α-glucosidase and α-amylase inhibitory activities of phloroglucinal derivatives from edible marine...

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Research ArticleReceived: 20 November 2008 Revised: 19 January 2009 Accepted: 17 March 2009 Published online in Wiley Interscience: 29 April 2009

(www.interscience.wiley.com) DOI 10.1002/jsfa.3623

α-Glucosidase and α-amylase inhibitoryactivities of phloroglucinal derivatives fromedible marine brown alga, Ecklonia cavaSang-Hoon Lee,a† Yong-Li ,a,b† Fatih Karadeniz,a Moon-Moo Kimc andSe-Kwon Kima,d∗

Abstract

BACKGROUND: Diabetes mellitus (DM) is a chronic metabolic disorder characterized by defects in insulin secretion and action,which can lead to damaged blood vessels and nerves. With respect to effective therapeutic approaches to treatment of DM,much effort has being made to investigate potential inhibitors against α-glucosidase and α-amylase from natural products. Theedible marine brown alga Ecklonia cava has been reported to possess various interesting bioactivities, which are studied here.

RESULTS: In this study, five phloroglucinal derivatives were isolated from Ecklonia cava: fucodiphloroethol G (1), dieckol (2),6,6′-bieckol (3), 7-phloroeckol (4) and phlorofucofuroeckol A (5); compounds 1, 3 and 4 were obtained from this genus for thefirst time and with higher yield. The structural elucidation of these derivatives was completely assigned by comprehensiveanalysis of nuclear magnetic spectral data. The anti-diabetic activities of these derivatives were also assessed using an enzymaticinhibitory assay against rat intestinal α-glucosidase and porcine pancreatic α-amylase. Most of these phlorotannins showedsignificant inhibitory activities in a dose-dependent manner, responding to both enzymes, especially compound 2, with thelowest IC50 values at 10.8 µmol L−1 (α-glucosidase) and 124.9 µmol L−1 (α-amylase), respectively. Further study of compound 2revealed a non-competitive inhibitory activity against α-glucosidase using Lineweaver-Burk plots.

CONCLUSION: These results suggested that Ecklonia cava can be used for nutritious, nutraceutical and functional foods indiabetes as well as for related symptoms.c© 2009 Society of Chemical Industry

Keywords: anti-diabetes; α-glucosidase; α-amylase; Ecklonia cava; phlorotannins

INTRODUCTIONDiabetes mellitus (DM) is a chronic metabolic disorder charac-terized by defects in both insulin secretion and insulin action,causing raised blood glucose levels, which in turn can damagemany systems in the body, such as blood vessels and nerves.1 DMaffects about 5% of the global population,2 and is becoming thefifth leading cause of death, according to a recent survey of globalmortality.3 The disorder is still maintaining a surprising preva-lence, and unfortunately the current treatments are only limitedto a weak control of exacerbation, and in addition have variousside effects. Based on the latest studies, DM can be classified intoseveral types, including type I and type II. The most common typeis type II DM, which accounts for over 90% of all diabetes suf-ferers. In the digestive process of dietary complex carbohydrate,α-glucosidase and α-amylase play a significant role. Inhibition ofboth enzymes can retard digestion of oligosaccharides and disac-charides, and delay glucose absorption as well as reduce glucoselevels in plasma, finally resulting in suppression of postprandialhyperglycemia.2

Therefore decreasing the level of postprandial hyperglycemiahas been considered one of most effective therapeutic approaches,with fewer disadvantages than other approaches in the earlyperiod of DM. With respect to suppression of glucose production

from carbohydrate and glucose absorption from the intestine,increasing efforts have being made to investigate and findpotential inhibitors of α-glucosidase and α-amylase in naturalproducts.4 – 6 In particular, the unique marine environment is beingturned to for the development of effective candidates.

As part of our continuous research in bioactive nature productsfrom marine sources, Ecklonia cava (EC), a marine edible brownseaweed widely distributed along the coast of Jeju Island ofSouth Korea, was selected as our target. EC has been reported topossess various interesting bioactivities such as radical scavenging

∗ Correspondence to: Se-Kwon Kim, Marine Bioprocess Research Center, PukyongNational University, Busan 608-737, South Korea. E-mail: [email protected]

† These authors contributed equally to this work.

a Department of Chemistry, Pukyong National University, South Korea

b Resource Institute, Academy of Sciences of Traditional Chinese Medicine of JilinProvince, Chang-Chun, 130021, People’s Republic of China

c Department of Chemistry, Dong-Eui University, Busan 614-714, South Korea

d Marine Bioprocess Research Center, Pukyong National University, Busan 608-737, South Korea

J Sci Food Agric 2009; 89: 1552–1558 www.soci.org c© 2009 Society of Chemical Industry

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Anti-diabetes activity of phloroglucinal derivatives www.soci.org

O

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Figure 1. Chemical structures of phlorotannins from Ecklonia cava.

J Sci Food Agric 2009; 89: 1552–1558 c© 2009 Society of Chemical Industry www.interscience.wiley.com/jsfa

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www.soci.org S-H Lee et al.

activity,7 – 9 anti-inflammation activity,10 matrix metalloproteinaseinhibitory activity11 and anticoagulant activity.12 The chemicalstudy of EC revealed that phlorotannins, oligomeric polyphenolsof the phloroglucinol unit, are the main constituents responsiblefor the biological activity of EC;9,11,12 they include eckol, dieckol,8,8′-bieckol and 8,4′′′-bieckol (inhibitory activity on HIV-1 reversetranscriptase and protease, angiotensin converting enzyme andtyrosinase inhibitory activity).13 – 15 In our previous study, the mainphlorotannin 6,6′-bieckol isolated from EC also showed interestinganti-HIV-1 activity by a unique pathway.16

Phenolic derivatives from different sources have been reportedin many research fields with regard to various bioactivities.17 – 20

In this study, five phlorotannins from EC methanolic extract aredescribed and completely characterized by mass spectrometricand nuclear magnetic resonance (NMR) techniques. Among thesephlorotannins (Fig. 1), three compounds (1, 3, and 4) wereisolated from this genus for the first time, along with the othertwo phloroglucinal derivatives (2 and 5). Moreover, to developpotential candidates from marine natural products with activityagainst diabetes, these phlorotannins were also investigated withregard to their inhibitory effects on both α-glucosidase and α-amylase. The result revealed that most of these phlorotanninsshowed significant activities. Further study of the most effectivecompound (2) for an inhibitory mechanism indicated a non-competitive inhibition against α-glucosidase using the method ofLineweaver–Burk plots. These results suggested that EC can beused for nutritious and functional foods for diabetes as well asrelated symptoms.

MATERIALS AND METHODSGeneral materialsThe marine edible brown seaweed Ecklonia cava was collectedalong the coasts of Jeju Island of Korea. Fresh EC was washedthree times with tap water to remove salt and lyophilized. Ratintestinal acetone powder and porcine pancreatic α-amylase weresupplied by Sigma Aldrich Korea Co. (Seoul, Korea). 1H NMR(400 MHz) and 13C NMR (100 MHz) spectra were recorded ona JEOL JNM-ECP 400 NMR spectrometer (JEOL, Tokyo, Japan),using DMSO-d6 solvent peak (2.50 ppm in 1H and 39.5 ppm in13C NMR) as an internal reference standard. For some signals,chemical shifts were approximated to the third decimal place. Thisis to distinguish between signals of very close value but whichcould nevertheless be clearly differentiated by visual inspectionof the spectra. Mass spectra were obtained on a JEOL JMS-700 spectrometer. Extraction of EC was performed using anextraction unit (Dongwon Scientific Co., Seoul, Korea). Columnchromatography was carried out using silica gel 60 (230–400mesh, Merck, Darmstadt, Germany) and Sephadex LH-20 (Sigma,St Louis, MO, USA). Thin-layer chromatography (TLC) was run onpre-coated Merck Kieselgel 60 F254 plates (0.25 mm), and the spotson the TLC plate were detected under a UV lamp (254 and 365 nm)using CHCl3 –MeOH–H2O–acetic acid (65 : 25 : 4 : 3, v/v/v/v) as adevelopment solvent system.21 Vanillin–H2SO4 was employed asthe detecting agent for phenolic compounds.22 All the solventand chemicals used in this study were of reagent grade fromcommercial sources.

Extraction, isolation and purification of phlorotanninsThe lyophilized EC was ground into powder before extraction. Thedried EC powder (10 kg) was extracted by stirring the extraction

unit with MeOH (3 × 5 L) for 10 days. The extract (273 g) was sus-pended in water and partitioned with EtOAc. The EtOAc fraction(24.87 g), which exhibited the most effective α-glucosidase andα-amylase inhibitory activities, was subjected to silica gel flashchromatography and eluted with hexane–EtOAc–MeOH (gradi-ent) to yield 10 subfractions (F1–F10). F5 and F6 (976.235 mg),with the highest activities, were further purified by Sephadex LH-20 with MeOH to yield the phloroglucinal derivatives compounds1 (47.62 mg), 2 (118.78 mg), 3 (127.51 mg), 4 (132.96 mg) and 5(53.71 mg), respectively.

Assay for α-glucosidase inhibitory activityThe α-glucosidase inhibitory assay was performed according tothe method described by Kim et al.23 with slight modification.1 µL α-glucosidase solution (2 units mL−1, 0.1mol L−1 potassiumphosphate buffer, pH 6.8) was pre-mixed with 1 µL of samplesolution at different concentrations (in 10% DMSO) in 95 µL of0.1 mol L−1 potassium phosphate buffer (pH 6.8) in 96-well plates.Following incubation for 20 min at 37.5 ◦C, 3 µL p-nitrophenylglucopyranoside (pNPG, 3 mmol L−1) as substrate was addedto the mixture to start the reaction. The reaction mixture wasincubated at 37.5 ◦C for 20 min, followed by addition of 1 molL−1 Na2CO3 solution to terminate the reaction. The amount ofreleased product (p-nitrophenol) was measured at 410 nm usinga UV microplate reader (Tecan Austria GmbH, Grodig, Austria)to estimate enzymatic activity. The IC50 value was defined asthe concentration of inhibitor required to inhibit 50% of the α-glucosidase inhibitory activity. For all tests, the inhibition assaywas performed in triplicate.

Assay for α-amylase inhibitory activityThe α-amylase inhibitory assay was performed according to themethod described by Kim et al.23 with slight modification. 10 µLα-amylase solution (1 unit mL−1, distilled water (DW)) was pre-mixed with 1 µL of sample solution at different concentrations(in 10% DMSO). Following incubation for 15 min, 180 µL of 1%starch solution in 20 mmol L−1 sodium phosphate buffer (pH6.9) was added to start the reaction. The reaction was carriedout at 37.5 ◦C for 5 min and terminated by addition of 500 µL ofthe DNS reagent (1% 3,5-dinitrosalicylic acid, 12% Na-K tartratein 0.4 mol L−1 NaOH). The reaction mixture was placed in awater bath at boiling point for 15 min and then cooled down toroom temperature. α-Amylase activity was determined at 540 nmby using a spectrophotometer (Model U-3210, Hitachi Co., Tokyo,Japan). The IC50 value was defined as the concentration of inhibitorrequired to inhibit 50% of the α-amylase inhibitory activity. For alltests, the inhibition assay was performed in triplicate.

Determination of the inhibition pattern on α-glucosidaseDifferent concentrations of phrolotannin derivatives were addedto each reaction mixture and the enzyme activity was measuredwith different concentrations of the substrate. The α-glucosidaseinhibitory pattern in the presence of compound 2 was determinedusing Lineweaver–Burk plots.

Statistical analysisThe data were expressed as the mean of three replicate determi-nations and standard deviation (SD); statistical comparisons weremade with Student’s t-test (P < 0.01).

www.interscience.wiley.com/jsfa c© 2009 Society of Chemical Industry J Sci Food Agric 2009; 89: 1552–1558

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RESULTS AND DISCUSSIONPostprandial hyperglycemia is a prominent and early defectin the development of type 2 DM. The treatment goal forpatients with type 2 DM is to maintain near-normal levels ofglycemic control, both in the fasting and postprandial states.24

α-Amylase breaks down starch, glycogen and oligosaccharidesby catalyzing hydrolysis of α-1,4-glucosidic linkages, and α-glucosidase further breaks down the disaccharides into simplersugars, readily available for intestinal absorption.6 Inhibitors ofthese enzymes delay carbohydrate digestion and prolong overallcarbohydrate digestion time, causing a reduction in the rate ofglucose absorption and consequently blunting the postprandialplasma glucose rise.25

In this study anti-diabetic potential was investigated usingEC, which is an edible brown seaweed and abundant along thecoast of Jeju Island in South Korea. The EtOAc fraction of ECmethanolic extract showed the strongest inhibitory activity. Itsfurther isolation resulted in bioactive phlorotannin derivatives,which are polymers of phloroglucinol units and found especiallyin marine brown algae.26 It has been reported that phlorotanninderivatives are responsible for biological activities such as HIV-1 reverse transcriptase and protease, angiotensin convertingenzyme and tyrosinase inhibitory activity.13 – 15 However, thereare no reports on the α-glucosidase and α-amylase inhibitoryeffects of phlorotannin derivatives isolated from EC. In our presentstudy, we established the anti-diabetic potential of EC in themanagement of type 2 DM and revealed that active compoundsresponsible for this activity should be phlorotannin derivatives.

Screening of EC extracts on α-glucosidase and α-amylaseMany α-glucosidase inhibitory extracts from various sourceshave recently been reported, such as Punica granatum flowerextract (IC50, 1.8 µg ml−1), French maritime pine bark extract (IC50,5.34 µg ml−1) and green tea extract (IC50, 19.74 µg ml−1).27,28

The methanolic extract of EC showed considerable inhibitoryactivity for only α-glucosidase. The methanolic extract of EC,however, showed low inhibitory activity (IC50, 37.07 µg ml−1)against α-glucosidase. But after partitioning the extract whichwas suspended in water with EtOAc, the EtOAc-soluble fractiondemonstrated over eight times higher inhibitory activity (IC50,4.34 µg ml−1) than the water fraction (IC50, 35.37 µg ml−1) on α-glucosidase and its IC50 value on α-amylase was 345.0 µg ml−1

(Table 1). Broadhurst et al.29 reported that polyphenolic extractsfrom a number of plants were found to be effective inhibitorsof intestinal α-glucosidase activity and also α-amylase activity,which may prove to be synergistic in their potential therapeuticeffect on the postprandial plasma glucose rise. The EtOAc fractionexhibiting the most effective inhibitory activities on α-glucosidaseand α-amylase was selected for further investigation in orderto isolate the effective constituents responsible for the aboveinhibitory activity.

Isolation and identification of phlorotannin derivatives fromECIn the continuing investigation, five phlorotannins were isolatedfrom the EtOAc fraction of EC methanolic extract. Among thesephlorotannins clearly elucidated by mass spectrometry and one-dimensional NMR, together with comparison to those datapublished previously, three compounds were isolated for thefirst time from EC: fucodiphloroethol G (1),30 6,6′-bieckol (3)31

and 7-phloroeckol (4);32 the other two phlorotannins were alsodetermined as dieckol (2)32 and phlorofucofuroeckol A (5).31

Table 1. 13C and 13C-DEPTa NMR (100 MHz) data for phlorotannins(1–5) in DMSO-d6

Position 1 2 3 4 5

1 123.3 (s) 122.1 (s) 123.5 (s) 122.1 (s) 122.5 (s)

2 154.5 (s) 146.09 (s) 145.4 (s) 145.9 (s) 147.2 (s)

3 95.7 (d) 98.3 (d) 97.7 (d) 98.2 (d) 98.3 (d)

4 153.0 (s) 141.8 (s) 141.4 (s) 141.9 (s) 142.1 (s)

4a 123.19 (s) 121.9 (s) 123.1 (s) 122.7 (s)

5 92.3 (s)

5a 142.4 (s) 141.3 (s) 142.3 (s) 134.1 (s)

6 150.3 (s) 93.5 (d) 99.7 (d) 93.6 (d) 103.3 (s)

7 154.2 (s) 151.3 (s) 154.5 (s) 103.5 (s)

8 97.9 (d) 97.8 (d) 98.1 (d) 146.6 (s)

9 145.96 (s) 144.5 (s) 146.0 (s) 99.2 (d)

9a 123.9 (s) 122.7 (s) 123.9 (s)

10a 137.2 (s) 137.2 (s) 137.1 (s) 150.5 (s)

1′ 121.9 (s) 160.3 (s) 160.4 (s) 160.3 (s)

2′ 151.1 (s) 93.6 (d) 93.7 (d) 93.6 (d) 120.0 (s)

3′ 94.7 (d) 158.8 (s) 158.8 (s) 158.8 (s) 149.6 (s)

4′ 154.6 (s) 96.2 (d) 96.1 (d) 96.2 (d) 151.0 (s)

5′ 94.7 (d) 158.8 (s) 158.8 (s) 158.8 (s) 94.8 (d)

6′ 151.1 (d) 93.6 (d) 93.7 (d) 93.6 (d) 144.9 (s)

1′′ 156.5 (s) 122.2 (s) 122.5 (s) 126.4 (s)

2′′ 101.6 (s) 145.92 (s) 151.2 (s) 137.0 (s)

3′′ 157.7 (s) 98.2 (d) 94.8 (d) 160.2 (s)

4′′ 96.8 (d) 141.9 (s) 154.8 (s) 93.7 (d)

4a′′ 123.11 (s) 158.8 (s)

5′′ 157.8 (s) 94.8 (d) 96.2 (d)

5a′′ 142.6 (s) 158.8 (s)

6′′ 93.3 (d) 93.9 (d) 151.2 (s) 93.7 (d)

7′′ 153.1 (s) 159.9 (s)

8′′ 98.5 (d) 93.4 (d)

9′′ 146.06 (s)

9a′′ 122.6 (s)

10a′′ 137.1 (s)

1′′′ 101.0 (s) 155.9 (s)

2′′′ 157.1 (s) 94.5 (d)

3′′′ 94.7 (d) 151.2 (s)

4′′′ 157.2 (s) 124.2 (s)

5′′′ 94.7 (d) 151.2 (s)

6′′′ 157.1 (s) 94.5 (d)

a DEPT, distortionless enhancement by polarization transfer.

Compound 1 (fucodiphloroethol G, 47.62 mg): off-white pow-der; 1H NMR (DMSO-d6, 400 MHz) δ 5.83 (1H, d, J = 2.8 Hz, H-3),5.52 (1H, d, J = 2.8 Hz, H-5), 5.84 (2H, br s, H-3′, 5′), 5.98 (1H, d,J = 2.3 Hz, H-4′′), 5.85 (1H, d, J = 2.3 Hz, H-6′′), 5.90 (2H, br s,H-3′′′, 5′′′), 9.11 (3H, s, OH-2, 2′, 6′), 8.99 (1H, s, OH-4), 8.93 (2H, s,OH-4, 6′′′), 8.95 (2H, s, OH-3′′, 5′′), 8.57 (1H, s, OH-2′′′), 8.47 (1H, s,OH-4′′′); 13C NMR (DMSO-d6, 100 MHz); see Table 1. Low-resolutionelectron impact mass spectrometry (LREIMS) m/z 499.08, [M + H]+

(C24H18O12).Compound 2 (dieckol, 118.78 mg): light-brown powder

(lyophilized), 1H NMR (DMSO-d6, 400 MHz) δ 9.71 (1H, s, OH-9),9.61 (1H, s, OH-9′′), 9.51 (1H, s, OH-4′′), 9.46 (1H, s, OH-4), 9.36 (2H,s, OH-3′′, 5′′), 9.28 (1H, s, OH-2′′), 9.23 (1H, s, OH-2), 9.22 (1H, s,OH-7′′), 9.15 (2H, s, OH-3′, 5′), 6.17 (1H, s, H-3′′), 6.14 (1H, s, H-3),6.02 (1H, d, J = 2.7 Hz, H-8), 5.98 (1H, d, J = 2.7 Hz, H-8′′), 5.95 (1H,


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Table 2. Inhibitory activities of EC extracts and phlorotannins againstα-glucosidase and α-amylase

Samplea α-Glucosidase IC50 α-Amylase IC50

MeOH extract 37.07 µg mL−1 >1 mg mL−1

DW fr. 35.57 µg mL−1 >1 mg mL−1

EtOAc fr. 4.34 µg mL−1 345.00 µg mL−1

Fucodiphloroethol G (1) 19.52 µmol L−1 >500 µmol L−1

Dieckol (2) 10.79 µmol L−1 124.98 µmol L−1

6,6′-Bieckol (3) 22.22 µmol L−1 >500 µmol L−1

7-phloroeckol (4) 49.49 µmol L−1 250.02 µmol L−1

Phlorofucofuroeckol A (5) 19.71 µmol L−1 >500 µmol L−1

a MeOH extract, methanolic extract; DW fr., water fraction; EtOAc fr.,ethyl acetate fraction.

s, H-2′′′, 6′′′), 5.82 (1H, d, J = 2.7 Hz, H-6), 5.81 (1H, d, J = 2.7 Hz,H-6′′), 5.80 (1H, t, J = 2.0 Hz, H-4′), 5.78 (2H, d, J = 2.0 Hz, H-2′, 6′);13C NMR data; see Table 1, LREIMS m/z 743.10 [M]+ .

Compound 3 (6,6′-bieckol, 127.51 mg): light-brown powder(lyophilized), 1H NMR (DMSO-d6, 400 MHz) δ 9.29 (1H, s, OH-9),9.16 (2H, s, OH-3′, 5′), 9.15 (1H, s, OH-2), 9.09 (1H, s, OH-4), 8.65 (1H,s, OH-7), 6.09 (1H, s, H-3), 6.05 (1H, s, H-8), 5.80 (1H, d, J = 2.2 Hz,H-4′), 5.75 (2H, d, J = 2.2 Hz, H-2′, 6′), 13C NMR data, see Table 1;LREIMS m/z 743.12 [M]+ .

Compound 4 (7-phloroeckol, 132.96 mg): light-brown powder(lyophilized), 1H NMR (DMSO-d6, 400 MHz) δ 9.65 (1H, s, OH-9),9.45 (1H, s, OH-4), 9.24 (1H, s, OH-2), 9.16 (2H, s, OH-3′, 5′), 9.15(2H, s, OH-2′′, 6′′), 9.03 (1H, s, OH-4′′), 6.14 (1H, s, H-3), 5.77 (1H, d,J = 2.6 Hz, H-6), 6.00 (1H, d, J = 2.6 Hz, H-8), 5.85 (2H, s, H-3′′, 5′′),5.71 (2H, J = 1.8 Hz, H-2′, 6′), 5.79 (1H, t, J = 1.8 Hz, H-4′); 13C NMRdata, see Table 1; LREIMS m/z 496.13 [M]+ .

Compound 5 (phlorofucofuroeckol A, 53.71 mg): light-brownpowder (lyophilized); 1H NMR (DMSO-d6, 400 MHz) δ 10.17 (1H, s,OH-14), 9.92 (1H, s, OH-4), 9.88 (1H, s, OH-10), 9.48 (1H, s, OH-2),9.23 (2H, s, OH-3′′,5′′), 9.21 (2H, s, OH-3′, 5′), 8.23 (1H, s, OH-8), 6.71(1H, s, H-13), 6.42 (1H, s, H-9), 6.29 (1H, s, H-3), 5.82 (2H, t, J = 2.2Hz, H-4′, 4′′), 5.75 (2H, d, J = 2.2 Hz, H-2′, 6′), 5.71 (2H, d, J = 2.2Hz, H-2′′, 6′′); 13C NMR (DMSO-d6, 100 MHz); see Table 1. LREIMSm/z 603.12 [M + H]+ (C30H18O14).

Inhibitory effects of phlorotannins on α-glucosidase and α-amylaseα-Glucosidase and α-amylase inhibitory activities of phlorotannins(1–5) are shown in Table 2. In general, the inhibitory profiledemonstrated that phlorotannins showed greater inhibitoryactivity on α-glucosidase than on α-amylase. In the case of α-glucosidase, the IC50 value of compound 2 (10.79 µmol L−1)was about 4.5 times higher than the IC50 of compound 4(49.49 µmol L−1), which has the lowest activity among theisolated compounds (Table 2). Compounds 1, 3 and 5 alsoexhibited remarkable α-glucosidase inhibitory activities, with IC50

values at 19.52 µmol L−1 (1), 22.22 µmol L−1 (3) and 19.71 µmolL−1 (5), respectively (Table 2). In the case of α-amylase, onlycompounds 2 and 4 showed considerable α-amylase inhibitoryactivities, with IC50 values at 124.98 µmol L−1 and 250.02 µmolL−1, respectively. Compounds 1, 3 and 5, however, did not showsignificant inhibitory effects on α-amylase with over 500 µmolL−1 of IC50 value. Although not all of the isolated phlorotanninsexhibited significant α-amylase inhibitory effects, the observeddata clearly indicated the potential of these compounds as

α-gl

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Figure 2. Effect of dieckol (2) on α-glucosidase (A) and α-amylase (B) atdifferent concentrations. Results are means ± standard error of threeindependent experiments.

inhibitors for α-glucosidase; in particular, these phlorotanninderivatives showed higher IC50 values than that of the therapeuticdrug acarbose (60.8 µmol L−1).33 Compound 2 exhibited themost potent inhibitory activity among the tested phlorotanninderivatives, with dose-dependent inhibitory effects on both α-glucosidase and α-amylase (Fig. 2). Acarbose, which is a knownsugar derivative, inhibits intestinal α-glucosidases followed bya diminished and delayed rise in blood glucose after a meal,finally resulting in a reduction in postprandial hyperglycemia.34

Most of the α-glucosidase inhibitors are sugars or derivativesof sugar moieties, and there are only few non-saccharidecompounds which effectively inhibit glucosidases.35 Bharathamet al.36 reported that non-saccharide compounds can effectivelyinhibit α-glucosidase, such as sulfonamide chalcone derivatives,and their crucial inhibition role is due to the direct binding tothe active site by hydrophobic interactions. Phlorotannins aresecondary metabolites of polyphenol mainly produced by brownand red algae, which can be complexed with macromoleculesthrough hydrophobic interactions and through formation ofhydrogen, covalent or ionic bonds.37 It has been reported thatphlorotannin derivatives have been isolated from EtOAc fractionand are responsible for the biological activities of EC,9,11,12 andpolyphenolic compounds such as phlorotannins can alter glucoseutilization in mammals, causing insulin-like effects.29


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Figure 3. Lineweaver–Burk plot ofα-glucosidase in the presence of dieckol(2). Inhibitory activity of α-glucosidase was determined in the presence orabsence of dieckol (2) (�, 10 µmol L−1; �, 5 µmol L−1; ♦, control) at variousincubation times using pNPG as enzyme substrate. S, Z-Arg-Arg-MCAconcentration; V, mucin-like carcinoma-associated antigen (MCA) release.Results are means ± standard error of three independent experiments.

Determination of inhibition pattern of dieckol (2)The α-glucosidase inhibition pattern of compound 2, whichshowed the highest inhibitory activity on α-glucosidase, wasestimated using Lineweaver–Burk plots (Fig. 3). Plots of theinitial velocity versus enzyme concentration with different con-centrations of compound 2 gave a family of straight lines anddisplayed noncompetitive inhibition. Polyphenols are knownto bind proteins through hydrogen bonding and hydrophobicinteractions.38,39 Increasing the concentration of compound 2 re-sulted in the lowering of the slope of the line, indicating thatcompound 2 can combine with an enzyme molecule to produce adead-end complex, regardless of whether a substrate molecule isbound or not.

Compound 2 showed potential inhibitory activity against α-glucosidase with IC50 values at 10.97 µmol L−1. The mechanism ofaction may be suggested with the present better understandingof relevant publications. α-Glucosidase (EC 3.2.1.20, α-D-glucosideglucohydrolase), which catalyzes the liberation of α-glucose fromthe non-reducing end of the substrate, is an exo-type carbohydrase.It is widely present in various sources such as plants. Based onrecent research, most inhibitors act by mimicking the pyranosylmoiety ofα-glucosidase. Polyphenolic compounds, such as tanninsfrom terrestrial plants and phlorotannins from marine algae,are known to be associated with a variety of proteins to formcomplexes.40 The hydroxyl groups in phlorotannin derivativesmay play an important role in promoting inhibitory activity. Forexample, o-quinones derived from catechols are covalently boundto protein amino acid and thiol groups. Therefore, phlorotanninsshould bind to active or binding sites of the enzymes, resulting ininhibition of the enzyme activity.

In conclusion, this study investigated the potential anti-diabeticactivity of phlorotannin derivatives isolated from EtOAc frac-tion of Ecklonia cava, focusing on the inhibitory effects onboth α-glucosidase and α-amylase enzymes. Among these testedphlorotannin derivatives, compound 2 (dieckol) showed the high-est inhibitory activity against α-glucosidase and α-amylase, withIC50 values at 10.97 and 124.98 µmol L−1, respectively. Further-more, its inhibitory pattern was evaluated as a noncompetitive

α-glucosidase inhibitor. The other tested compounds (1, 3, 4 and5) along with EtOAc fraction also exhibited considerable potentialenzyme inhibitory activity. This study suggests that phlorotan-nin derivatives and EtOAc fraction from Ecklonia cava could bepotential candidates in developing medicinal preparations andnutraceutical or functional foods for DM and related symptoms.

ACKNOWLEDGEMENTSThis research was supported by a grant from the Marine BioprocessResearch Center as part of the Marine Bio 21 Project funded bythe Ministry of Land, Transport and Maritime Affairs, Republic ofKorea.

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α-glucosidase and α-amylase inhibitory activities of phloroglucinal derivatives from edible marine...

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