The inhibitory potential of Montmorency tart cherry on key enzymes relevant to type 2 diabetes and cardiovascular disease

Accepted Manuscript The inhibitory potential of Montmorency tart cherry on key enzymes relevant to type 2 diabetes and cardiovascular disease Ara Kirakosyan, Enrique Gutierrez, Beatriz Ramos Solano, E. Mitchell Seymour, Steven F. Bolling PII: DOI: Reference:

S0308-8146(18)30092-X https://doi.org/10.1016/j.foodchem.2018.01.084 FOCH 22273

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

18 September 2017 7 December 2017 11 January 2018

Please cite this article as: Kirakosyan, A., Gutierrez, E., Solano, B.R., Seymour, E.M., Bolling, S.F., The inhibitory potential of Montmorency tart cherry on key enzymes relevant to type 2 diabetes and cardiovascular disease, Food Chemistry (2018), doi: https://doi.org/10.1016/j.foodchem.2018.01.084

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The inhibitory potential of Montmorency tart cherry on key enzymes relevant to type 2 diabetes and cardiovascular disease

Ara Kirakosyana, Enrique Gutierrezb, Beatriz Ramos Solanob, E. Mitchell Seymoura, Steven F. Bollinga

a

Cardiovascular Research Center, University of Michigan, Ann Arbor, MI, USA

b

Pharmaceutical and Health Sciences Department, Faculty of Pharmacy, Universidad San Pablo-CEU

Universities, Boadilla del Monte, Madrid, Spain.

ABSTRACT The inhibitory potential of Montmorency tart cherry on glycemia regulation and other enzymes relevant to inflammation were evaluated. Tart cherry has superior inhibitory potential against key enzymes associated with carbohydrate digestion linked to hypertension. In particular, α-amylase activity was significantly inhibited (IC50=3.46±0.06 mg/ml), whereas we observed mild inhibition of α-glucosidase (IC50=11.64±0.65 mg/ml). Angiotensin I-converting enzyme inhibition was also strong by about 89%. Tart cherry extract showed strong to moderate inhibitions of cyclooxygenase-1 (65%), lipoxygenase (64%), cyclooxygenase-2 (38%) and xanthine oxidase (26%), respectively. Anthocyanins, cyanidin 3-rutinoside and cyanidin 3-glucoside, were strong inhibitors of α-amylase and α-glucosidase. Kaempferol showed relatively potent inhibition on COX and XO. It was revealed that some pairs of metabolites manifest positive or negative interactions against XO enzyme inhibition. Inhibition of all these enzymes provides a strong biochemical basis for management of type 2 diabetes and cardiovascular disease by controlling glucose absorption, reducing associated hypertension and inflammation. 1

1. Introduction Tart cherries and the anthocyanin compounds present in them have gained much interest due to their beneficial health implications. Anthocyanins have been reported to be strong antioxidants, as well as potential anti-inflammatory and anticancer agents (Kirakosyan, Seymour, Urcuyo Llanes, Kaufman, & Bolling, 2009; Seymour et al., 2009; Wang, & Stoner, 2008; Ghosh, 2005). In addition to these effects, anthocyanins act as a vasoprotectors (Doyle, & Cashman, 2003) and possibly have anti-obesity effects (Seymour et al., 2009; Wu et al., 2006). The potential dietary intake of anthocyanins was considered to be among the greatest of the various classes of flavonoids (Wu et al., 2006). However, much remains to be elucidated before a comprehensive understanding of the effects of anthocyanins and other flavonoids and their functions emerge. People with metabolic syndrome are exposed to increased risks of type-2 diabetes and cardiovascular diseases. Foods high in soluble carbohydrates can result in hyperglycemia, leading to elevation of postprandial glucose. α-glucosidase and α-amylase are key enzymes of dietary carbohydrate digestion in humans. To assess the diet-related prophylaxis of type 2 diabetes mellitus, several natural products have already been examined for inhibitory effect upon α-amylase or α-glucosidase (Loizzo, Di Lecce, Boselli, Menichini, & Frega, 2011). Thus, several polyphenolic constituents were identified as strong inhibitors of these two enzymes (Loizzo et al., 2011). Another important enzyme connected to hyperglycemia and linked to hypertension is angiotensin I-converting enzyme (ACE), which plays an essential role in the regulation of blood pressure in humans (Villiger, Sala, Suter, & Butterweck, 2015). Recent guidelines encourage use of renin-angiotensin-aldosterone system (RAAS) blockers for patients with diabetes and hypertension (Shih, Chu, Ou, & Chen, 2015). This category includes ACE inhibitors and 2

angiotensin II receptor blockers (Shih et al., 2015). Inhibition of ACE is therefore considered an important approach in the treatment of hypertension related to metabolic syndrome. In fact, several other enzymes play central roles in common metabolic reactions, appearing in developmentally regulated processes and as responses to abiotic and biotic stresses. Concerning other aspects of metabolic syndrome, there is a reduced incidence of hyperglycemia and hyperlipidemia with a higher intake of berries and fruits (Seymour et al., 2008; Seymour et al., 2011; Paquette et al., 2017). One of these enzymes, known as xanthine oxidase (XO), catalyzes the oxidation of heterocyclic compounds, such as endogenous and exogenous purines and pyrimidines. XO is considered a major contributor of free radicals in various pathological conditions. More specifically, XO has been implicated in several diseases including gout disease, ischemia-reperfusion injury, myocardial infarction, hypertension, atherosclerosis, diabetes and cancer (Kushiyama et al., 2016). Lipoxygenases (LOXs) are a family of enzymes which dioxygenate unsaturated fatty acids, thus initiating lipoperoxidation of membranes and the synthesis of several signaling molecules. Consequently, these induce structural and metabolic changes in the cell in a number of pathophysiological conditions (Kuhn, Banthiya, & van Leyen, 2015). Another important enzyme related to inflammation and cellular metabolism is cyclooxygenase (COX). COX enzymes catalyze the first step in the biosynthesis of prostaglandins (PGs) and thromboxanes (France´s et al., 2013). The COX-1 isoform is constitutively expressed in many tissues, whereas COX-2 is induced by growth factors, tumor promoters and cytokines (France´s et al., 2013). Additionally, the role of some PGs in several cardiovascular risk factors, diabetes, and obesity has recently been revealed (France´s et al., 2013). It is important to understand how phytochemical-rich tart cherry positively affects the prevention and retardation of oxidative stress-mediated physiological problems, such as inflammation associated with metabolic syndrome, hyperglycemia, and hypertension (Seymour 3

et al., 2008; Seymour et al., 2009). Unravelling the inhibitory potential of tart cherry, and its most bioactive constituents, against the above-mentioned key enzymes may shed light on the possible mechanisms of tart cherry health effects. We aimed to demonstrate the superior ability of tart cherry fruits in vitro in exerting health benefit properties. To this purpose, in the present study the inhibitions of two major carbohydrate digestive enzymes, α-glucosidase and α-amylase, and hypertension-modifying ACE enzyme have been investigated.

In addition, the inhibitory potential of tart cherry on

regulation of XO, COX, and lipoxygenase activities were evaluated. All of these enzymes are connected to metabolic syndrome and directly or indirectly involved in the initiation and propagation of inflammation.

2. Materials and methods 2.1. Anthocyanin and total phenolic analysis of tart cherry IQF powder Tart cherry powder was prepared by VanDrunen Farms (Momence, IL USA). The powder’s nutrient analysis was conducted by VanDrunen Farms and its subsidiary, Futureceuticals. The quantification of anthocyanins and total phenolics of tart cherry powder have been evaluated by our group using the same method as we reported previously (Kirakosyan et al., 2010). Briefly, 0.5 g of powder was extracted with 10 ml methanol:water:acetic acid (85:15:0.5 v/v/v) for anthocyanins and 10 ml methanol:water (80:20 v/v) for other flavonoids in 15-ml screw-cap tubes at 4oC and placed on a shaker overnight in the dark. The samples were then vortexed and sonicated. After filtration through a 0.22 µm filter, the extracts were ready for analysis. Obtained results were validated by liquid chromatography-mass spectrometry analysis (Kirakosyan et al., 2010). The anthocyanin and key flavonoid contents of tart cherry powder are presented in Table 1. 4

2.2. In vitro α-glucosidase inhibition assay Alpha-glucosidase (α-glucosidase from Saccharomyces cerevisiae, Sigma Chemical Co., St. Louis, MO) inhibition was assessed using the method described by Schmidt and coworkers (Schmidt, Lauridsen, Dragsted, Nielsen, & Staerk, 2012). In brief, 90 µl of 0.1 M phosphate buffer (pH 7.5), 10 µl test sample dissolved in DMSO, and 80 µl of enzyme solution (well concentration 0.08 U/ml) were added to each well. The mixture was incubated at 28°C for 10 min before adding 4-Nitrophenyl-α-D-glucopyranoside (PNPG) (Sigma Chemical Co., St. Louis, MO) to a final volume of 200 µl. The blank was carried out in a similar manner, with the test sample replaced by solvent. The hydrolysis rate of PNPG to release p-nitrophenolate was monitored at 405 nm every 30 s for 35 min. Incubation and absorbance measurements were performed with a BioTek Eon™ microplate reader with a built-in incubator, controlled by Gene5 2.0 software. The α-glucosidase inhibitory activity was expressed as percentage inhibition and was calculated using the following formula: % inhibition = (Slope blank – Slope sample)/ Slope blank X100.

2.3. In vitro α-amylase inhibition assay The α-amylase (α-amylase from porcine pancreas, Sigma Chemical Co., St. Louis, MO) inhibition assay was conducted according to the method described by Trinh and coworkers (Trinh, Staerk, & Jäger, 2016). In brief, 80 µl of 0.1M phosphate buffer (pH 6.0), 20 µl test sample dissolved in DMSO, and 80 µl of the enzyme solution (well concentration 0.05 U/ml) were added to each well. After incubation at 37°C for 10 min, the reaction was started by adding 2-Chloro-4-nitrophenyl-α-D-maltotrioside (CNP-G3) (Sigma Chemical Co., St. Louis, MO) to a final volume of 200 µl. The blank was carried out in a similar manner, with the test sample replaced by solvent. Absorbance was measured at 405 nm every third minute for 30 min on the 5

same instrument as described above. The α-amylase inhibitory activity was expressed as % inhibition and was calculated using the following formula: % inhibition = (Slope blank–Slope sample)/ Slope blank X 100.

2.4. Angiotensin I-converting enzyme (ACE) inhibition assay ACE inhibition was assayed by a slightly modified method than previously described (Kwon, Apostolidis, & Shetty, 2008). The substrate, hippuryl-histidyl-leucine (HHL), and angiotensin Iconverting enzyme (ACE) from rabbit lung were purchased from Sigma (Sigma Chemical Co., St. Louis, MO). 40 µl of extract (10 mg/ml tart cherry powder dissolved in 80% MeOH) were incubated with 60 µl of 1.0 M NaCl-borate buffer (pH 8.3) containing 1.0 mU ACE solution at 37oC for 10 min. After pre-incubation, 20 µl of 5.0 mM substrate (HHL) solution was added to reaction mixture. Test solutions were incubated with agitation at 37oC for 30 min. The reaction was stopped with 250 µl of 0.5 M hydrogen chloride. The hippuric acid (HA) formed was detected and quantified by HPLC method (Shimadzu HPLC equipped with a photodiode array detector (SPD-10A system from Shimadzu, Japan). The HPLC conditions were as follows: a Phenomenex LunaTM column (Torrance, CA USA) (5 micron pore size, C-18, 150 mm. x 4.60 mm), flow rate of 0.4 ml per min. The isocratic solution of 75% H2O + 0.1% TFA and 25% of C2H3N was used. Detection was set at 226 nm. Pure hippuric acid was used to calibrate the standard curve and retention time. The % inhibition was calculated by formula: % inhibition = (A–B)/ A x 100 A is the peak area of HA without addition of ACE inhibitors; B is the peak area of HA with addition of ACE inhibitors.

2.5. Lipoxygenase activity inhibition assay

6

The inhibition of lipoxygenase was conducted using a Lipoxygenase Inhibitor Screening Assay Kit (Cayman Chemical, Ann Arbor, MI USA). The kit provides an accurate and convenient method for screening lipoxygenase inhibitors. This assay measures the hydroperoxides generated from the incubation of a lipoxygenase (15-LO) with arachidonic acid. Absorbance measurements were performed with a BioTek Eon™ microplate reader with a built-in incubator, controlled by Gene5 2.0 software. Percent enzyme inhibition was calculated as 100 × [(∆A1 − ∆A2)/∆A1], where ∆A1 and ∆A2 are values for increase in absorbance at 500 nm for sample without test substance and with test substance, respectively. Quercetin was used as a positive control.

2.6. Xanthine oxidase inhibition assay The XO (Xanthine Oxidase, Buttermilk; Calbiochem, San Diego, CA) inhibitory activity with hypoxanthine (Sigma Chemical Co., St. Louis, MO) as the substrate was measured spectrophotometrically (Shimadzu, Japan). The assay mixture consisted of 50 µl of test compound (dissolved in 80% MeOH) or for a blank 80% MeOH alone, 150 µl of 50 mM sodiumpotassium phosphate buffer (PBS) [pH = 7.5] and 50 µl of enzyme solution (0.5 U/ml in PBS). Substrate solution 250 µl (0.15M hypoxanthine in the same buffer) was added. The increase in absorbance at 295 nm was measured over a period of 5 min. % enzyme inhibition was calculated as 100 × [(∆A1 − ∆A2)/∆A1], where ∆A1 and ∆A2 are values for the increase in A295 for the sample without test substance and with test substance, respectively.

2.7. Cyclooxygenase (COX 1 and COX 2) inhibition assay COX inhibitor activity was measured using the commercially available COX Activity Assay (Cayman Chemical, Ann Arbor, MI USA) following the manufacturer´s instructions. The Kit measures the peroxidase activity of COX 1 and COX 2 simultaneously, assayed colorimetrically 7

by monitoring the appearance of oxidized N, N, N, N-tetramethyl-p-phenylenediamine (TMPD); samples were measured in triplicate at 590 nm using a BioTek Eon™ microplate reader.

2.8. Determination of synergistic effect of selected compounds in tart cherry To assess synergy among tart cherry phytochemicals, we examined the dose-dependent impact of single and combined tart cherry constituents on xanthine oxidase inhibitory activity. According to our previous study with tart cherry phytochemicals (Kirakosyan et al., 2010) selected targeted compounds have been employed for their synergistic types of interactions at the proper dose ratios (e.g., 1:1; 1:2; and 1:4). Combination index (CI) for three ratios was determined using CombiTool (Jena, Germany).

2.9. Statistical analysis Results were expressed as means and standard deviations from three independent experiments run in duplicate (n=6). Data were subjected to one-way ANOVA, and the significance of differences between means calculated by Duncan’s multiple range test using SPSS for Windows, and significance accepted at P<0.05.

3. Results 3.1.

Inhibition of glycemia regulating enzymes

The results presented in Table 2 show that tart cherry extract tended to be a strong inhibitor of glycemia regulating enzymes. In particular α-amylase activity showed significant inhibition (IC50=3.46±0.06 mg/ml). On the contrary, α-glucosidase inhibition is moderate (IC50=11.64±0.65 mg/ml). ACE inhibition was strong by about 89%. In addition, tart cherry extract exhibited strong lipoxygenase 8

inhibitory activity (64% inhibition). The approach of mild inhibition of α-amylase and strong inhibition of α-glucosidase is considered ideal for managing blood glucose. In fact, tart cherry extract strongly inhibited α-amylase and ACE. Lipoxygenase inhibition by almost 64% is also considered strong based on our comparison with red raspberry, grape and blueberry inhibitory potentials (Figure 1). Several individual compounds naturally present in tart cherry were used to test their inhibitory effects on these enzymes. We excluded ACE enzyme for the individual compounds study due to the complexity of ACE inhibition assayed by HPLC analysis. Results in Table 3 show that almost all individual compounds possess strong inhibitory activity on α-amylase and α-glucosidase, excluding melatonin (α-amylase inhibition only).

Very strong inhibition of α-amylase was

observed with cyanidin 3-rutinoside and cyanidin 3-glucoside (IC50=0.004±0.001 mg/ml and IC50=0.007±0.001 mg/ml, respectively). α-glucosidase inhibition was also significant with the use of these two anthocyanin compounds. It is interesting to note that anthocyanins do not show lipoxygenase inhibition. Quercetin was the most potent inhibitor of lipoxygenase, by about 67%.

3.2.

The COX enzyme inhibitory activity

It is well known that COX-1 is constitutively expressed in most tissues and believed to be responsible for normal physiological functions. On the contrary, COX-2 is involved in many inflammatory processes and is induced in various carcinomas, suggesting that COX-2 plays a key role in inflammation and tumorigenesis. We therefore examined the inhibitory effects of tart cherry crude extract on both COX enzymes (Table 4). Our results revealed that tart cherry extract inhibited COX-1 enzyme activity by 65%. On the other hand, COX-2 enzyme inhibition was moderate, only by 38%. The inhibitory potentials of tart cherry individual constituents showed different patterns of COX-1 and COX-2 inhibitions (Table 4). The most active compound 9

for COX-2 inhibition was isorhamnetin-3-rutinoside at 50 µg/ml concentration. Kaempferol appeared to be most bioactive for COX-1 inhibition at the same concentration.

3.3.

Xanthine oxidase inhibitory activity

Tart cherry crude extract showed moderate inhibition of XO (26%). We also examined XO inhibition pattern by tart cherry individual constituents. Data in Table 4 shows that kaempferol has the highest inhibitory activity on xanthine oxidase (23.3% of inhibition) among other phytochemicals, followed by cyanidin 3-rutinoside (21.5%). In a follow up experiment, we conducted a combination study of XO inhibition by several tart cherry phytochemicals. The XO assay was easy to carry out; in addition, we assumed that low percentage of inhibition by tart cherry individual constituents may reveal even negligible synergistic or antagonistic effects. The isobolographic analysis (CombiTool) revealed that the most important combination of two different constituents is isorhamnetin 3-rutinoside and kaempferol (XO inhibition by 38 % at 2:1 ratio). Some examples of additive effects (kaempferol : melatonin) and antagonistic interaction (cyanidin 3-rutinoside : melatonin) were observed as well (Table 5).

4. Discussion Our in vitro results of α-amylase inhibition support the view that tart cherry may reduce the glucose peaks that can occur after a meal. This is consistent with our other tart cherry studies in vivo (Seymour et al., 2008; Seymour et al., 2009). This is of particular importance in people with diabetes, where low insulin levels prevent extracellular glucose being cleared from organism. It is well known that hyperglycemia often leads to hypertension, a macrovascular complication in which ACE is stimulated. Within the enzyme cascade of the renin-angiotensin system, ACE 10

removes histidyl-leucine from angiotensin I to form the physiologically active octapeptide angiotensin II, one of the most potent known vasoconstrictors (Shih et al., 2015). On the other hand, ACE is an important enzyme in maintaining vascular tension through the inactivation of bradykinin (a vasodilator substance). (Villiger et al., 2015; Fernández, & Labra, 2013). The beneficial effect of ACE inhibitors in hypertension may relate, at least in part, to their capacity to interfere with bradykinin metabolism (Tom, Dendorfer, & Jan Danser, 2003). ACE inhibition by tart cherry extract support our view that it may have great implication for a future dietary approach to prevent hypertension in humans. Tart cherries contain sugar, but they paradoxically reduce glycemic stress following a meal compared to other foods (Seymour et al., 2009). In addition, slow conversion of starch to monosaccharides may positively affect postprandial stress as well. In a study reported by Tahsini and Heydari (Tahsini, & Heydari, 2012), tart cherry extracts showed an anti-diabetic effect in alloxan induced diabetic rats. The study revealed the hypoglycemic activity of tart cherry. However, neither phytochemical composition nor possible mechanisms of action of the cherry extracts were reported in this study. They concluded that tart cherry extracts may possibly reduce the number of released free radicals, which are the causative agents for the tissue distraction and insulin resistance. Such hypoglycemic effect of tart cherry extracts still remains speculative. Diverse phytochemicals in tart cherry may be directly involved in several therapeutic actions by altering specific gene transcription and translation, thereby improving physiological resistance to oxidative and inflammatory stressors. Several individual compounds have shown a strong inhibitory effect in different assays. This is particularly relevant to anthocyanins, and other flavonoids. In fact, such effects are neglected because their content in tart cherry fruits is relatively low. Furthermore, in dietary approach more attention has been given to whole fruits and their nutritional values. However, here, we are not excluding possible synergistic interaction 11

of several bioactive compounds even though their occurrence in tart cherry fruit is minimal. When tart cherry individual constituents were used in combination at relevant doses, some pairs of metabolites manifested positive or negative interactions. This synergistic or antagonistic concept is still not well understood; however, the observed effects of interactions may be explained by enhanced uptake, absorption, metabolism and finally reduced excretion. Another possibility of such effects may be based on enhanced effectiveness (binding to receptor molecules like bioactive proteins that enhance protein–protein or protein–ligand interactions) at the target site(s) of action. Although this is an in vitro study, the results indicate the positive effect of tart cherry and its individual compounds on glycemia regulation and other enzymes relevant to inflammation. It is well known that in vitro bioassays sometimes may not be relevant to the real picture of pharmacological and nutritional effects of single compound(s) or whole food consumption. However, in vitro studies can only be considered misleading if pharmacokinetic or pharmacodynamic properties of studied compounds were ignored, or if compounds were used in a higher dosage than physiologically accepted (Villiger et al., 2015). We have shown that tart cherry extract and its bioactive constituents possess activity for potential health benefits as inhibitors of key enzymes relevant to type 2 diabetes and cardiovascular disease. The difference in activity between anthocyanins and other compounds, to our knowledge, has not been reported previously. No systematic investigations of tart cherry extracts on all studied enzymes have been reported previously. Moreover, dietary approach with tart cherry fruits has intensively been employed by our group using several rat models and human subjects. Of note is that all present and previous data are well correlated. The current study with tart cherry fruit extract revealed several important mechanistic concepts that may also hold true for edible fruits of other plants. These insights could help target the addition of tart cherry as a part of an overall comprehensive dietary management of 12

metabolic syndrome and its complications. Moreover the local tart cherry-growers will get the benefit of the great increase in favour of this fruit as a part of the daily diet.

Conflict of interest All authors declare that they have no conflict of interest.

Acknowledgements This study was funded by an unrestricted Grant from the Cherry Research Committee of the Cherry Marketing Institute (Lansing, MI, USA). The Cherry Marketing Institute did not participate in data analysis or manuscript preparation.

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Schmidt, J.S., Lauridsen, M.B., Dragsted, L.O., Nielsen, J., & Staerk, D. (2012). Development of a bioassay-coupled HPLC-SPE-ttNMR platform for identification of αglucosidase inhibitors in apple peel (Malus ×domestica Borkh.). Food Chemistry, 135, 16921699. Seymour, E.M., Singer, A.A., Kirakosyan, A., Urcuyo-Llanes, D.E., Kaufman, P.B., & Bolling, S.F. (2008). Altered hyperlipidemia, hepatic steatosis, and hepatic peroxisome proliferator-activated receptors in rats with intake of tart cherry. Journal of Medicinal Food 11, 252-259. Seymour, E.M., Lewis, S.K., Urcuyo-Llanes, D.E., Tanone, I.I., Kirakosyan, A., Kaufman, P.B., & Bolling S.F. (2009). Regular tart cherry intake alters abdominal adiposity, adipose gene transcription, and inflammation in obesity-prone rats fed a high fat diet. Journal of Medicinal Food, 12, 935-942. Seymour, E.M., Tanone, I.I., Urcuyo-Llanes, D.E., Lewis, S.K., Kirakosyan, A., Kondoleon, M.G., Kaufman, P.B., & Bolling, S.F. (2011). Blueberry intake alters skeletal muscle and adipose tissue peroxisome proliferator-activated receptor activity and reduces insulin resistance in obese rats. Journal of Medicinal Food, 14, 1511-1518. Shih, C.-J., Chu, H., Ou, S.-M., & Chen, Y.-T. (2015). Comparative effectiveness of angiotensin-converting enzyme inhibitors and angiotensin II receptor blockers on major adverse cardiac events in patients with newly diagnosed type 2 diabetes: A nationwide study. International Journal of Cardiology, 199, 283-289. Tahsini, L., & Heydari, R. (2012). Anti diabetic effect of cherries in alloxan induced diabetic rats. Recent Patents on Endocrine, Metabolic & Immune Drug Discovery, 6, 67-72. Tom, B., Dendorfer, A., & Jan Danser, A.H. (2003). Bradykinin, angiotensin-(1–7), and ACE inhibitors: how do they interact? The International Journal of Biochemistry & Cell Biology, 35 792–801.

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Trinh, B.T.D., Staerk, D., & Jäger, A.K. (2016). Screening for potential α-glucosidase and α-amylase inhibitory constituents from selected Vietnamese plants used to treat type 2 diabetes. Journal of Ethnopharmacology, 186, 189-195. Villiger, A., Sala, F., Suter, A., & Butterweck, V. (2015). In vitro inhibitory potential of Cynara scolymus, Silybum marianum, Taraxacum officinale, and Peumus boldus on key enzymes relevant to metabolic syndrome. Phytomedicine, 22, 138-144. Wang, L.-S. & Stoner, G.D. (2008). Anthocyanins and their role in cancer prevention. Cancer Letters, 269, 281–290. Wu, X., Beecher, G.R., Holden, J.M., Haytowitz, D., Gebhardt, S.E., & Prior, R.L. (2006). Concentrations of anthocyanins in common foods in the United States and estimation of normal consumption. Journal of Agricultural and Food Chemistry, 54, 4069-4075.

16

70

c

60

% inhibition

50 40 b 30 20 10 a

a

0 Red Raspberry

Blueberry

Grape

Tart Cherry

Figure 1. Comparison of lipoxygenase inhibitory potentials of red raspberry, blueberry, grape and tart cherry extracts. Values showing the same index in the same row are not significantly different (P<0.05).

17

Table 1. Anthocyanin and other flavonoid contents in tart cherry powder Anthocyanins and other flavonoids Cyanidin 3-sophoroside Cyanidin 3-glucosylrutinoside Cyanidin 3-glucoside

µg/g of dry weight 2.8 ± 0. 5 325.9 ± 57.2 3.1 ± 0.7

Cyanidin 3-rutinoside 5- β-D-glucoside

120.2 ± 20.5

Cyanidin 3-rutinoside

274.2 ± 43.2

Peonidin 3-glucoside

7.4 ± 1.1

Peonidin 3-rutinoside

40.4 ± 7.7

Pelargonidin

1.2 ± 0.4

Isorhamnetin rutinoside

176.6 ± 22.3

Quercetin-3-rutinoside

292.6 ± 35.4

Quercetin-3-glucoside

5.4 ± 0.4

Kaempferol-3-rutinoside

85.9 ± 9.8

Table 2. Inhibitory potential of tart cherry extract on glycemia regulating enzymes

18

Assay

Value

α-amylase IC50 (mg/ml)

3.46±0.06

α-glucosidase IC50 (mg/ml)

11.64±0.65

Lipoxygenase (% inhibition)

63.97±2.07

ACE (% inhibition)

88.70±0.08

Table 3. α-glucosidase, α-amylase and lipoxygenase enzyme inhibition by tart cherry individual compounds* 19

Individual Compounds

α-glucosidase

α-amylase

Lipoxygenase

IC50 (mg/ml)

IC50 (mg/ml)

(%) inhibition**

Isorhamnetin 3-rutinoside

0.548±0.064c

0.164±0.023c

15.263±3.375b

Melatonin

0a

0.150±0.001c

15.904±0.739b

Quercetin

0.093±0.001bc

0.063±0.002b

67.419±1.406d

Kaempferol

0.155±0.002c

0.063±0.002b

29.738±1.846c

Cyanidin 3-rutinoside

0.027±0.002ab

0.004±0.001a

0a

Cyanidin 3-glucoside

0.029±0.003ab

0.007±0.001a

0a

*Values showing the same index in the same row are not significantly different (P<0.05). **Individual compound concentration (0.1mg/ml).

Table 4. Inhibition of COX-1, COX-2 and XO (% inhibition) by tart cherry extract and its individual compounds (0.1mg/ml)*

20

COX-1

COX-2

XO

Cyanidin 3-glucoside

47.0±0.31b

36.3±0.22b

19.8±2.8b

Cyanidin 3-rutinoside

44.6±0.48a

37.6±0.29b

21.6±3.3c

Quercetin

52.8±0.61c

42.3±0.38c

10.0±1.9a

Isorhamnetin 3-rutinoside

45.4±0.47a

44.5±0.39c

20.7±2.8b

Kaempherol

53.1±0.62c

34.8±0.28a

23.4±3.3d

Melatonin

44.7±0.39a

37.4±0.41b

10.6±1.8a

Tart cherry

65.1±0.72

37.8±0.35

26.0±3.5

*Values showing the same index in the same row are not significantly different (P<0.05).

Table 5. XO inhibitory capacities with a combination of two sets of metabolites (0.1mg/ml) at relevant dose ratios (1:1 versus 1:2 versus 1:4) Interacting compounds Cyanidin 3-rutinoside : Melatonin

Dose ratio 1:1 2:1 4:1 1:2 21

% inhibition 2 1 3.8 18.9

1:4 Type of Interaction Cyanidin 3-rutinoside : Isorhamnetin 3-rutinoside

Type of Interaction Kaempferol : Melatonin

1:1 2:1 4:1 1:2 1:4 1:1 2:1 4:1 1:2 1:4

Type of Interaction Isorhamnetin 3-rutinoside : Kaempferol

1:1 2:1 4:1 1:2 1:4

Type of Interaction Kaempferol : Cyanidin 3-glucoside

1:1 2:1 4:1 1:2 1:4

Type of Interaction Cyanidin 3-glucoside : Quercetin

1:1 2:1 4:1 1:2 1:4

Type of Interaction Cyanidin 3-rutinoside: Quercetin

1:1 2:1 4:1 1:2 1:4

Type of Interaction

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26.9 Antagonistic 2 3 11.4 4.7 31.3 Additive 35.8 36.7 31.3 14.5 32.2 Additive 32.7 38.0 32.7 36.7 35.3 Synergistic 24.2 19.5 18.9 17.1 16.7 Additive 36.7 32.7 28.7 31.3 28.7 Synergistic 10.0 12.7 14.5 13.6

16.2 Antagonistic

Highlights:

Tart cherry has superior inhibitory potential against glycemia regulating enzymes

α-amylase activity was significantly inhibited (IC50=3.46±0.06 mg/mL)

Tart cherry showed strong to moderate inhibitions of cyclooxygenase-1, lipoxygenase, cyclooxygenase-2 and xanthine oxidase, respectively.

Cyanidin 3-rutinoside and cyanidin 3-glucoside are strong inhibitors of α-amylase and αglucosidase. Kaempferol showed relatively potent inhibition on COX and XO.

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