In Vitro Inhibitory Effect on Digestive Enzymes and Antioxidant

Article pubs.acs.org/JAFC

In Vitro Inhibitory Effect on Digestive Enzymes and Antioxidant Potential of Commonly Consumed Fruits Anna Podsędek,* Iwona Majewska, Małgorzata Redzynia, Dorota Sosnowska, and Maria Koziołkiewicz Institute of Technical Biochemistry, Department of Biotechnology and Food Sciences, Lodz University of Technology, Stefanowskiego 4/10, 90-924 Łódź, Poland ABSTRACT: Dietary inhibitors of fats and carbohydrates degrading enzymes can reduce obesity and type 2 diabetes. In this study, we screened crude extracts from 30 commonly consumed fruits to test their in vitro inhibitory effect against key enzymes relevant for obesity (pancreatic lipase) and type 2 diabetes (α-glucosidase and α-amylase), total phenolic content (Folin− Ciocalteu method), and antioxidant capacity (ABTS and FRAP). The IC50 values of the fruits tested varied from 39.91 to >400 mg/mL, from 1.04 to >80 mg/mL, and from 0.72 to 135.07 mg/mL against α-glucosidase, α-amylase, and pancreatic lipase, respectively. Antioxidant capacity ranged from 0.66 to 124.66 μmol of TE/g of fruit and strongly correlated with phenolic content, while the enzyme inhibition was poorly correlated with total phenolic and antioxidant capacity. Among fruits tested, blue honeysuckle and red gooseberry exhibited the highest inhibitory activity with respect to the carbohydrate degrading enzymes, while lingonberry had the strongest anti-lipase activity. KEYWORDS: fruits, enzyme inhibition, pancreatic lipase, α-glucosidase, α-amylase, antioxidant capacity, phenolic compounds

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approach to preventing type 2 diabetes. Pancreatic α-amylase [EC 3.2.1.1; α-1,4-glucan-4-glucanohydrolase] is an endoglucosidase that is delivered into the intestinal lumen as a constituent of pancreatic juices and catalyzes the hydrolysis of starch to maltose and maltotriose.12 α-Glucosidase [EC 3.2.1.20] is a membrane-bound enzyme located in the epithelium of the small intestine, and it catalyzes the cleavage of glycosidic bonds and releases of glucose from disaccharides and oligosaccharides.13 The inhibitory activity of plant origin phenolic compounds against carbohydrate degrading enzymes is being increasingly documented. For example, cocoa,11 colored grains14 and almond seed skin15 as well as herbal polyphenolic-rich fractions16 have shown inhibitory effects on α-amylase and/or α-glucosidase activities in vitro. It is reasonable to hypothesize that a phenolic-rich diet may reduce absorption of fats and sugars via an inhibition of the enzymes degrading these substrates. For this reason the interest in studying the inhibition of digestive enzymes by these phytochemicals has recently increased. It is well-known that fruits are very important components of the human diet as well as the sources of dietary phenolics. There is some evidence that polyphenols from fruits and fruit products can also inhibit digestive enzymes. McDoughall et al.17 reviewed that berry anthocyanins, ellagitannins, and proanthocyanidins inhibit α-glucosidase, α-amylase, and pancreatic lipase, respectively. Additionally, strawberry, raspberry, blueberry, rowanberry, blackcurrant, and red grape extracts showed inhibitory effects on carbohydrate degrading enzymes.18−23 Some information is also available about antilipase potential of fruit extracts, because such inhibitory activity

INTRODUCTION Overweight and obesity are the fifth leading risk of global deaths. In addition, 44% of the diabetes burden, 23% of the ischemic heart disease burden, and between 7% and 41% of certain cancer burdens are attributable to overweight and obesity (http://www.who.int). According to the World Health Organization (WHO) overweight and obesity as well as their related diseases can be reduced by limiting energy intake from total fats and sugars, engaging in regular physical activity, and increasing consumption of fruits, vegetables, legumes, whole grains, and nuts. Dietary intervention seems to be a reasonable option for treatment of obesity and type 2 diabetes. Inhibition of dietary fat and sugar absorption from the intestine seems to be an effective way to prevent obesity and type 2 diabetes. In the continuing search for novel effective antiobesity and antidiabetes agents, medicinal plants or inedible parts of plants have been screened for potential lipase, αamylase, or α-glucosidase inhibitory activities.1−5 Pancreatic lipase (EC 3.1.1.3; triacylglycerol acyl hydrolase) produced by the pancreatic acinar cells hydrolyzes triglycerides mainly into 2-monoacylglycerols and free fatty acids, and it is responsible for the hydrolysis of 50−70% of total dietary fats in the intestinal lumen.1,6 The plant derived pancreatic lipase inhibitors are classified into the following classes of chemical compounds: saponins, terpenes, and phenolics.1 However, it is increasingly recognized that among different classes of phytochemicals phenolic compounds are the most active lipase inhibitors. Consistently with this statement, several in vitro studies have shown that white, green, oolong, and black tea polyphenols7−9 as well as green coffee and cocoa polyphenols10,11 inhibit pancreatic lipase. Phenolic compounds affect also digestive enzymes involved in the hydrolysis of dietary carbohydrates. The inhibition of αamylase and α-glucosidase by polyphenolic-rich plant extracts or isolated phenolic compounds may offer a natural dietary © 2014 American Chemical Society

Received: Revised: Accepted: Published: 4610

February 17, 2014 May 1, 2014 May 2, 2014 May 2, 2014 dx.doi.org/10.1021/jf5008264 | J. Agric. Food Chem. 2014, 62, 4610−4617

Journal of Agricultural and Food Chemistry

Article

was demonstrated for blueberry,24 lingonberry, Arctic bramble, cloudberry, strawberry, raspberry,25 and Cornelian cherries.4 Therefore, it is reasonable to hypothesize that consumption of fruits may decrease α-glucosidase, α-amylase, and/or pancreatic lipase activities and may be an important support in the management of postprandial hyperglycemia and obesity. The objective of this study was to screen the different fruits commonly consumed in Poland as well as all over the world for their pancreatic lipase, α-glucosidase, and α-amylase inhibitory activities and determine the correlation of the inhibitory effect to antioxidant capacity and phenolic content.

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(EC 3.1.1.3) from porcine pancreas type II, p-nitrophenyl-α-Dglucopyranoside (pNPG), p-nitrophenyl acetate, 4-methylumbelliferyl oleate, p-nitrophenyl-α-D-glucopiranoside, 2,2′-azinobis(3-ethyl-benzthiazoline-6-sulfonic acid) (ABTS), 2,4,6-tris-2-pyridyl-s-triazine (TPTZ), potassium persulfate, trolox, TRIS-base, dimethyl sulfoxide (DMSO), and acetonitrile were obtained from Sigma-Aldrich. Acetone and sodium carbonate were purchased from Chempur (Piekary ́ s̨ kie, Poland). Folin−Ciocalteu reagent, potato starch, hydrochloric Sla acid, sodium chloride, calcium chloride, and ferric chloride of analytical grade were purchased from POCH (Gliwice, Poland). Ultrapurity water was prepared in the laboratory using a Simplicity Water Purification System (Millipore, Marlborough, MA, USA). Preparation of Fruit Extracts. Extraction from the edible, mixed part of fruits (20 g) was done with 70% acetone (200 mL) at room temperature for 60 min with constant stirring. After centrifuging at 4000 rpm for 10 min, and filtration through Munktell filter AB, the supernatants were concentrated at 40 °C (vacuum rotary evaporator RII, Büchi, Switzerland) to remove the acetone and the aqueous phase was diluted to 25 mL with water. For further analytical and biological activity assays, a gradient of concentrations was prepared via serial dilution of the fruit extracts in pure water. Finally, 1 mL of extract corresponded to 0.8 mg of fruit. Pancreatic Lipase Inhibition in Vitro Assay. The inhibition of lipase activity was determined by measuring the release of 4methylumbelliferone from 4-methylumbelliferyl oleate or p-nitrophenol from p-nitrophenyl acetate by using a fluorimetric or spectrophotometric method, respectively. The amount of the inhibitor (expressed as mg of fruit per 1 mL of reaction mixture under assay conditions) required to inhibit 50% of the enzyme activity is defined as the IC50 value. The IC50 of the fruits tested was obtained from the line of the plot of the fruit concentration in 1 mL of reaction mixture versus the % inhibition. All samples were assayed in triplicate. Fluorimetric assay using 4-methylumbeliferyl oleate as substrate (4MUO) was carried out according to previous papers26,27 with some modification. Lipase from porcine pancreas type II was dissolved in buffer consisting of 20 mM Tris-base, 150 mM NaCl, and 1.3 mM CaCl2 (pH 7.4) at 1 mg/mL, and then the supernatant was used after centrifugation at 13 000 rpm for 5 min. The 0.1 mM substrate solution was prepared by dissolving 1.1 mg of 4-MUO in 0.5 mL of DMSO and diluting 50-fold with the above Tris buffer. For the pancreatic lipase assay, in a 96-well plate 25 μL of the varying concentrations of the fruit extracts were combined with 25 μL of enzyme solution, mixed, and incubated at 37 °C for 5 min. Then, 50 μL of 0.1 mM 4-MUO solution was added, mixed, and incubated at 37 °C for 20 min. The amount of 4-methylumbelliferone released by lipase was measured with a microplate reader (Synergy2, BioTek Instruments Inc.) at an excitation wavelength of 360 nm and at an emission wavelength of 460 nm. The sample blank and the control blank (without substrate) were performed in the same way. The inhibition (%) was calculated by using the following formula:

MATERIALS AND METHODS

Materials. The list of fruits used in this study is given in Table 1. Gallic acid, intestinal acetone powder from rat source of α-glucosidase (EC 3.2.1.20), α-amylase (from porcine pancreas type VI-B, lipase

Table 1. Popular Name, Scientific Name, Origin, and Purchased Place of Fruits Tested fruit sample

scientific name

origin

place of purchase

apple cv. Golden Delicious banana

Malus domestica

Poland

local farmers

Musa

local supermarket

bilberry black currant blackberry blueberry cv. Toro blue honeysuckle cv. Wojtek cranberry chokeberry gooseberry green gooseberry red grape pink grapefruit red

Vaccinium myrtillus Ribes nigrum Rubus f ruticosus Vaccinium corymbosum Lonicera caerulea var..kamtschatica Vaccinium oxycoccus Aronia melanocarpa Ribes uva-crispa Ribes uva-crispa Vitis vinifera Citrus paradis

not known Poland Poland Poland Poland

kiwi lingonberry mandarine

Actinidia chinensis Vaccinium vitis-idaea Citrus reticulata

orange

Citrus sinensis

peach

Prunus persica

pear cv. Lukasówka pear cv. Nashi pineapple

Pyrus communis

plum cv. Renkloda plum cv. Weg̨ ierka Zwykła pomegranate pomelo raspberry cv. Polka red currant sour cherry strawberry cv. Onebor sweet cherry cv. Regina

Poland Poland Poland Poland Poland Chile not known Chile Poland not known not known not known Poland

local market local market local farmers Research Institute of Horticulture Research Institute of Horticulture local market local market local market local market local supermarket local supermarket local supermarket local market local supermarket local supermarket local supermarket local supermarket

inhibition (%) = Pyrus communis Ananas sativus

local supermarket local supermarket

Prunus domestica

Chile Costa Rica Poland

Prunus domestica

Poland

local market

Punica granatum Citrus maxima

local supermarket local supermarket

Rubus idaeus

Tunisia not known Poland

Ribes rubrum Prunus cerasus Fragaria x ananassa

Poland Poland Poland

local market local farmers local farmers

Prunus avium

Poland

local farmers

(Fcontrol − Fcontrol blank ) − (Fsample − Fsample blank ) (Fcontrol − Fcontrol blank ) × 100

where Fcontrol and Fcontrol blank are a fluorescence value of the solvent control with and without 4-MUO, respectively; Fsample and Fsample blank are a fluorescence value of the extract with and without 4-MUO, respectively. The procedure of Umezawa et al.28 for lipase activity determination using p-nitrophenyl acetate as substrate was modified. The 50 mM stock solution of p-nitrophenyl acetate in DMSO was prepared. Pure water was added to reach a final concentration of 10 mM. Lipase from porcine pancreas type II was dissolved in water at 10 mg/mL, and then centrifuged at 13 000 rpm for 5 min. The composition of the reaction mixture was as follows: 0.2 mL of fruit extract, 0.2 mL of lipase solution, 1.4 mL of 0.1 M Tris buffer (pH 7.4), and 0.2 mL of 10 mM p-nitrophenyl acetate solution. The absorbance at 400 nm was read immediately against water after incubation at 37 °C for 10 min. In the control sample, the extract was replaced with 0.2 mL of water. A blank

local market

local farmers

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dx.doi.org/10.1021/jf5008264 | J. Agric. Food Chem. 2014, 62, 4610−4617


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Table 2. Pancreatic Lipase, α-Amylase, and α-Glucosidase Inhibitory Activities of Fruitsa enzyme inhibition IC50 (mg of fresh fruit/mL) pancreatic lipaseb

fruit apple banana bilberry black currant blackberry blueberry blue honeysuckle cranberry chokeberry grape pink green gooseberry kiwi lingonberry mandarine orange peach pear Lukasówka pear Nashi pineapple plum Renkloda plum Weg̨ ierka Zwykła pomegranate pomelo raspberry red currant red gooseberry red grapefruit sour cherry strawberry sweet cherry

14.01 32.54 0.82 1.73 6.23 1.33 8.03 1.04 1.24 0.93 2.01 9.53 0.72 29.53 15.06 41.52 58.10 135.07 20.05 20.03 9.81 4.24 23.24 0.91 0.78 1.42 7.83 6.42 5.72 14.22

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.53 1.30 0.12 0.14 0.58 0.09 0.44 0.10 0.05 0.07 0.13 0.36 0.03 1.83 0.26 1.69 1.69 3.40 0.73 0.37 0.44 0.31 4.10 0.06 0.04 0.03 0.31 0.26 0.05 0.07

pancreatic lipasec

f j a a bcd a de a a a a e a i f k l m g g e b h a a a de cd bc f

>80 >80 25.12 7.53 23.13 14.53 15.53 9.03 >80 21.13 19.52 16.12 4.07 61.56 26.52 >80 >80 >80 37.21 76.53 60.06 18.53 34.13 22.54 9.54 20.06 21.16 16.54 73.04 >80

± ± ± ± ± ±

0.09 0.04 0.38 0.17 0.08 0.07

k b j d e c

± ± ± ± ± ±

0.10 0.20 0.09 0.07 0.64 0.47

i h ef a p l

± ± ± ± ± ± ± ± ± ± ±

0.27 1.11 0.23 0.12 1.26 0.58 0.08 0.60 0.58 0.36 0.60

n s o g m j c h i f r

α-amylase >200 >200 29.57 3.88 18.47 46.40 50.20 7.99 1.18 18.92 1.92 >200 32.71 >200 >200 >200 >200 >200 >200 >200 >200 32.15 >200 17.25 1.14 1.04 >200 >200 18.18 >200

± ± ± ± ± ± ± ± ±

0.80 0.05 1.02 1.08 1.29 0.26 0.09 0.56 0.09

α-glucosidase

f b de h i c a e a

± 0.86 g

± 2.17 g ± 1.01 d ± 0.02 a ± 0.04 a

± 0.29 de

385.70 399.10 75.76 85.79 219.73 68.18 39.91 204.15 204.55 98.93 63.36 319.98 102.68 >400 >400 264.44 >400 369.11 >400 >400 167.88 191.88 >400 241.04 159.76 99.76 >400 258.18 156.36 91.51

± ± ± ± ± ± ± ± ± ± ± ± ±

23.12 lm 8.14 lm 4.83 bcd 1.10 cde 17.68 h 1.91 bc 2.54 a 9.83 gh 19.14 gh 2.13 e 0.63 b 2.26 k 8.98 e

± 3.66 j ± 7.10 l

± 8.22 f ± 0.23 g ± 17.70 i ± 5.33 f ± 6.49 e ± 21.20 ij ± 5.99 f ± 3.56 de

Values are means ± standard deviations. Mean values with different letters within the same column are statistically different (p < 0.05). b4Methylumbelliferyl oleate based assay. cp-Nitrophenyl acetate based assay. a

sample without the p-nitrophenyl acetate was measured for each extract. The results were expressed as inhibition (%) of lipase activity according to the following formula: inhibition (%) =

Acontrol − (A sample − A sample blank ) Acontrol

added after stopping of the reaction with 0.1 M sodium carbonate solution. All samples were assayed in triplicate. The rate of α-glucosidase inhibition activity was calculated as a percentage of the control by the following equation:

⎡ (AA − AB) ⎤ inhibition (%) = 100 × ⎢1 − ⎥ (A C − AD) ⎦ ⎣

× 100

Alpha-Glucosidase Inhibition Assay. The inhibition of αglucosidase activity was determined by measuring the amount of glucose hydrolyzed from p-nitrophenyl-α-D-glucopyranoside. An αglucosidase enzyme solution was prepared by dissolving 0.5 g of intestinal acetone powder from rat in 10 mL of saline (0.9% w/v) and sonicated 12 times at regular intervals (30 s sonications and 30 s off) in an ice bath. After centrifugation at 3000 rpm for 30 min at 4 °C, the resulting supernatant was diluted two times with 0.1 M potassium phosphate buffer (pH 6.9) and was used as the enzyme solution. For the α-glucosidase assay, in a 96-well plate 50 μL of the fruit extract was combined with 50 μL of a fresh prepared enzyme solution and incubated for 10 min. The enzyme reaction was initiated by adding 50 μL of 5 mM p-nitrophenyl-α-D-glucopyranoside solution in the above buffer (pH 6.9). The mixtures were incubated at 37 °C for 20 min. Finally, 100 μL of 0.1 M sodium carbonate solution was added and the absorbance (AA) was read at 405 nm (Synergy2, BioTek Instruments Inc.). Blank and α-glucosidase controls were also included. αGlucosidase control (AC) consisted of buffer, substrate, and enzyme. Blank (AB) consisted of the fruit extract, substrate, and enzyme added after stopping of the reaction with 0.1 M sodium carbonate solution. Blank to control (AD) consisted of the buffer, substrate, and enzyme

where AC, AA, AB, and AD were the absorbance of control, sample, blank sample, and blank control, respectively. The inhibitory activity was expressed as the IC50 value. Alpha-Amylase Inhibition Assay. The α-amylase inhibitory effect of the fruit extracts was assayed according to the procedure described previously by Xiao et al.,29 with slight modifications. Briefly, 40 μL of 0.2% (w/v) potato starch solution, 20 μL of the fruit extract, or 0.1 M phosphate buffer containing 6 mM CaCl2 (pH 6.9) (control) was mixed with 20 μL of α-amylase from porcine pancreas type VI-B dissolved in the above buffer in a 96-well plate to start the enzyme reaction. After incubation at 37 °C for 10 min, the reaction was stopped by the addition 80 μL of 0.4 M HCl. After that, 100 μL of 5 mM I2 in 5 mM KI was added, and the absorbance was read at 600 nm (Synergy2, BioTek Instruments Inc.). All samples were assayed in triplicate. The α-amylase inhibitory activity was calculated as follows:

⎡ (AB − AA ) ⎤ inhibition (%) = 100 × ⎢1 − ⎥ (AD − A C) ⎦ ⎣ where AA and AB were the absorbance of incubated mixture containing fruit extract and starch with or without amylase, respectively. AC and 4612



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AD were the absorbance of incubated mixture containing starch and amylase or only starch. The inhibitory activity was expressed as the IC50 value. Determination of Antioxidant Capacity. The fruit extracts were assessed for antioxidant capacity by the following two methods: 2,2′azinobis(3-ethylbenzthiazoline-6-sulfonic acid) radical scavenging activity (ABTS method) and ferric reducing antioxidant power (FRAP method). In both methods, trolox (6-hydroxy-2,5,7,8tetramethychroman-2-carboxylic acid) was used as a standard and the antioxidant capacity was expressed as μmol of trolox equivalents (TE) per 1 g fresh weight of fruit. All samples were assayed in triplicate. ABTS Method. ABTS•+ radical cation scavenging activity was determined according to the procedure described by Re et al.30 2,2′Azinobis(3-ethylbenzthiazoline-6-sulfonic acid) radical cation (ABTS•+) was produced by the reacting of 7 mM ABTS water solution with 2.45 mM potassium persulfate (final concentration) followed by an incubation of the mixture in the dark for 12−16 h at room temperature. Stock solution of ABTS•+ was diluted with methanol until an absorbance of 0.76 ± 0.02 at 734 nm was reached. Then, 20 μL of fruit extract or water (control) was mixed with 1 mL of diluted ABTS•+ solution, and after 6 min at 30 °C absorbance of this mixture was measured at 734 nm. FRAP Method. The FRAP assay developed by Benzie and Strain31 was performed with some modification. Briefly, 2.7 mL of FRAP reagent, prepared freshly and warmed to 30 °C, was mixed with water (0.27 mL) and the fruit extract (0.09 mL). The FRAP reagent was prepared by mixing 2.5 mL of a 10 mM solution of 2,4,6-tris-2-pyridyls-triazine (TPTZ) in 40 mM HCl with 2.5 mL of 20 mM FeCl3 and diluting with 25 mL of 0.3 mM acetate buffer (pH 3.6). Absorbance at 593 nm was recorded after 10 min incubation of the solution at 30 °C. Total Phenolic Content. The content of the total phenolics was evaluated by using the Folin−Ciocalteu method. Each fruit extract (0.1−1.0 mL) was mixed with 25 mL of distilled water followed by 0.5 mL of Folin−Ciocalteu reagent and 5 mL of 20% (w/w) Na2CO3. The volume of reaction mixture was adjusted to 50 mL with distilled water. The absorbance was then measured at 760 nm after incubation at room temperature for 20 min. Total phenolic content was expressed as mg of gallic acid equivalent (GAE) per 100 g of fresh weight of fruit. All samples were assayed in triplicate. Phenolic Profile. Phenolic profiles were determined using a highperformance liquid chromatography system (Waters, Milford, MA) that consisted of a gradient pump (1525), photodiode array detector (2998), autoinjector (2707) and Breeze 2 system controller equipped with a 250 × 4.6 mm i.d., 5 μm Symmetry C18 column (Waters). The mobile phase was a binary gradient with A, water/formic acid (90:10, v/v), and B, water/acetonitrile/formic acid (40:50:10, v/v/v), with a flow rate of 1 mL/min.32 The binary gradient was as follows: 12% B (0 min), 12−30% B (0−26 min), 30−100% B (26−40 min); 100% B (40−43 min), 12% B (43−48 min), and 12% B (48−50 min). Phenolics were qualified into one of the four subclasses and quantified on the base of maximum of UV−vis absorption and spectra recorded from 200 to 600 nm. The hydroxybenzoic acid derivatives and flavanols were quantified at 280 nm and expressed as gallic acid equivalents, hydroxycinnamic acids at 320 nm as chlorogenic acid equivalents, flavonols at 360 nm as rutin equivalents, and anthocyanins at 520 nm as cyanidin 3-glucoside equivalents. The results were expressed as mg/100 g fresh weight of fruit. All samples were assayed in triplicate. Statistical Analysis. The results are given as the means ± standard deviations. Data were analyzed by means of a one-way ANOVA using Statistica Ver. 6.0 (USA), and Duncan’s post hoc test was used to assess the differences between the means with significance level p < 0.05. Pearson’s correlation coefficients were determined using Microsoft Excel XP.

fat, so its inhibition can lead to beneficial effects on overweight and obesity. In the present work the anti-lipase activity of fruits was investigated using p-nitrophenyl acetate (spectrophotometric method) and 4-methylumbelliferyl oleate (fluorimetric method) as substrates. Significant differences (p < 0.05) were found among the analyzed fruits in the inhibitory activities toward pancreatic lipase type II (Table 2). The IC50 values varied from 0.72 to 135.07 mg of fruit/mL in the fluorimetric method and from 4.07 to more than 80 mg of fruit/mL in the spectrophotometric method. Among the 30 fruits examined, only lingonberry and red currant strongly inhibited pancreatic lipase activity in both methods. They showed IC50 values less than 0.8 mg/mL in the fluorimetric assay, and less than 10 mg/ mL in the spectrophotometric assay. In addition, bilberry, pink grape, raspberry, cranberry, chokeberry, blueberry, red gooseberry, and black currant showed a relatively high inhibitory effect with their IC50 values less than 2 mg/mL in the fluorimetric assay. Black currant and cranberry showed also high inhibitory activity with IC50 less than 10 mg/mL in the assay of p-nitrophenol release. On the other hand, seven fruits used at 80 mg/mL concentration (the maximum dose of fruit extract in the assay condition) inhibited in this assay pancreatic lipase activity less than 50%. Few reports have been published about pancreatic lipase inhibitory activity of crude fruit extracts in vitro. Slanc et al.33 reported that 20 mg/mL of blueberry and apple fruits provided 70% inhibitory activity, while raspberry and banana extracts provided between 40 and 70% inhibitory activity against pancreatic lipase, measured with p-nitrophenyl palmitate. Methanolic extract of the Carols muscadine inhibited pancreatic lipase activity with IC50 34.41 mg/mL.34 Roh and Jung4 demonstrated a relatively high anti-lipase activity (34.8%) for Cornelian cherries at a concentration of 0.1 mg/mL when pnitrophenyl butyrate was used as a substrate. On the other hand, seven raspberry cultivars examined by Zhang et al.23 did not show anti-lipase activity measured by fluorimetric assay. Other in vitro studies also demonstrated that lipase activity was effectively inhibited by phenolic-rich extracts of raspberry, strawberry, cloudberry, and Arctic bramble, while black currant and rowanberry extracts had no effect in p-nitrophenyl lauratebased assay.25 These opposite effects can result from the use of different analytical methods (different type and concentration of synthetic substrate, concentration of lipase) and type of extraction solvent. For this reason, there is a need to standardize the method, and further studies with the use of dietary lipids as substrates should be carried out to compare and/or confirm anti-lipase activity of fruit extracts evaluated using artificial substrates. α-Glucosidase and α-Amylase Inhibitory Activity. In human, dietary carbohydrates are hydrolyzed by pancreatic αamylase and intestinal α-glucosidase enzymes responsible for the breakdown of oligosaccharides and disaccharides into monosaccharides suitable for absorption.3 The inhibition of these enzymes is specifically useful for the treatment of noninsulin-dependent diabetes because it will slow down the release of glucose in the blood. In the present work the antiglucosidase or anti-amylase activity of fruits was investigated using p-nitrophenyl-α-D-glucopyranoside or potato starch as substrates, respectively. Significant differences (p < 0.05) were found among the analyzed fruits in the inhibitory activities toward α-glucosidase and α-amylase (Table 2). The inhibition of α-glucosidase was in a wide range with the IC50 values from 39.91 to more than 400 mg fruit/mL. Blue honeysuckle showed

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RESULTS AND DISCUSSION Pancreatic Lipase Inhibitory Activity. Pancreatic lipase is the most important enzyme responsible for digestion of dietary 4613



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Table 3. Antioxidant Capacity and Total Phenolic Content of Fruitsa antioxidant capacity (μmol of TE/g fruit) fruit apple banana bilberry black currant blackberry blueberry blue honeysuckle cranberry chokeberry grape pink green gooseberry kiwi lingonberry mandarine orange peach pear Lukasówka pear Nashi pineapple plum Renkloda plum Weg̨ ierka Zwykła pomegranate pomelo raspberry red currant red gooseberry red grapefruit sour cherry strawberry sweet cherry a

ABTS 8.77 1.83 54.17 35.75 28.91 27.09 51.54 20.43 124.66 10.38 18.56 1.97 34.82 3.54 4.80 1.59 1.46 1.65 4.02 0.84 2.03 14.02 3.25 20.36 23.45 24.39 3.69 10.70 16.56 6.00

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.86 0.11 4.50 2.60 1.14 2.05 4.02 1.14 8.27 0.55 1.21 0.10 3.85 0.28 0.11 0.17 0.06 0.14 0.18 0.09 0.20 0.88 0.07 0.50 1.44 1.78 0.13 0.28 0.84 0.33

FRAP de ab m l k jk m h n e gh ab l abc bc ab ab ab abc a ab f abc h i ij abc e fg cd

4.98 1.15 41.70 29.93 23.36 16.86 49.52 12.74 94.24 6.10 12.28 1.46 22.28 1.66 3.50 0.79 0.81 0.76 4.01 0.66 1.49 7.62 2.22 16.81 16.68 16.99 3.08 9.12 9.95 4.34

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.43 0.06 2.88 1.33 1.29 0.80 4.69 0.37 6.87 0.30 0.45 0.08 2.57 0.11 0.06 0.06 0.06 0.07 0.20 0.06 0.08 0.32 0.19 0.20 1.25 0.53 0.09 0.53 0.78 0.11

total phenolics (mg of GAE/100 g) def ab m l k j n i o ef hi abc k abc abcde a a a bcde a abc fg abcd j j j abcd g gh cde

126.80 30.61 487.45 473.64 288.82 337.05 540.04 239.01 1423.74 117.71 245.99 46.40 410.73 64.93 86.27 24.83 23.76 27.20 62.14 22.78 36.55 183.32 60.18 183.98 261.95 319.63 105.49 120.80 192.95 77.53

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

4.99 g 1.27 ab 9.27 m 12.44 m 20.65 j 20.92 k 27.73 n 23.35 i 87.83 o 5.40 g 21.13 i 2.75 abcd 14.53 l 1.56 cde 2.75 ef 1.42 a 1.39 a 1.37 a 2.64 bcde 1.35 a 2.49 abc 10.05 h 2.20 bcde 10.42 h 31.89 ij 12.94 k 4.03 fg 11.37 g 19.67 h 4.87 def

Values are means ± standard deviations. Mean values with different letters within the same column are statistically different (p < 0.05).

pancreatic α-amylase. Contrarily, in our study raspberry crude extract inhibited activity of both enzymes at the moderate level. This observation is consistent with the report of McDougall et al.18 that the raspberry extract inhibited activity of pancreatic αamylase. In addition, α-glucosidase inhibitory activity was also demonstrated for Carols muscadine34 and apple,36 while αamylase inhibitory effect was demonstrated for rowanberry, cloudberry, and Arctic bramble.22 Antioxidant Capacity. The contribution of fruits to health improvement has been partly attributed to their antioxidant capacity. Antioxidant properties of fruits tested were estimated as scavenging potential toward ABTS•+ radical cation and the potential to reduce ferric to ferrous ion (FRAP), and were expressed as trolox (soluble analogue of vitamin E) equivalents (TE): Table 3. Significant differences (p < 0.05) were found among the analyzed fruits in the antioxidant capacity. The trolox equivalents (TE) values varied from 0.84 to 124.66 μmol/g of fresh fruit and from 0.66 to 94.24 μmol/g in ABTS and FRAP methods, respectively. Chokeberry had the highest antioxidant capacity, while plum Renkloda showed the lowest TE values, regardless of assay method. Antioxidant capacities of the fruits have been studied in vitro by many authors,37−39 and our results are consistent with published data. Total Phenolic Content and Phenolic Profile. The health benefits of fruits are mainly attributed to their vitamins, carotenoids, and phenolic compounds. Phenolic compounds range from simple, low molecular weight, single aromatic

the strongest α-glucosidase inhibitory activity with an IC50 value of 39.91 mg/mL, and the order of the most potent αglucosidase inhibitors was as follows: blue honeysuckle > green gooseberry >blueberry > bilberry > black currant > sweet cherry > pink grape > red gooseberry. Although the fruits tested appeared to have anti-αglucosidase activities, our experiments have shown that they are weak inhibitors of pancreatic α-amylase. Among 30 fruits tested, half of them at concentration 200 mg/mL inhibited αamylase activity below 50%. The findings showed that red and green gooseberries, chokeberry, and red currant were the most effective inhibitors of this enzyme with IC50 values less than 2 mg/mL. α-Amylase and/or α-glucosidase inhibitory activities of raspberry, strawberry, lingonberry, black currant, pomegranate, and blueberry extracts have been confirmed by other authors,18,22 although the inhibitory effect ranks are different from those obtained in our experiments. Similarly to our results, Da Silva Pinto et al.35 reported red and black currants and red and green gooseberries as the good inhibitors of αglucosidase and α-amylase, while red currant was the strongest inhibitor of both enzymes. In the present study red currant has indicated very high α-amylase inhibitory activity, but a moderate inhibitory effect against α-glucosidase. Zhang and co-workers23 observed strong α-glucosidase inhibitory effect for the phenol-rich extracts from raspberries, while none of the seven extracts showed detectable inhibitory effect against 4614



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Table 4. Pearson’s Correlation Coefficients (r) between Total Phenolic Content, Antioxidant Capacities, and Digestive Enzymes Inhibitory Activities of Thirty Fruits total phenolics α-amylase α-glucosidase pancreatic lipasea pancreatic lipaseb ABTS FRAP a

total phenolics

α-amylase

α-glucosidase

pancreatic lipasea

pancreatic lipaseb

ABTS

FRAP

1 −0.2482 −0.3322 −0.3469 −0.5302 0.9920 0.9792

1 −0.1331 0.5322 −0.0253 −0.2054 −0.1600

1 0.5312 0.0788 −0.3372 −0.3405

1 0.6254 −0.3434 −0.3290

1 −0.4726 −0.4384

1 0.9917

1

4-Methylumbelliferyl oleate based assay. b-p-Nitrophenyl acetate based assay.

Table 5. Phenolic Profiles Determined by HPLCa phenolics (mg/100 g) fruit

HBA and flavanols

blue honeysuckle lingonberry red gooseberry

58.62 ± 2.43 106.67 ± 3.55 77.38 ± 0.23

b

HCA

c

55.04 ± 0.81 26.16 ± 1.72 12.48 ± 2.47

flavonolsd

anthocyaninse

26.33 ± 3.08 5.94 ± 0.83 3.12 ± 0.48

335.37 ± 4.35 25.82 ± 0.75 4.15 ± 0.37

Values are means ± standard deviations. bDetermined at 280 nm as gallic acid equivalents. HBA: hydroxbenzoic acids. cDetermined at 320 nm as chlorogenic acid equivalents. HCA: hydroxycinnamic acids. dDetermined at 360 nm as rutin equivalents. eDetermined at 520 nm as cyanidin 3glucoside equivalents.

a

ringed compounds to large and complex flavonoids and tannins, with a wide range of biological activities. In this study, the total extractable phenolic contents of 30 different fruits were determined by colorimetric method with Folin− Ciocalteu reagent, and expressed as gallic acid equivalents (GAE). Although the Folin−Ciocalteu reagent is nonspecific to phenolic compounds, and can react with a wide range of thiol derivatives, some vitamins, especially vitamin C, and aromatic amino acids, it is still the most widely used reagent to determine total phenolic content.40 Moreover, the responses of phenolic compounds to this assay were affected by both the position and degree of hydroxylation in the phenolic molecule. In the present study significant differences (p < 0.05) were found among the analyzed fruits in the total phenolic content. The phenolic contents for the fruits studied ranged from 22.78 to 1423.74 mg of GAE/100 g fresh weight (Table 3). Among the fruits, chokeberry contained the highest amount of phenolics followed by blue honeysuckle, bilberry, black currant, and lingonberry, whereas the lowest levels (less than 30 mg of GAE/100 g) were found in plum Renkloda, pears Lukasówka and Nashi, and peach. In general, the present findings are consistent with literature data,39,41−43 wherein phenolic content in fruits may be affected by a large number of factors such as climatic conditions, agrotechnical processes, cultivars, harvest time, and storage conditions. Evaluation of Parameters across Tests. In the present study a highly positive correlation between phenolic content and antioxidant capacities measured by ABTS and FRAP assays was reported with a Pearson’s correlation coefficient of r ≥ 0.97 when used for 30 fruit extracts (Table 4). Others researchers also noticed good correlation between total phenolic content and antioxidant potential of fruits as well as a strong correlation between ABTS and FRAP assays.38,44−46 Some authors postulated that the Folin−Ciocalteu (FC) assay should not be viewed as a measure of total phenolic content, but rather a measure of overall antioxidant capacity.46 Therefore, it could be expected to find excellent linear correlations between the total phenolic content obtained by the FC method and the antioxidant activity determined by FRAP and ABTS assays.

We also observed a high positive correlation between antioxidant capacities measured by ABTS and FRAP assays with a Pearson’s correlation coefficient of r ≥ 0.992. In contrast to the high correlation between phenolic content and antioxidant capacity, a weaker correlation was observed between phenolic content and inhibition of pancreatic lipase, αglucosidase, or α-amylase (r < −0.530), as well as between antioxidant capacity and the inhibitory effect (r < −0.473); Table 4. Our results are consistent with the data for blueberry anthocyanin extract for which α-amylase inhibitory activity correlated poorly with both total phenolic content and FRAP.24 Lack of correlation between α-glucosidase inhibitory activity and antioxidant activity and total phenolic content has been reported for the phenol-rich extracts from seven raspberry cultivars.23 In contrast, Wang and co-workers20 noted a positive correlation (averaging r = 0.89) between α-glucosidase inhibitory activity and scavenging activity for peroxyl and hydroxyl radicals as well as for hydrogen peroxide and singlet oxygen in blueberry peel tissue. Flores et al.24 also observed high correlation between α-glucosidase inhibition by blueberry anthocyanin extract and both its total phenolic content and FRAP. Results of the present and previous studies suggest that the type of phenolics is more important for the inhibitory effects against digestive enzyme than total phenolic amounts. Moreover, phenolic compounds present in the crude extracts may show synergistic and/or antagonist effects which can influence results of measurements in the assay system. Remarkable differences not only in content but also in composition of phenolic compounds for fruits affect their biological activity. Moreover, a large diversity in the structure between the different subclasses of phenolic compounds as well as within the group influences their stability, solubility, and bonding ability with the digestive enzymes. For example, significant differences in anti-lipase activity were dependent upon the procyanidin degree of polymerization.47 Previous study also demonstrated that anthocyanidin has much stronger anti-lipase and anti-glucosidase activities than the glycoside form.48 Among nine phenolic acids tested by Zhang et al.23 only 4615



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(2) Kumar, S.; Narwal, S.; Kumar, V.; Prakash, O. α-Glucosidase inhibitors from plants: A natural approach to treat diabetes. Pharmacogn. Rev. 2011, 5, 19−29. (3) De Sales, P. M.; Souza, P. M.; Simeoni, L. A.; Silveira, D. αAmylase inhibitors: a review of raw material and isolated compounds from plant source. J. Pharm. Pharm. Sci. 2012, 15, 141−183. (4) Roh, C.; Jung, U. Screening of crude plant extracts with antiobesity activity. Int. J. Mol. Sci. 2012, 13, 1710−1719. (5) Yun, J. W. Possible anti-obesity therapeutics from naturea review. Phytochemistry 2010, 71, 1625−1641. (6) Mukherjee, M. Human digestive and metabolic lipasesa brief review. J. Mol. Catal. B: Enzym. 2003, 22, 369−376. (7) Gondoin, A.; Grussu, D.; Stewart, D.; McDougall, G. J. White and green tea polyphenols inhibit pancreatic lipase in vitro. Food Res. Int. 2010, 43, 1537−1544. (8) He, Q.; Lv, Y.; Yao, K. Effects of tea polyphenols on the activities of α-amylase, pepsin, trypsin and lipase. Food Chem. 2007, 101, 1178− 1182. (9) Uchiyama, S.; Taniguchi, Y.; Saka, A.; Yoshida, A.; Yajima, H. Prevention of diet-induced obesity by dietary black tea polyphenols extract in vitro and in vivo. Nutrition 2011, 27, 287−292. (10) Almoosawi, S.; McDougall, G. J.; Fyfe, L.; Al-Dujaili, E. A. S. Investigating the inhibitory activity of green coffee and cacao bean extracts on pancreatic lipase. Nutr. Bull. 2010, 35, 207−212. (11) Gu, Y.; Hurst, W. J.; Stuart, D. A.; Lambert, J. D. Inhibition of key digestive enzymes by cocoa extracts and procyanidins. J. Agric. Food Chem. 2011, 59, 5305−5311. (12) Damager, I.; Numao, S.; Chen, H.; Brayer, G. D.; Withers, S. G. Synthesis and characterisation of novel chromogenic substrates for human pancreatic α-amylase. Carbohydr. Res. 2004, 339, 1727−1737. (13) Li, Y.; Wen, S.; Kota, B. P.; Peng, G.; Li, G. Q.; Yamahara, J.; Roufogalis, B. D. Punicagranatum flower extract, a potent alphaglucosidase inhibitor, improves postprandial hyperglycemia in Zucker diabetic fatty rats. J. Ethnopharmacol. 2005, 99, 239−244. (14) Yao, Y.; Sang, W.; Zhou, M.; Ren, G. Antioxidant and alphaglucosidase inhibitory activity of colored grains in China. J. Agric. Food Chem. 2010, 58, 770−774. (15) Tsujita, T.; Shintani, T.; Sato, H. α-Amylase inhibitory activity from nut seed skin polyphenols. 1. Purification and characterization of almond seed skin polyphenols. J. Agric. Food Chem. 2013, 61, 4570− 4576. (16) Wongsa, P.; Chaiwarit, J.; Zamaludien, A. In vitro screening of phenolic compounds, potential inhibition against α-amylase and αglucosidase of culinary herbs in Thailand. Food Chem. 2012, 131, 964− 971. (17) McDougall, G. J.; Kulkarni, N. N.; Stewart, D. Current developments on the inhibitory effects of berry polyphenols on digestive enzymes. BioFactors 2008, 34, 73−80. (18) McDougall, G. J.; Shpiro, F.; Dobson, P.; Smith, P.; Blake, A.; Stewart, D. Different polyphenolic components of soft fruits Inhibit αamylase and α-glucosidase. J. Agric. Food Chem. 2005, 53, 2760−2766. (19) Johnson, M. H.; Lucius, A.; Meyer, T.; de Mejia, E. G. Cultivar evaluation and effect of fermentation on antioxidant capacity and in vitro inhibition of α-amylase and α-glucosidase by highbush blueberry (Vaccinium corombosum). J. Agric. Food Chem. 2011, 59, 8923−8930. (20) Wang, S. Y.; Camp, M. J.; Ehlenfeldt, M. K. Antioxidant capacity and α-glucosidase inhibitory activity in peel and flesh of blueberry (Vaccinium spp.) cultivars. Food Chem. 2012, 132, 1759−1768. (21) Cheplick, S.; Kwon, Y.-I.; Bhowmik, P.; Shetty, K. Phenoliclinked variation in strawberry cultivars for potential dietary management of hyperglycemia and related complications of hypertension. Bioresour. Technol. 2010, 101, 404−413. (22) Grussu, D.; Stewart, D.; McDougall, G. J. Berry polyphenols inhibit α-amylase in vitro: identifying active components in rowanberry and raspberry. J. Agric. Food Chem. 2011, 59, 2324−2331. (23) Zhang, L.; Li, J.; Hogan, S.; Chung, H.; Welbaum, G. E.; Zhou, K. Inhibitory effect of raspberries on starch digestive enzyme and their antioxidant properties and phenolic composition. Food Chem. 2010, 119, 592−599.

ellagic acid inhibited α-glucosidase activity, and according to You et al.34 ellagic acid was a stronger inhibitor of this enzyme than catechin and quercetin. Therefore, phenolic composition may have a significant importance for inhibition of digestive enzymes by fruits. For the above-mentioned reason, the three extracts with the highest inhibitory activity against enzymes tested were analyzed for the phenolic profile by HPLC (Table 5). The most abundant phenolic compounds found in the blue honeysuckle extract (the highest α-glucosidase inhibitory activity) were anthocyanins. The inhibitory effectiveness of anthocynins against α-glucosidase has also been reported for other colored fruits such as black currant, strawberry, raspberry, and blueberry fruit extracts.18,20 This may suggest that anthocyanins are important constituents regarding anti-glucosidase activity. Although chokeberries are known as the most rich source of anthocyanins among fruits49 in the present study they showed higher IC50 value compared to blue honeysuckle. This difference in the IC50 values may result from different anthocyanin profile for these two fruits. The major anthocyanin in chokeberry is cyanidin 3-galactoside (66.7% of total anthocyanins),42,49 while cyanidin 3-glucoside dominates in the blue honeysuckle fruit (71−89% of total anthocyanins).50 According to Bräunlich et al.51 cyanidin-3-galactoside possessed 1.8-fold lower α-glucosidase inhibitory activity than cyanidin-3glucoside. So, inhibitory activity of anthocyanins is strongly influenced by the sugar units linked to the anthocyanidin. Phenolics with maximum at 280 nm, i.e., flavanols and/or hydroxybenzoic acids, are the main phenolic compounds in red gooseberries which exhibited the highest α-amylase inhibitory activity (Tables 2 and 5). According to literature data52 proanthocyanidins are important phenolics in gooseberry fruit. α-Amylase inhibitory activity of strawberry and red raspberry was associated with tannins, especially ellagitannins.35,53 However, a high level of ellagitannins in yellow raspberries did not increase amylase inhibition.22 Probably the presence of anthocyanins potentiated the enzyme inhibition by ellagitannins. The authors also suggested that high α-amylase inhibitory activity of rowanberry is related to proanthocyanidins. The high pancreatic lipase inhibitory activity of lingonberries may be caused by proanthocyanidins.25 In the present study the flavanols were the main phenolic compounds in these berries (Table 5). Sugiyama et al.54 suggested that the oligomeric procyanidins present in apple polyphenol extract inhibited triglyceride absorption by inhibiting pancreatic lipase activity in mice and humans.

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AUTHOR INFORMATION

Corresponding Author

*Tel: 48 426313435. Fax: 48 42 6366618. E-mail: anna. [email protected]. Funding

The work was funded by the National Science Centre allocated on the basis of Decision No. DEC-2011/01/B/NZ9/02046 and grant No. N N312 340740. Notes

The authors declare no competing financial interest.

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REFERENCES

(1) Birari, R. B.; Bhutani, K. K. Pancreatic lipase inhibitors from natural sources: unexplored potential. Drug Discovery Today 2007, 12, 879−889. 4616



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In Vitro Inhibitory Effect on Digestive Enzymes and Antioxidant

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