anti-diabetic - 복사본

Original article

Characterisation of the hypoglycaemic activity of glycoprotein

purified from the edible brown seaweed, Undaria pinnatifida

S.M. Rafiquzzaman,1 Jong Min Lee,1 Raju Ahmed,1 Jong-Hee Lee,2 Jin-Man Kim3 & In-Soo Kong1*

1 Department of Biotechnology, Pukyong National University, Busan 608-737, Korea

2 World Institute of Kimchi, Gwangju 503-360, Korea

3 Department of Biotechnology and Chemical Engineering, Chonnam National University, Yeosu 550-749, Korea

(Received 18 May 2014; Accepted in revised form 11 August 2014)

Summary Seaweed has been reported to control postprandial hyperglycaemia in various ways. We recently reported

the characterisation of a glycoprotein from Undaria pinnatifida (UPGP) with antioxidant activities. In this

study, we characterised the hypoglycaemic effect of UPGP through monitoring a-glucosidase inhibition

and glucose transport across yeast cell. Dose-dependent inhibitions of UPGP against yeast and rat intesti-

nal a-glucosidase were observed with IC50 values of 0.11 and 0.29 mg mL�1, respectively. UPGP showed

stable inhibition following incubation at different temperatures and metal ions. Regarding bioaccessibility,

the inhibition was decreased slightly during the gastric phase compared to undigested UPGP, with an

increase during the duodenal phase. Kinetics and membrane dialysis revealed mixed and reversible

inhibition, respectively. Furthermore, UPGP with acarbose showed synergistic inhibition against a-gluco-sidase, and UPGP increased the rate of glucose transport across the yeast cell. In conclusion, our study

demonstrated that UPGP may be used as bioaccessible food additives for controlling postprandial

hyperglycaemia.

Keywords Glucose transport, glycoprotein, hypoglycaemic, Undaria pinnatifida, yeast cell, a-glucosidase.

Introduction

Therapeutic agents from natural sources are gainingpopularity for the treatment of chronic diseases includ-ing cancer, heart disease and diabetes due to their lowincidence of side effects and cost (Ventakesh et al.,2003). Several epidemiological studies suggest thatfruits, vegetables, grains, nuts, seaweeds and less-pro-cessed staple foods provide the best protection againstthe development of chronic diseases (Ness & Powles,1997). Among chronic diseases, diabetes is now consid-ered a serious global problem. The global prevalenceof diabetes is estimated to increase from 177 millionpeople in 2000 to at least 366 million by 2030 (Wildet al., 2004). Diabetes mellitus is a complex metabolicdisorder categorised into type I and type II, resultingfrom either insulin insufficiency or insulin dysfunction,respectively. Type II diabetes is the more commonform of diabetes, accounting for 90% of the diabeticpopulation.

In individuals with type II diabetes, nutrient uptakerelated to the first-phase insulin response is severely

diminished, resulting in elevated postprandial glucoselevels. Thus, controlling postprandial glucose levels iscritical for the early treatment of diabetes. Hydrolysisof starch and carbohydrate is the major source ofglucose in the blood. Human a-glucosidase is locatedin the brush border of the surface membrane ofintestinal cells and is a key enzyme for the hydrolysisand absorption of carbohydrate in the human body(Nichols et al., 2002). It is believed that inhibitinga-glucosidase can delay the breakdown of carbohy-drate in the small intestine and ultimately decreaseblood glucose levels (Van de Laar, 2008).Currently, several antidiabetic drugs, including acar-

bose, miglitol and voglibose, which inhibit a-glucosi-dase activity are available (Van de Laar, 2008). Whilethese treatments can control postprandial glucoselevels, their continuous use is often associated withundesirable side effects, including liver toxicity andadverse gastrointestinal problems (Hanefield et al.,1991). Thus, there is a need for natural a-glucosidaseinhibitors with no adverse secondary effects. It hasbeen reported that seaweeds and their organic extractscontain a wide array of bioactive substances withdiverse health effects (Rindi et al., 2012). Previously,*Correspondent: E-mail: [email protected]

International Journal of Food Science and Technology 2015, 50, 143–150

doi:10.1111/ijfs.12663

© 2014 Institute of Food Science and Technology

143

the majority of studies related to seaweed have focusedon the isolation of polysaccharides and characterisa-tion of their biofunctional activity. Previous studieshave reported that fucoidan and fucoxanthin fromUndaria pinnatifida elicit a wide range of therapeuticeffects, including anti-inflammatory, antiviral and anti-coagulant activities (Cumashi et al., 2007). However,our studies found that glycoprotein from U. pinnatifidaand Saccharina japonica showed potent antioxidantand DNA protective activities (Kim et al., 2012;Rafiquzzaman et al., 2013). Moreover, seaweeds havebeen shown to exhibit antidiabetic properties by inhib-iting carbohydrate-hydrolysing enzymes (Jung et al.,2012). Accordingly, it is known that seaweed extractsand their fractions can act as functional ingredients infoods to control hyperglycaemia.

Therefore, the objective of this study was to cha-racterise the antihyperglycaemic effect of UPGP usingin vitro measurements such as a-glucosidase inhibitionand promotion of glucose transport across the yeastcell membrane. We also performed further characteri-sation of UPGP, including determination of tempera-ture and metal ion stabilities, bioaccessibility using invitro digestion models, kinetics, and the nature ofinhibition using membrane dialysis. Furthermore, thecombined effect of UPGP and another a-glucosidaseinhibitor (acarbose) was investigated.

Materials and methods

Materials

Undaria pinnatifida was collected from the local mar-ket in Busan, Korea, and preserved within a plasticbox under dried conditions for experimental use. Yeasta-glucosidase (EC 3.2.1.20), rat intestinal acetonepowder, p-nitrophenyl-a-D-glucopyranoside (PNPG),porcine pepsin, taurodeoxycholate, taurocholate, gly-codeoxycholate and pancreatin were purchased fromSigma-Aldrich (Busan, Korea). All other chemicalsused, including solvents, were of analytical grade.

Purification of UPGP

Glycoprotein (MW: ~10 kDa) was purified fromU. pinnatifida and identified using sodium dodecyl sul-phate–polyacrylamide gel electrophoresis (SDS-PAGE)followed by Coomassie Brilliant Blue (CBB), silverand periodic acid-Schiff (PAS) staining, as reportedpreviously (Rafiquzzaman et al., 2013).

Yeast a-glucosidase inhibitory activity assay

The enzyme inhibitory activity of UPGP was evaluatedspectrophotometrically using the procedure reportedby Li et al. (2005). Briefly, 60 lL of a reaction mixture

containing 20 lL of 100 mM phosphate buffer (pH6.8), 20 lL of 2.5 mM p-nitrophenyl-a-D-glucopyrano-side (PNPG) and 20 lL of the sample (test concentra-tion ranging from 0.1 to 1.3 mg mL�1) dissolved indistilled water (DW) was added to each well, followedby the addition of 20 lL of a-glucosidase [0.2 U mL�1

in 10 mM phosphate buffer (pH 6.8)]. The plate wasincubated at 37 °C for 15 min, after which 80 lL of0.2 M sodium carbonate solution was added to stopthe reaction. The absorbance was then recorded at405 nm using a microplate spectrophotometer (Biotek,USA, ELx-800). Acarbose dissolved in distilled waterwas used as a positive control. The percentage inhibi-tion (%) was obtained using the following equation:% inhibition = (Ac–As)/Ac 9100, where Ac is theabsorbance of the control and As is the absorbance ofthe sample.

Rat intestinal a-glucosidase inhibition assay

Rat intestinal a-glucosidase was prepared as describedby Jo et al. (2009) with minor modifications. Briefly,rat intestinal acetone powder (200 mg) was dissolvedin 4 mL of 50 mM ice-cold phosphate buffer and soni-cated for 15 min at 4 °C. After vigorous vortexing for20 min, the suspension was centrifuged (10 000 g,4 °C, 30 min) and the resulting supernatant was usedfor the assay. A reaction mixture containing 50 lL ofphosphate buffer (50 mM; pH 6.8), 10 lL of rat intesti-nal a-glucosidase (1 U mL�1) and 20 lL of UPGP atvarying concentrations was pre-incubated for 5 min at37 °C, after which 20 lL of 1 mM PNPG was addedto the mixture as substrate. After further incubation at37 °C for 30 min, the reaction was stopped by adding50 lL of Na2CO3 (0.1 M). The absorbance was thenrecorded at 405 nm using a microplate spectrophotom-eter (Biotek, ELx-800). The percentage inhibition wasobtained using the same equation as used in the yeasta-glucosidase assay.

Temperature and metal ion stability of UPGP

The stability of UPGP at various temperatures and inthe presence of different metal ions in terms of ratintestinal a-glucosidase inhibition was investigated asdescribed by Oh & Lim (2008) with minor modifica-tions. Briefly, to investigate temperature stability,UPGP was incubated at different temperatures(0–100 °C) for 1 h. Following incubation, UPGP wasused for a-glucosidase inhibition assays as describedabove. To investigate the effect of metal ions, 100 lLof UPGP (1 mg mL�1) were reacted with each buffer(CaCl2, MnCl2, MgCl2, KCl, NaCl, CoCl2 or ZnCl2)containing 2 mM of metal ions for 2 h at room tem-perature (RT). The reaction solutions were thensubjected to a-glucosidase inhibition assays.

© 2014 Institute of Food Science and TechnologyInternational Journal of Food Science and Technology 2015

Hypoglycemic activity of glycoprotein S. M. Rafiquzzaman et al.144

Bioaccessibility of UPGP measured using the in vitrodigestion model

The bioaccessibility of UPGP was measured using thein vitro digestion model as described previously (Raf-iquzzaman et al., 2013) with minor modifications.Briefly, UPGP solutions (1 mg mL�1) were acidified topH 2 with 1 mL of porcine pepsin preparation (0.04 gpepsin in 0.1 mol L�1 HCl) and incubated at 37 °C ina shaking water bath at 95 rpm for 1 h. After gastricdigestion, the pH was increased to 5.3 with 0.9 M

sodium bicarbonate, and then 200 lL of the bile saltsglycodeoxycholate (0.04 g in 1 mL of saline), taur-odeoxycholate (0.025 g in 1 mL of saline) and tau-rocholate (0.04 g in 1 mL of saline), as well as 100 lLof pancreatin (0.04 g in 500 mL saline) were added tothe solution. The pH of each sample was increased topH 7.4 with 1 M NaOH, and the samples were incu-bated at 37 °C in a shaking water bath at 95 rpm for2.5 h to complete the intestinal phase of the in vitrodigestion process. After the intestinal digestion phase,2 mL of each sample was extracted and stored at�20 °C. Samples were analysed within 2 weeks.

Analysis of enzyme kinetics

To determine the kinetic mechanism, the Lineweaver& Burk (1934) plot method was used. Using a Linewe-aver–Burk (LB) plot, we determined the enzyme kinet-ics of rat intestinal a-glucosidase using 1, 2 and 3 mM

PNPG as a substrate in the absence or presence of1.5 mg mL�1 (filled circles), 1.0 mg mL�1 (open cir-cles) or 0.5 mg mL�1 (filled inverted triangles) UPGP.The mode of inhibition of UPGP was determined byanalysing data calculated following Michaelis–Mentonkinetics.

Dialysis for reversibility of UPGP action

Rat intestinal a-Glucosidase (100 U mL�1) was incu-bated with UPGP (10 mg mL�1) in 0.5 mL of sodiumphosphate buffer (50 mM, pH 6.7) for 2 h at 37 °Cand dialysed against sodium phosphate buffer (5 mM,pH 6.7) at 4 °C for 24 h, changing the buffer every12 h. Another premixed enzyme solution (0.5 mL) wasmaintained 4 °C for 24 h without dialysis for the con-trol experiment. Reversibility of UPGP was deter-mined by comparing the residual enzyme activity afterdialysis with that of a nondialysed control (Lee, 2000).

Combined inhibitory effect of UPGP and acarbose

This assay was performed as described previously(Adisakwattana et al., 2011). The combined inhibitoryeffect was evaluated in solution containing acarbosealone or in a mixture with UPGP to evaluate the

synergistic effect of UPGP and acarbose on rat intesti-nal a-glucosidase. The reaction was performed asdescribed above.

Effect of UPGP on glucose transport by yeast cells

Yeast (Saccharomyces cerevisiae) cells were preparedaccording to the method of Cirillo (1962). Briefly, yeastcells were washed by repeated centrifugation (3000 g;5 min) in distilled water, and a 10% (v/v) suspensionwas prepared in distilled water. Various concentrationsof extracts (1–5 mg) were added to 1 mL of glucosesolution (5–20 mM) and incubated together for 10 minat 37 °C. The reaction was initiated by adding 100 lLof yeast suspension, vortexed and further incubated at37 °C for 60 min. After 60 min, the tubes were centri-fuged (2500 g; 5 min) and glucose concentrations inthe supernatant were determined using the glucose oxi-dase method. The percentage increase in glucoseuptake by yeast cells was calculated using the followingformula: Increase in glucose uptake (%) = (Abs. con-trol–Abs. sample/Abs. control) 9100, where Abs con-trol is the absorbance of the control reaction(containing all reagents except the test sample) andAbs sample is the absorbance of the test sample.

Statistical analysis

All experiments were performed in triplicate, and dataare presented as means � standard deviation. Analysisof variance (ANOVA) was performed, and comparisonsof means were conducted using Turkey’s multiple com-parison tests using the SPSS program (version 16.0 forwindows, SPSS Inc.). Values were considered to differsignificantly if the P value was <0.05.

Results

a-Glucosidase inhibitory activity assay

a-Glucosidase of S. cerevisiae (EC 3.2.1.20) was usedto investigate the inhibitory activity of purified UPGP.The inhibitory activity against a-glucosidase was deter-mined using PNPG as substrate. UPGP significantlyinhibited yeast a-glucosidase in a dose-dependentmanner (Fig. S1a). The highest inhibitory activity(95.03%) was found at 0.9 mg mL�1 UPGP, whichinhibited yeast a-glucosidase with an IC50 value of0.11 mg mL�1. Acarbose (positive control) showed anIC50 value of 0.69 mg mL�1 (Table 1) under similarconditions. Similarly, rat intestinal a-glucosidase wasused to evaluate the inhibitory activity of UPGP.UPGP and acarbose inhibit a-glucosidase with IC50 val-ues of 0.29 and 0.04 mg mL�1, respectively (Table 1).UPGP also showed dose-dependent inhibitory activitiesagainst rat intestinal a-glucosidase (Fig. S1b).

© 2014 Institute of Food Science and Technology International Journal of Food Science and Technology 2015

Hypoglycemic activity of glycoprotein S. M. Rafiquzzaman et al. 145

Stability at various temperatures and in the presence ofdifferent metal ions

UPGP showed stable rat intestinal a-glucosidase inhi-bition activity in the presence of different metal ions.For example, the activities of UPGP in the presence ofCa2+, Mn2+ and Zn2+ were highly stable at 80–90%inhibition, whereas Co2+ showed the lowest inhibitionactivity, at 75% (Fig. 1a). As shown in Fig. 1b, theinhibition by UPGP of yeast a-glucosidase was stableat a wide range of temperatures (0–100 °C). The high-est yeast a-glucosidase inhibition activity occurred at37 °C and the inhibitory activity varied little from 0 to100 °C; all temperatures examined showed over 80%inhibition compared to the control.

Bioaccessibility of UPGP measured using the in vitrodigestion model

The rat intestinal a-glucosidase inhibitory activity ofUPGP was measured before and after the in vitrodigestion model to determine its bioaccessibility. Therewas little variation in inhibitory activity betweendigested and undigested UPGP. However, the a-gluco-sidase inhibitory activity of UPGP after the gastricand duodenal phases of in vitro digestion was 9.24%and 7.7% lower, respectively, compared to that ofundigested UPGP (Fig. 2).

Enzyme kinetics

To investigate the inhibition mode of UPGP, a LBplot was generated using data obtained from kinetic

studies, which was calculated using the Michaelis–Menton equation to confirm the inhibition pattern. Inthe LB plot, the designated line of various concentra-tions intersected the axis on the left side of the zeropoint, indicative of its ability to act as a mixed-typeinhibitor (Fig. 3).

Reversibility of UPGP action

The rat intestinal a-glucosidase activity was not com-pletely recovered after dialysis, as shown by theenzyme-mixed inhibitor curve (EID) that reachedlevels corresponding to 70% compared to the enzymecontrol without dialysis (EC) (Fig. S2). Proximal run-ning of ED as an experimental control along with ECand EID ensured that dialysis alone did not signifi-cantly affect enzyme activity. However, the nondialy-sed mixture of enzyme and inhibitor (EIC) showedinhibitory activity.

Combined effect

The combined effect of UPGP and acarbose on a-glu-cosidase inhibition is shown in Fig. 4. The percentageof a-glucosidase inhibition increased upon addition ofUPGP to the mixture containing acarbose. Thus,

Table 1 Inhibitory effects of UPGP and acarbose against yeast and

rat intestinal a-glucosidase

Enzyme type

IC50 (mg mL�1)*

UPGP Acarbose†

Yeast a-glucosidase 0.11 � 0.08 0.69 � 0.13

Rat intestinal a-glucosidase 0.29 � 0.10 0.04 � 0.002

*IC50, concentration of inhibitor to inhibit 50% of its activity.†A positive control.

(a) (b)Figure 1 Effect of metal ions (a) and tem-

perature (b) on the inhibitory activities of

UPGP against rat intestinal a-glucosidase.UPGP (1 mg mL�1) was incubated at differ-

ent temperature (0–100 °C) and metal ions,

and UPGP was then used for a-glucosidaseinhibition. Results are expressed as

mean � SD (n = 3). *P ≤ 0.05 compared

with untreated UPGP.

Figure 2 Bioaccessibility of UPGP on the inhibitory activities

against rat intestinal a-glucosidase measured by in vitro digestion

model. Results are expressed as mean � SD (n = 3). Different letters

in superscripts indicate significant differences (P ≤ 0.05) compared

with untreated UPGP.



UPGP and acarbose had an additive inhibitory effecton a-glucosidase.

Effect of UPGP on glucose transport by yeast cells

The rate of glucose transport across the cell membranewas investigated using an in vitro system with S. cere-visiae suspended in 5–20 mM glucose solutions in thepresence/absence of UPGP (Fig. 5). The amount ofglucose remaining in the medium after a specific timeserved as an indicator of glucose uptake by yeast cells.UPGP moderately increased glucose transport in yeastcells up to 50%, which was directly proportional tothe sample concentration and inversely proportional tothe glucose concentration.

Discussion

Phenolics, flavonoids, fucoidan and fucoxanthin fromseaweed show diverse biofunctional activities, includ-

ing antidiabetic effects (Rindi et al., 2012; Lordanet al., 2013). However, no antidiabetic activity hasbeen reported for glycoprotein purified from seaweed.In our recent study, we reported the purification anddetection of a glycoprotein from U. pinnatifida, as wellas its chemical characterisation, and determination ofits antioxidant and DNA-protecting activity (Rafiquzz-aman et al., 2013). As part of our continuous effort inexploring biofunctional activity, we evaluated the anti-hyperglycaemic effect of UPGP in terms of its a-gluco-sidase inhibition activity and effect on glucose uptakeby yeast cells. Yeast a-glucosidase is used to identifyinhibitory activity in the extracts of, or active com-pounds purified from, medicinal plants or food items(Hogan et al., 2010). However, a-glucosidase preparedfrom rat intestinal acetone powder closely mimics themammalian system (Ohta et al., 2002) and may be abetter model to identify, design and develop antihyper-glycaemic agents, particularly for the management ofpostprandial hyperglycaemia in diabetes. Therefore, wetested both systems. The present study reports thedose-dependent antihyperglycaemic effect of purifiedUPGP, which is mediated by inhibition of a-glucosi-dase (Fig. S1a,b). The inhibitory activity of UPGP ona-glucosidase from the rat intestine was lower thanthat of acarbose. However, UPGP showed consider-ably higher (P ≤ 0.05) inhibitory activity against yeasta-glucosidase compared with acarbose (Table 1). Thedifferences in inhibitory activities may be due tostructural differences between these two enzymes(Chiba, 1997). These results indicated that UPGP is anovel type of inhibitor that inhibits a-glucosidase fromyeast or the rat intestine. We used a common substrate(PNPG) for both enzymes to ensure that the experi-mental protocols were identical. Regarding furthercharacterisation of UPGP such as stability, bioaccessi-bility, kinetics, reversibility and synergistic inhibition

Figure 4 The synergistic effect of acarbose (AC) and UPGP at dif-

ferent concentrations on rat intestinal a-glucosidase inhibition.

Results are expressed as mean � SD (n = 3). *P ≤ 0.05 compared

with acarbose alone.

Figure 3 Lineweaver–Burk plots for the rat intestinal a-glucosidaseinhibition by UPGP. Enzyme was treated with various concentration

of PNPG (0.5, 1.0 and 1.5 mM) in the presence of UPGP

[0.5 mg mL�1 (▼); 1.0 mg mL�1 (○); 1.5 mg mL�1 (●)] and found

mixed type of inhibition. Figure 5 Effect of UPGP on glucose transport by yeast cells at dif-

ferent glucose concentrations. Results are expressed as mean � SD

(n = 3). *P ≤ 0.05 compared with UPGP (1 mg mL�1) alone.



with acarbose, we used rat intestinal a-glucosidase asit closely mimics the mammalian system (Ohta et al.,2002). Furthermore, we also used yeast a-glucosidasefor those studies and found little variation in terms ofinhibition (data not shown).

When exploring possible industrial use as a foodadditive, we investigated the stability of UPGP at 0–100 °C and in the presence of various metal ions.UPGP was highly stable (P ≤ 0.05) at these tempera-tures and in the presence of various metal ions(Fig. 1a,b). This result is in agreement with the find-ings of Kim et al. (2005) and suggested that UPGPwould be easier to handle during processing or manu-facturing steps. Thus, UPGP may have industrial usesas a food additive. In other words, we determined thebioaccessibility of UPGP using an in vitro digestionmodel to provide an indication of its biofunctionalactivities in a biological system, as the model isdesigned to simulate in vivo digestion. The overallinhibitory activities of UPGP were lower (P ≤ 0.05)during the gastric phase than in the undigested andduodenal phases (Fig. 2). The differences between thegastric and duodenal phases were due to changes inthe pH of the UPGP solution, as pH affects enzymeactivity. The inhibitory activity remained after the gas-tric phase of digestion, indicating that structural defor-mation or conformational change might not takeplace. This result is similar to previous reports (Raf-iquzzaman et al., 2013) and suggested that purifiedUPGP is stable under simulated digestion conditions.However, some findings regarding the stability of nat-ural compounds subjected to changes in pH con-tradicted previous reports (D’Archivio et al., 2010).

To identify the type of enzymatic inhibition, kineticanalyses were performed at different substrate andinhibitor concentrations. LB plots were drawn to con-firm the inhibition pattern; UPGP showed a mixedinhibition type (Fig. 3). It is possible that UPGP pos-sesses more than one a-glucosidase inhibitor, which issupported by the findings of Kim et al. (2005). Thisassumption was further supported by previous reportsthat UPGP contained a number of protein and carbo-hydrate, which is linked with O-glycans (Rafiquzz-aman et al., 2013). It is also possible that UPGPattaches to a wide region of a-glucosidase or that itattaches to a unique region and causes structural mod-ifications. We next performed a dialysis experimentand found that the inhibitory activity of UPGP wasalmost reversible; that is, the enzyme activity wasrecovered after dialysis due to removal of inhibitors(Fig. S2). A similar result was found by Lee (2000)who reported that dibutyl phthalate from Streptomycesmelanosporofaciens was an almost reversible inhibitor.Reversible inhibition is a useful property of a-glucosi-dase inhibitors because the enzyme remains intact evenafter elimination of the inhibitor. In contrast, when

the inhibitor binds irreversibly to the enzyme, it maysuffer from hypoglycaemia due to chronic carbohy-drate malabsorption (Shihabuddin et al., 2011).Acarbose has been used clinically to treat type II

diabetes mellitus. The lowest dose of acarbose with aclinical effect is 150 mg per day (Rodier et al., 1998).However, recent reports have shown that acarbosetreatment is associated with many adverse effects,such as flatulence, meteorism and abdominal disten-sion, which occur in a dose-dependent manner (Hane-field et al., 1991). In general, acarbose may be usedin combination with other agents such as sulfonylureaand metformin or as a monotherapy for patients withdiabetes (Adisakwattana et al., 2011). Our presentstudy shows that the combination of acarbose andUPGP results in additive inhibition (P ≤ 0.05)(Fig. 4), suggesting that it may have significant clini-cal benefits for delaying postprandial hyperglycaemiaand hyperinsulinaemia. This could lead to the devel-opment of a novel combined therapy for patientswith diabetes. Therefore, the dosage of acarbose canbe reduced by combining with UPGP, which wouldlikely reduce the adverse effects of acarbose inpatients with diabetes.Other than the inhibition of a-glucosidase by

UPGP, several other mechanisms for the hypoglycae-mic effect of phytochemicals have been proposed, suchas manipulation of glucose transporters, b-cell regener-ation and enhancement of insulin release (Tiwari &Rao, 2002). Our findings demonstrated that glucosetransport across the yeast cell membrane significantlyincreased (P ≤ 0.05) in the presence of UPGP (Fig. 5).The rate of glucose transport was dependent on theexternal glucose concentration as well as the sampleconcentration. This finding is in agreement with Ah-med & Urooj (2010). It is generally known that glu-cose is transported across the yeast cell membrane byfacilitated diffusion. Facilitated carriers are specificcarriers that transport solutes down the concentrationgradient. Therefore, effective transport is possible onlyif intracellular glucose is removed (Teysink et al.,1998).We performed GC-MS analysis of UPGP; the

results suggested that the glycoside moiety contributedto the a-glucosidase inhibitory effect (data not shown).Glycosides are considered promising natural inhibitorsof a-glucosidase (Akkarachiyastit et al., 2010). Thehydroxyl group of glycosides may interact with theactive site of the enzyme through covalent or nonco-valent interactions, which could modulate a-glucosi-dase inhibition (Lo Piparo et al., 2008). In addition, ithas been reported that glycoproteins differ from anonglycosylated proteins in terms of their large ter-tiary structure (Nelson & Cox, 2000). This structuraldiversity may result in the hypoglycaemic effect ofUPGP.



Conclusions

Our results confirm the hypoglycaemic properties ofUPGP based on various in vitro methods. This effectwas mediated by inhibiting a-glucosidase and promot-ing glucose transport across the yeast cell membrane,as revealed using an in vitro yeast cell model. Based onour results, UPGP may be applicable as a nutraceuti-cal or functional food to control hyperglycaemia.

Acknowledgments

This work was supported by a Research Grant ofPukyong National University (2014 year).

References

Adisakwattana, S., Lerdsuwankij, O., Poputtachai, U., Minipun, A.& Suparpprom, C. (2011). Inhibitory activity of cinnamon barkspecies and their combination effect with acarbose against intesti-nal a-glucosidase and pancreatic a-amylase. Plant Foods forHuman Nutrition, 66, 143–148.

Ahmed, F. & Urooj, A. (2010). In vitro studies on the hypoglycemicpotential of Ficus racemosa stem bark. Journal of the Science ofFood and Agriculture, 90, 397–401.

Akkarachiyastit, S., Charoenlertkul, P., Yibchok-anun, S. & Adisak-wattana, S. (2010). Inhibitory activities of cyanidin and its glyco-sides and synergistic effect with acarbose against intestinal a-glucosidase and pancreatic a-amylase. International Journal Molec-ular Science, 11, 3387–3396.

Chiba, S. (1997). Molecular mechanisms in a-glucosidase and glu-coamylase. Bioscience Biotechnology Biochemistry, 61, 1233–1239.

Cirillo, V.P. (1962). Mechanism of glucose transport across the yeastcell membrane. Journal of Bacteriology, 84, 485–491.

Cumashi, A., Ushakova, N.A. & Preobrazhenskaya, M.E. (2007). Acomparative study of the anti-inflammatory, anticoagulant, antian-giogenic, and antiadhesive activities of nine different fucoidansfrom brown seaweeds. Glycobiology, 17, 541–552.

D’Archivio, M., Filesi, C., Var�ı, R., Scazzocchio, B. & Masella, R.(2010). Bioavailability of the polyphenols: status and controversies.International Journal of Molecular Science, 4, 1321–1342.

Hanefield, M., Fischer, S., Schulze, J. et al. (1991). Therapeuticpotentials of acarbose as first-line drug in NIDDM insufficientlytreated with diet alone. Diabetes Care, 14, 732–737.

Hogan, S., Zhang, L., Li, J., Sun, S., Canning, C. & Zohu, K.(2010). Antioxidant rich grape pomace extract suppresses postpran-dial hyperglycemia in diabetic mice by specifically inhibiting alpha-glucosidase. Nutrion Metabolism (Lond), 7, 71.

Jo, S.H., Ka, E.H., Lee, H.S., Jang, H.D. & Kwon, Y.I. (2009).Comparison of antioxidant potential and rat intestinal a-glucosid-ases inhibitory activities of quercetin, rutin, and isoquercetin. Inter-national Journal Applied Research, 4, 52–60.

Jung, H.A., Islam, M.N., Lee, C.M. et al. (2012). Promising antidia-betic potential of fucoxanthin isolated from the edible brown algaeEisenia bicyclis and Undaria pinnatifida. Fisheries Science, 78,1321–1329.

Kim, Y.M., Jeong, Y.K., Wang, M.H., Lee, W.Y. & Rhee, H.I.(2005). Inhibitory effect of pine extract on a-glucosidase activityand postprandial hyperglycemia. Nutrition, 21, 756–761.

Kim, E.Y., Kim, Y.R., Nam, T.J. & Kong, I.S. (2012). Antioxidantand DNA protection activities of a glycoprotein isolated from aseaweed, Saccharina japonica. International Journal of Food Scienceand Technology, 5, 1020–1027.

Lee, D.S. (2000). Dibutyl phthalate, an a-glucosidase inhibitor fromStreptomyces melanosporofaciens. Journal of Bioscience and Bioengi-neering, 3, 271–273.

Li, T., Zhang, X.D., Song, Y.W. & Liu, J.W. (2005). A microplate-based screening method for a-glucosidase inhibitors. Chinese Jour-nal of Clinical Pharmacology Therapy, 10, 1128–1134.

Lineweaver, H. & Burk, D. (1934). The determination of enzyme dis-sociation constants. Journal of American Chemical Society, 56,658–666.

Lo Piparo, E., Scheib, H., Frei, N., Williamson, G., Grigorov, M. &Chou, C.J. (2008). Flavonoids for controlling starch digestion:structural requirements for inhibiting human alpha-amylase. Jour-nal of Medicinal Chemistry, 51, 3555–3561.

Lordan, S., Smyth, T.J., Soler-Vila, A., Stanton, C. & Ross, P.R.(2013). The a-amylase and a-glucosidase inhibitory effects of Irishseaweed extracts. Food Chemistry, 141, 2170–2176.

Nelson, D.L. & Cox, M.M. (2000). Carbohydrates and glycobiology.In: Lehninger Principles of Biochemistry, 3rd edn. (edited by M.Fisher). Pp. 311–318. New York, NY: Worth publishers.

Ness, A.R. & Powles, J.W. (1997). Fruit and vegetables, and cardio-vascular disease: a review. International Journal of Epidemiology,26, 1.

Nichols, B.L., Avery, S.E., Karnsakul, W. et al. (2002). Congenitalmaltase-glucoamylase deficiency associated with lactase and sucrosedeficiencies. Journal of Pediatric and Gastroenterology Nutrition, 35,573–579.

Oh, P.S. & Lim, K.T. (2008). Antioxidant activity of Dioscorea bata-tas Decne glycoprotein. European Food Research and Technology,226, 375–387.

Ohta, T., Sasaki, S., Oohori, T., Yoshikawa, S. & Kurihara, H.(2002). a-glucosidase inhibitory activity of 70% methanol extractfrom Ezoishige (Pelvetia babingtonii de Toni) and its effects on theelevation of blood glucose level in rats. Bioscience BiotechnologyBiochemistry, 66, 1552–1554.

Rafiquzzaman, S.M., Kim, E.Y., Kim, Y.R., Nam, T.J. & Kong,I.S. (2013). Antioxidant activity of glycoprotein purified from Un-daria pinnatifida measured by an in vitro digestion model. Interna-tional Journal Biological Macromolecules, 62, 265–272.

Rindi, F., Soler-Vila, A. & Guiry, M.D. (2012). Taxonomy ofmarine macroalgae used as source of bioactive compounds. In:Marine bioactive compounds: sources, characterization andapplications. (edited by M. Hayes). Pp. 1–53 New York, NY:Springer.

Rodier, M., Richard, J.L., Monnier, L. & Mirauze, J. (1998). Effectof long term acarbose (Bay g 5421) therapy on metabolic controlon non-insulin-dependent (type II) diabetes mellitus. Diabetes andMetabolism, 14, 12–14.

Shihabuddin, H.M.S., Priscilla, D.H. & Thirumurugan, K. (2011).Cinnamon extracts inhibits a-glucosidase activity and dampenspostprandial glucose excursion in diabetic rats. Nutrition andMetabolism, 8, 46.

Teysink, B., Jasper, A., Hans, D.V., Van Dam, W.K. & Walsh,M.C. (1998). Intracellular glucose concentration in derepressedyeast cells consuming glucose is high enough to reduce the glucosetransport rate by 50%. Journal of Bacteriology, 180, 556–562.

Tiwari, A.K. & Rao, J.M. (2002). Diabetes mellitus and multipletherapeutic approaches of phytochemicals: present status andfuture prospects. Current Science, 23, 30–33.

Van de Laar, F.A. (2008). Alpha-glucosidase inhibitors in the earlytreatment type 2 diabetes. Journal of Vascular Health and RiskManagement, 4, 1189–1195.

Ventakesh, S., Reddy, G.M., Reddy, B.M., Ramesh, M. & AppaRao, A.V.N. (2003). Antihyperglycemic activity of Carallumaattenuate. Fitoterapia, 74, 274–279.

Wild, S., Roglic, G., Green, A., Sicree, R. & Kinh, H. (2004). Glo-bal prevalence of diabetes estimates for the year 200 and projec-tions for 2030. Diabetes Care, 27, 1047–1053.



Supporting Information

Additional Supporting Information may be found inthe online version of this article:

Figure S1. Inhibition of yeast a-glucosidase (a) andrat intestinal a-glucosidase (b) as a function of concen-tration of UPGP and acarbose.

Figure S2. Reversibility of UPGP action against ratintestinal a-glucosidase. Reversibility of UPGP wasdetermined by comparing the residual enzyme activityafter dialysis with that of nondialysed one. Results areexpressed as mean � SD (n = 3).



anti-diabetic - 복사본

Documents