reduced green tea flavonols bioaccessibility.

r 162 Comprehensive Reviews in Food Science and Food Safety Vol. 13, 2014 C 2014 Institute of Food Technologists® Bioavailability of bioactive compounds . . .

However, certain transport mechanisms such as unidentified Mart´ın and others 2012), pigs (Lesser and others 2006; Walton stomach active transport or the transport in the small intestine of and others 2006), and dogs (Reinboth and others 2010). This way, flavonoids through interaction with the sodium-dependent glu- Gonthier and others (2003) did not detect parent compounds or cose transporter are not considered with the in vitro digestion catechin derivatives in the plasma of rats given purified procyani- method (Bermudez-Soto´ and others 2007). For this reason, other dins. Crespy and others (2002) also used Wister rats to determine methods have been developed. that quercetin, but not its glycosides, was absorbed from the rat Bioaccessibility studies using Caco-2 cells have been con- stomach. Disparity in the results between in vitro data and epi- ducted (Dupas and others 2006; Fernandes and others 2012). demiological studies are likely attributed to the physicochemical Glucuronidation, sulfation, and methylation processes carried out characteristics of polyphenols. Bioavailability in rodent studies has by polyphenols can be studied using these cells. Yi and others been estimated to be over 10% of ingested dose, ranging from 2% (2006) found that by growing on Transwell membranes Caco-2 to 20%. Interestingly, quantification of the flavonol quercetin and cells, anthocyanins could be degraded and demethylated during its main methylated metabolites (isorhamnetin and tamarixetin) by absorption and transport. Epigallocatechin was minimally uptaken Surco-Laos and others (2011)inaCaenorhabditis elegans model rein the human intestinal Caco-2 cell model (Vaidyanathan and vealed that higher levels of quercetin plus metabolites were present Walle 2003), in accordance with Hong and others (2002), who in the worm’s organism than those of isorhamnetin or tamarixetin observed a poor uptake of epigallocatechin gallate by HT-29 hu- plus their respective metabolites. This observation suggests that man colon adenocarcinoma cells. Neilson and others (2010) used greater capacity of quercetin uptake than of methylated derivatives this method to compare the efficiency of dimer absorption com- by the nematode exists, although quercetin is further transformed pared to monomers of catechin. In addition, an assessment of by C. elegans to a greater extent than isorhamnetin or tamarixetin. proanthocyanidin transport showed that oligomers of 6 units were With reference to human studies, these are limited as large transported approximately 10-fold less across a layer of Caco-2 population sizes are necessary. Nevertheless, Manach and others cells than radiolabeled monomers, dimers, and trimers (Deprez´ (2005) reported plasma concentrations of phenolic metabolites of and others 2000). A deeper study was carried out by Wang and 0to4μmol/L after 97 human volunteers ingested 50 mg aglycone others (2013) in which grape seed phenolic extract was subjected equivalents. Russell and others (2009) recovered in the urine 26% to in vitro GI digestion and ileal fermentation, followed by Caco-2 to 27% of the major free benzoic acids (gentisic, protocatechuic, cells assay. Only microbial metabolites, but not original phenolic and ρ-hydroxybenzoic) and the major conjugated acid (syringic compounds passed through the Caco-2 cell layer. acid), detected in plasma within 5 h after consumption of a sin- Some polyphenols may be metabolized by Caco-2 cells, which gle dose of a portion of strawberries. Research carried out by must be taken into account. Ferulic acid-sulfate, synaptic acid- Hackman and others (2008) showed a rapid transport into blood sulfate, ρ-coumaric acid-sulfate, and methyl ferulate-sulfate were of metabolites, in a dose-dependent manner, with peak plasma generated after 24-h exposure of hydroxycinnamates to differen- concentrations at 1 to 2.5 h after ingesting a flavanol-rich food, tiated Caco-2 cells according to Kern and others (2003). Mean- reaching baseline levels within 8 h. Colonic microflora metabo- while, Yi and others (2006) suggested a degradation and demethy- lized most of the flavanols not absorbed in the small intestine to a lation of anthocyanins from blueberries during absorption and variety of derivatives of phenolic acid and valerolactone, able to be transport by Caco-2 cells. absorbed. After 48 h of incubation with human colonic microflora, Further assays include the use of the Ussing chamber, where procyanidins of 6 units were degraded into low-molecular-weight a small section of intestinal mucosa is situated between 2 cham- aromatic acids (Deprez and others 2000). The wide variability of bers with buffer solution, preserving the epithelial polarity (Clarke results obtained by Suarez´ and others (2011) indicated a high de- 2009). Not only passive diffusion but transporter-mediated, tran- pendence on the individual in the absorption and metabolism of scellular, paracellular, and endocytosis transport can be measured. olive oil phenols. Bergmann and others (2009) employed the Ussing chamber in or- Moreover, although some in vitro studies suggest the degradation der to study the intestinal transport of polyphenols in apples. They of anthocyanins in the intestinal phase, under in vivo conditions used monolayers of the T84 colon carcinoma cell line and found direct absorption of anthocyanins may take place in the stom- that the transport of various hydroxycinnamic acids and flavonoids ach (Manach and others 2004). Bioavailability of anthocyanins has depended on the polarity. Cardinali and others (2013) also used been demonstrated to be lower than that of other flavonoids, and colonic cells in a Ussing chamber and obtained a bioaccessibility according to Yang and others (2011), generally less than 1% of of 0.1% of the polyphenol verbascoside. Moreover, Erk and others the consumed amounts (180 to 215 mg/day) is absorbed. They (2013) observed that the absorption of coffee polyphenols in the are absorbed by different mechanisms in the stomach and small jejunum is governed by their physicochemical properties when intestine involving specific enzymes, such as bilitranslocase (Passa- they used pig jejunal mucosa in the Ussing chamber. montia and others 2002). They subsequently enter the circulatory In situ studies have also been carried out in the assessment system within 15 to 60 min, after passing through the liver, and of polyphenolic bioavailability. Wang and others (2011) followed are distributed to different tissues, with a maximum concentration this procedure in the study of total flavonoid extracts, with the of nanomolar levels. Mostly, anthocyanins reach the colon and are inclusion of liver perfusion, in order to determine flavonoid extensively metabolized there by bacteria, contributing therefore metabolism. This method was also used by Fong and others (2012) to their bioavailability (Hidalgo and others 2012). to study the metabolism and absorption of flavones from herbs us- Among the isoflavones, genistein, daidzein, and glycitein are ing rat intestines. This way they found out that acetaminophen, the most active compounds found in soybeans. Equol is a highly (-)-epicatechin, piperine, and mainly curcumin could significantly bioavailable metabolite that comes from diadzein and exhibits inhibit the intestinal metabolism of the flavone baicalein and sub- higher activity than the original isoflavone (Kanazawa 2011). sequently increase its absorption. Using a Caco-2 cell model, Simmons and others (2012) found Meanwhile, in vivo studies were carried out to test the bioac- that the lipid source and amount did not affect bioaccessibility of cessibility of polyphenols in rats (Dupas and others 2006; Mateos- isoflavones. However, transport across the monolayer was greater

r C 2014 Institute of Food Technologists® Vol. 13, 2014 Comprehensive Reviews in Food Science and Food Safety 163 Bioavailability of bioactive compounds . . . with shorter molecules. The in vivo human study carried out by glucosinolates was 32%. Lai and others (2010) effectuated an in Shinkaruk and others (2012) revealed that the bioavailability of vitro simulated digestion of glucoraphanin in the upper GI tract, glycitein from soy-based food was similar to that of daidzein and along with an ex vivo study using rat cecal microbiota and an in situ its urinary excretion was significantly higher than that of genistein. rat cecum assay. The in vitro study confirmed that glucoraphanin was not degraded by upper GI digestive enzymes, consequently Glucosinolates reaching the rat cecum intact. Meanwhile, in both in situ and ex Glucosinolates have gained much attention as food compounds vivo procedures, glucoraphanin was hydrolyzed to sulforaphane by of high dietary value due to its alleged beneficial effect in cancer F344 rat cecal microbiota and able to cross the cecal enterocyte prevention (Fimognari and others 2002). Nearly all of the bio- for systemic absorption. logical activities of these compounds may be attributed to their A dynamic computercontrolled in vitro large-intestinal model hydrolytic products, of which the isothiocyanates are prominent was designed by Krul and others (2002), with the inoculation of examples. Glucosinolates are hydrolyzed into isothiocyanates me- complex microflora of human origin, a semipermeable membrane, diated by myrosinase, which is still active in fresh vegetable prod- and pH continuously adjusted to 5.8. They observed peak levels of ucts, and by the bacterial microflora of the GI tract. Antibiotic allyl isothiocyanate 9 to 12 h after the addition of sinigrin, which treatment along with inactivation of the plant myrosinase (after accounts for 1% of the degraded sinigrin. Slightly higher values cooking, for example) causes a decrease in bioavailability, as indi- were obtained by Getahun and Chung (1999) who incubated cated by the fact that bioavailability is greater following ingestion of human feces with cooked watercress juice for 2 h. They found that myrosinase-containing compared with myrosinase-lacking prepa- 18% of total glucosinolates were hydrolyzed into isothiocyanates. rations (Dinkova-Kostova and Kostov 2012). One of the most ex- Intact glucosinolate and its metabolites in feces were lowly re- tensively studied isothiocyanates is sulforaphane whose glucosino- covered in animal studies, using different species, suggesting sub- late precursor is glucoraphanin, abundant in broccoli. In humans, stantial absorption and metabolism of these compounds (Slomin- metabolization of isothiocyanates occurs via the mercapturic acid ski and others 1988; Conaway and others 1999). In an in vivo pathway. animal study published by Hanlon and others (2008), rats were Simulated static GI digestion, dynamic GI digestion, Caco-2 administered sulforaphane in either a single intravenous dose (2.8 uptake, transport assays, and/or in vivo studies with animals and mmol/kg) or single oral doses of 2.8, 5.6, and 28 mmol/kg. with humans have all been used as glucosinolate bioaccessibility This compound was well and rapidly absorbed, with an absolute and bioavailability screening methods. However, colonic fermen- bioavailability of 82%, which decreased at higher doses, indicating tation is essential for the absorption of isothiocyanates, which must a dose-dependent pharmacokinetic behavior. be taken into account. Recent evidence (Penas˜ and others 2012) In an in vivo human study, Rouzaud and others (2004)ob- suggests that certain strains of Lactobacillus spp., L. mesenteroides served that isothiocyanates release was delayed when ingest- and L. plantarum, were capable of digesting in vitro glucosino- ing cooked cabbage and, therefore, suggested that glucosinolates lates. As a result, in human studies, degradation of glucosino- passed through the upper digestive tract without modification. lates to isothiocyanates exhibited high interindividual variation Furthermore, Riso and others (2009) carried out an in vivo hu- because of colonic microflora differences (Rungapamestryi and man crossover intervention study (broccoli diet compared with others 2007). It is important to note that urinary isothiocyanate cruciferous-free diet). They observed an increase of isothiocyanate metabolite (dithiocarbamate) excretion decreases from 47% to a plasma concentrations, while the intervention did not affect plasma negligible amount when bowel microflora is reduced by mechan- glucosinolate activity. Other in vivo studies discussed in Table 7 ical and antibiotics. firmly established that, compared to isothiocyanates, intake of glu- As a result of the importance of colonic fermentation, few cosinolates is associated with lower bioavailability, slower elimina- in vitro studies have been carried out toward the assessment of tion, and greater interindividual variation in excretion. Overall, glucosinolate bioaccessibility. Despite the fact that mastication of the large interindividual variability of conversion of glucosinolates cooked vegetables liberates glucosinolates, and mastication of fresh to urinary dithiocarbamates is evident following administration of plants additionally causes enzymatic hydrolysis of glucosinolates, either single or multiple doses of glucosinolates, and ranges be- no in vitro research has yet been conducted on the impact of tween 1% and more than 40% of the dose. Interestingly, there are these process. Vallejo and others (2004) carried out a simulated also diurnal variations: conversion of glucosinolates to dithiocar- in vitro digestion which consisted in a gastric phase followed by bamates is greater during the day, whereas conversion of isoth- an intestinal phase that included a cellulose dialysis tubing, as iocyanates to dithiocarbamates is more efficient during the night described previously by Gil-Izquierdo and others (2001)forde- (Fajey and others 2012). termining phenolic bioaccessibility. These authors reported a high loss of glucosinolates (69%) under gastric conditions of homoge- nized fresh broccoli inflorescence. However, Iori and others (2004) Vitamin E suggested that the previous article had underestimated the degrada- Vitamin E is actually a family of molecules, which include tive activity of myrosinase, still active in the uncooked broccoli. the tocopherols and the tocotrienols, all of them with important Consequently, stability of glucosinolates under pepsin digestion antioxidant properties and health benefits. is considered quite high, as reported by Maskell and Smithardt Alpha-tocopherol exhibits the highest biological activity and (1994) who obtained after simulated gastric digestion an over- molar concentration of lipid-soluble antioxidants in the human. all drop of total glucosinolates of only 14%. Progoitrin and glu- A handful of in vitro and in vivo assays have been conducted conapoleiferin showed greater susceptibility to peptic digestion on the determination of vitamin E bioaccessibility and bioavail- than gluconapin or glucobrassicin, and 4-hydroxyglucobrassicin ability, as shown in Table 8. It is important to note that during became undetectable. Differences between the results obtained digestion, vitamin E must be packaged into micelles to facilitate with the different inocula employed (Table 6) were minor. After absorption, the same as carotenoids. Therefore, Reboul’s simu- 4 h of small intestine simulated digestion, the loss of the total lated GI digestion procedure (Reboul and others 2006)employed

r 164 Comprehensive Reviews in Food Science and Food Safety Vol. 13, 2014 C 2014 Institute of Food Technologists® Bioavailability of bioactive compounds . . .

Table 6–Comparison of in vitro methods for glucosinolates bioaccessibility determination.

Method Maskell and Vallejo and Lai and Step Smithardt (1994) others (2004) others (2010) Food sample preparation Homogenization Oral phase – – Amylase, 3 min, 37 ◦C Gastric phase Porcine pepsin, pH 2, 4 h, 37 ◦C. Porcine pepsin, pH 2, 2 h, 37 ◦C Porcine pepsin, pH 2, 2 h, 37 ◦C Transition step Centrifugation, 1000 rpm, 20 min Small intestine phase Innocula of small intestine of pig Porcine bile extract, porcine Porcine bile extract, porcine fed with rapeseed pancreatin, lipase, pH 7, 2 h, pancreatin, pH 7.5, 2 h, 37 ◦C meal/soyabean meal or 37 ◦C commercial diet/Porcine pancreatin, pH 6, 1 to 4 h, 37 ◦C Separation Centrifugation, 1000 rpm, 20 min Dialysis in a semipermeable Hydrolyzation with myrosinase cellulose membrane simultaneously with intestinal phase in the assessment of carotenoids is also used to study vitamin E observed that ingesting diary 9.2 mmol (4 mg) of α-tocopherol bioaccessibility, with subsequent centrifugation and filtration steps. maintained plasma concentrations of α-tocopherol at 23 mmol/L, Desmarchelier and others (2013) followed Reboul’s in vitro di- suggesting that the dietary requirement for vitamin E may be less gestion, with palm oil as added fat. These authors showed that α- than that currently recommended. Johnson and others (2012), em- tocopheryl acetate was distributed between mixed micelles (36%), ploying mouse and human in vivo assays, discovered novel urinary liposomes (9%), and nonsolubilized food debris (52%). Further- metabolites: α-carboxyethylhydroxychroman (α-CEHC) glycine, more, they followed the in vitro digestion by uptake studies using α-CEHC glycine glucuronide, and α-CEHC taurine. Caco-2 cells. These cells were able to hydrolyze α-tocopheryl ac- Correlation between in vitro bioaccessibility data with bioavail- etate and to uptake α-tocopherol when α-tocopheryl acetate was ability determined by in vivo human assays was studied by Granado incorporated into mixed micelles but not into emulsions. Werner and others (2006). They observed no measurable difference in the and Bohm¨ (2011) extended Reboul’s method by an oral phase. case of broccoli in the plasma levels of α-tocopherol after a 7-d Overall, results obtained by these authors were highly dependent feeding intervention. on the amount of bile extract present in the digestive medium and Therefore, the great variety of methods employed in the assess- to a lesser extent on the simulated gastric pH and the incubation ment of tocopherol bioavailability provides different findings that time with digestive enzymes. Bioaccessibility of β-tocotrienol was will be important for future updates of intake recommendations found to be higher than that of α-tocotrienol. and will aid in understanding the disposition and roles of vitamin Depending on the dietary source, the bioaccessibility of vita- E in vivo. min E has been shown to vary widely. O’Callaghan and O’Brien (2010), who used in vitro simulated GI digestion coupled with Phytosterols Caco-2 cells, obtained bioaccessibility values of α-tocopherol Phytosterols have attracted much attention in recent years ranging from 11% in apple sauce to 86% in beef. Likewise, Reboul due to their health benefits, such as cholesterol lowering, anti- and others (2006) reported a 100% bioaccessibility of α-tocopherol inflammatory, antiatherogenicity, and anticancer potential. β- in bananas and bread, 29% and 22% in cheese and milk, respec- Sitosterol is the most common phytosterol found in leaf veg- tively, and as low as 0.5% in apples. These differences between etable natural products followed by campesterol, stigmasterol, and different food sources may be due to different sites and physico- sitostanol. chemical states of α-tocopherol, along with the presence of fiber, Granado-Lorencio and others (2011) applied the same in vitro fat, and phytosterols in the food source. method as the one used to study polyphenols bioaccesibility A dynamic gastric digestion model with nonhomogeneous gas- (Granado-Lorencio and others 2007) in the assessment of phy- tric mixing, shearing, and rate of delivery to the duodenum was tosterol bioaccesibility. Mandak and Nystrom¨ (2012) also used an employed by Mandalari and others (2013) in the assessment of in vitro digestion. These authors observed that bioaccessibility of tocopherols bioaccessibility of pistachios. They obtained a bioac- steryl ferulates (various plant sterols esterified to ferulic acid) was cessibility of almost 100% of tocopherols after duodenal digestion. found to be almost negligible. These findings suggest that intestinal Deat´ and others (2009) employed the TIMR procedure coupled enzymes immediately hydrolyze steryl ferulates and thus they are to Caco-2 cells. These authors showed that the absorption of practically unavailable for absorption in the small intestine, possi- α-tocopherol from a vitamin E-containing meal was significantly bly being bioactive in the gut. This was also shown in a further lower when compared to the pure compound. This finding reveals study (Mandak and Nystrom¨ 2013), where the low bioaccessibility that other components present in a meal may change the uptake of steryl ferulates (0.01% to 0.25%) was independent of the ce- behavior of vitamin E or compete in the absorption through the real matrix. A similar analytical method was applied by Alemany SR-BI transporter. and others (2013). These authors obtained a sterol bioaccessibility In vivo studies have also been used in the assessment of vita- of 2% to 6% in fruit-based milk beverages. However, a higher min E bioavailability. Nagy and others (2013) carried out a hu- bioaccessibility was observed for oxides of β-sitosterol, suggest- man study with healthy volunteers under maldigestion conditions. ing differences in the solubilization and absorption mechanism They found out that the acetylated form of α-tocopherol exhibited between plant sterols and their oxides. the same bioavailability as free α-tocopherol. A long-term human Yi and others (2012) carried out an in vivo rat study where study was also carried out by Novotny and others (2012). They oral bioavailability of sterols enhanced by Flammulina velutipes was

r C 2014 Institute of Food Technologists® Vol. 13, 2014 Comprehensive Reviews in Food Science and Food Safety 165 Bioavailability of bioactive compounds . . . urine. sprouts contributed significantly to bioavailability by boosting the glucosinolate- to-isothiocyanate conversion. S-transferase M1 allele) genotypes had a significant effect on the metabolism of sulforaphane. and erucin is dramatically lower and delayed in time when subjects consume broccoli supplements compared to fresh broccoli sprouts. myrosinase activity did not produce equivalent plasma concentrations of the bioactive isothiocyanate metabolites compared to broccoli sprouts. sulforaphane was eliminated in 24 h. glucoraphanin was recovered as sulforaphane metabolites. was far superior to glucoraphanin extracts. -No isothiocyanates were found in -Dithiocarbamates were the -Myrosinase activity in intact -GSTM1 (glutathione -Bioavailability of sulforaphane -70% of the administered -Bioavailability of sulforaphane theentirestudyin8-h collection intervals. tandem mass spectrometry was used to quantify sulforaphane and its thiol conjugates in plasma and urine. collected for 48 h duringphase each and analyzed for sulforaphane and erucin metabolites using LC–MS/MS. metabolites in overnight (roughly 12 h) urine samples Urine was collected throughout Liquid chromatography linked to Blood and urine samples were Sulforaphane and sulforaphane active myrosinase) and homogenates of boiled sprouts cultivars were prepared by cooking 100 g florets withmL 150 water for 90 spower on in high a 700-W microwave oven followed by homogenization. broccoli sprouts with active myosine and commercially available broccoli supplement and were designed to be indistinguishable from each lyophilized broccoli sprout powders rich in either devoid of myrosinase activitywith either glucosinolates only or isothiocyanates only. majorother. metabolites in urine. glucoraphanin or sulforaphane -Only 5% of -Broccoli the supplements administered devoid of Uncooked fresh sprouts (with volunteers of a hospital 16 Healthy subjects12 Healthy subjects Individual soup portions of the 2 Samples studied were fresh 50 Healthy subjects Rehydrated, previously Study desing Participants Sample preparation Measurements Major findings mmol of isothiocyanates (standard broccoli and high-glucosinolate broccoli). mmol of glucosinolates, with or without active myrosinase. of glucoraphanin or 150 mmol of sulforaphane for 7 d. Single dose study Inpatient and outpatient Single oral doses of 16 or 52 Single oral doses of 161 to 221 Multiple dose study of 800 mmol studies regarding glucosinolate bioavailability (%) of plant-derived products. In vivo ) ) ) ) 2001 2005 2011 2011 ( ( ( ( Table 7– Shapiro and others Gasper and others Clarke and others Egner and others

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Table 8–Comparison of in vitro methods for vitamin E bioaccessibility determination.

Method Reboul and O’Callaghan and Werner and Mandalari and Step others (2006) others (2010) Bohm¨ (2011) others (2013) Food sample Homogenization in saline + Homogenization Homogenization in saline + preparation pyrogallol pyrogallol Oral phase Amylase, pH 6.5, 5 min, 37 ◦C Amylase, pH 6.9 Gastric phase Porcine pepsin, pH 4, 30 min, Porcine pepsin, lipase, pH 4, Porcine pepsin, pH 3.5 to 4.5, Porcine pepsin, 37 ◦C 37 ◦C 1h,37◦C 0.5 h, 37 ◦C Transition step pH 5.4 Small intestine phase Porcine bile extract, porcine Porcine pancreatin, pH 7.8, Porcine bile extract, porcine Porcine bile extract, porcine pancreatin, pH 6, 30 min, 2h,37◦C pancreatin, pH 6.5 to 7, pancreatin, lecithin, 37 ◦C 0.5 h, 37 ◦C cholesterol, sodium taurocholate pH 7, 2 h, 37 ◦C Separation Centrifugation 20000 rpm, Ultracentrifugation 200000 Centrifugation 4000 rpm, 20 Centrifugation, 3700 rpm, 15 18 h, 10 ◦C + Filtration g,95min+ Filtration min, 10 ◦C + min, 7 ◦C. Centrifugation 14000 rpm, 5min,22◦C + Filtration Cell uptake Caco-2 cell demonstrated. Although in vitro and in vivo methods have been used employing spectrophotometric, fluorometric, and chemiluminescent to measure sterols bioaccesibility and bioavailabilty respectively, in methods. Food Anal Methods 6(1):317–32. vitro procedures have yet to be validated against human absorption Bergmann H, Rogoll D, Scheppach W, Melcher R, Richling E. 2009. The Ussing type chamber model to study the intestinal transport and modulation data. of specific tightjunction genes using a colonic cell line. Mol Nutr Food Res 1211(53):1211–25. Conclusions Bermudez-Soto´ MJ, Tomas-Barber´ an´ FA, Garc´ıa-Conesa MT. 2007. The wide range of options available to evaluate digestion and Stability of polyphenols in chokeberry (Aronia melanocarpa) subjected to in uptake in in vitro and model organisms has guaranteed a role for vitro gastric and pancreatic digestion. Food Chem 102:865–74. them in bioaccessibility and bioavailability studies for years to Borel P, Tyssandier V, Mekki N, Grolier P, Rochette Y, Alexandre-Gouabau MC, Lairon D, Aza¨ıs-Braesco V. 1998. Chylomicron β-carotene and retinyl come. Both in vitro and in vivo approaches are increasing our un- palmitate responses are dramatically diminished when men ingest derstanding of uptake of bioactive compounds from food products. β-carotene with medium-chain rather than long-chain triglycerides. J Nutr Nevertheless, more validation studies are needed which compare 128:1361–7. in vivo with in vitro results. It is noteworthy that none of the meth- Bouayed J, Hoffmann L, Bohn T. 2011. Total phenolics, flavonoids, anthocyanins and antioxidant activity following simulated gastro-intestinal ods presented in this report will absolutely predict how much of digestion and dialysis of apple varieties: bioaccessibility and potential uptake. a specific bioactive compound a human will absorb and utilize. Food Chem 128(1):14–21. In addition, the low bioavailability of the bioactive compounds Bouayed J, Deusser H, Hoffmann L, Bohn T. 2012. Bioaccessible and (in particular polyphenols), could imply the activation of some dialysable polyphenols in selected apple varieties following in vitro digestion alternative mechanisms that can justify their possible beneficial ef- vs. their native patterns. Food Chem 131:1466–72. Brown MJ, Ferruzzi MG, Nguyen ML, Cooper DA, Eldridge AL, Schwartz fect. Nonetheless, results obtained with in vivo assays enable the SJ, White WS. 2004. Carotenoid bioavailability is higher from salads prediction of the situation in humans quite accurately and may ingested with full-fat than with fat-reduced salad dressings as measured with help accelerate the study of phytochemical absorption for better electrochemical detection. Am J Clin Nutr 80(2):396–403. comprehension of their possible beneficial effects. Bugianesi R, Salucci M, Leonardi C, Ferracane R, Catasta G, Azzini E, Maiani G. 2004. Effect of domestic cooking on human bioavailability of naringenin, chlorogenic acid, lycopene and β-carotene in cherry tomatoes. Acknowledgments Eur J Nutr 43:360–6. Carbonell-Capella JM, Barba FJ, Esteve MJ, Fr´ıgola A. 2013a. High pressure This research project was supported by the Spanish Ministry processing of fruit juice mixture sweetened with Stevia rebaudiana Bertoni: of Science and Technology and European Regional Development optimal retention of physical and nutritional quality. Innov Food Sci Emerg Funds (AGL2010-22206-C02-01). J.M. 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