Journal of Chromatography A, 1340 (2014) 115–120

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Study on antidiabetic activity of wheat and barley starch using asymmetrical flow field-flow fractionation coupled with multiangle light scattering Haiyang Dou a,∗ , Bing Zhou b , Hae-Dong Jang b , Seungho Lee a,∗ a b

Department of Chemistry, Hannam University, Daejeon 305811, South Korea Department of Food and Nutrition, Hannam University, Daejeon 305811, South Korea

a r t i c l e

i n f o

Article history: Received 22 January 2014 Received in revised form 5 March 2014 Accepted 5 March 2014 Available online 12 March 2014 Keywords: Antidiabetic activity Wheat Barley Germination Asymmetrical flow field-flow fractionation Apparent density

a b s t r a c t The ability of asymmetrical flow field-flow fractionation (AF4) coupled online with multiangle light scattering (MALS) and refractive index detector (RI) (AF4-MALS–RI) for monitoring of change in molecular conformation of wheat and barley starch during germination process was evaluated. AF4 provides separation of starch molecules based on their hydrodynamic sizes, and MALS yields the molar mass and molecular size (radius of gyration, Rg ). In vitro and in vivo anti-hyperglycemic effect of germinated wheat and barley was studied. The relationship between antidiabetic activity and molecular conformation was, for the first time, investigated. The ratio of Rg to the hydrodynamic radius (Rh ) and the apparent density were proven to be important parameters as they offer an insight into molecular conformation. Results showed that, when geminated, the apparent density and the antidiabetic activity of barley were significantly increased, suggesting germination makes the molecules more compact which could contribute to enhancement of their antidiabetic activity. The information obtained by AF4-MALS–RI is valuable for understanding of germination mechanism, and thus for developing functional foods. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The structure and degradation of biomacromolecules, such as starch, have a bearing on some human diseases such as obesity, diabetes, and some colo-rectal cancers, which are reaching epidemic proportions in developed countries, and are growing rapidly in developing countries [1–3]. In 2011, approximately 366 million people of the global population aged 20–79 years were estimated to have diabetes [4]. There is an increasing demand for healthy diet, and the use of functional food and directed nutrition is nowadays presented as a way to stay fit and healthy. Wheat and barley are two of the most abundant and popular cereal materials and common sources of digestible carbohydrate in human diet. They provide minerals, dietary fiber and bioactive compounds [5]. The major carbohydrate in wheat and barley is starch, supplying from 20 to 50% of food energy, with higher fraction for “Asian” diet and also for diets of those in many developing countries.

∗ Corresponding authors at: Hannam University, Department of Chemistry, Daejeon 305811, South Korea. Tel.: +82 42 629 8822; fax: +82 42 629 8811. E-mail addresses: [email protected] (H. Dou), [email protected] (S. Lee). http://dx.doi.org/10.1016/j.chroma.2014.03.014 0021-9673/© 2014 Elsevier B.V. All rights reserved.

Starch consists of two types of polydisperse polysaccharides, amylose and amylopectin. The former consists of linear chains of (1 → 4)-␣-glucose linked residues having molar masses of 105 to 106 g/mol. The latter has a highly branched structure containing a mixture of (1 → 4) and (1 → 6)-␣-glucose linkages with molar mass reaching up to about 108 g/mol [6]. Wheat and barley starch and starch–derivatives have been widely used in a variety of industrial applications including food, beverage, pharmaceuticals, household products, cosmetics, paper and packaging [7,8]. It is known that germination significantly changes the nutritional quality of the cereal seeds, including the starch content [9,10]. On germination, protein, carbohydrates and minerals become more bio-available and bio-accessible [11]. Low-processed food (i.e. germinated seed) has become more popular for functional foods [12,13]. Although some researchers postulated that the enhancement in bioactivity of germinated seeds is also linked with modification of the starch structure and content, little is known about variation in physicochemical properties of starch (such as molar mass, molar mass distribution, conformation, and so on) during seed germination [14,15]. The major reason for a scarcity of information may be related to difficulties in studying the molar mass distribution of ultrahigh molar mass polymer by size exclusion chromatography

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(SEC). While SEC is a well-established method for analysis of polymers, difficulties in its application to large biomacromolecules such as amylopectin include exceeding the exclusion limit of the column, irreversible interaction of the sample with column packing and subsequently low recovery, and shear degradation [16–18]. During the last decade, an alternative separation technique, asymmetrical flow field-flow fractionation (AF4) coupled with multiangle light scattering (MALS) and refractive index detector (RI) has shown its applicability to the determination of the molar mass distribution of various ultrahigh molar mass polymers [19–21]. Unlike SEC, AF4 uses an open channel that requires no stationary phase or packing material. Thus in AF4, the shear forces during separation are minimized [22]. The viscosity of polysaccharide solution depends on the molar mass and conformation of the molecules and the ability to form aggregates [23]. Thus, characterization of the distributions of molar mass and related parameters (such as apparent density) of wheat and barley starch in aqueous solution is important for improvements in the human and animal nutrition. However, so far, little has been reported on the effect of germination on physicochemical properties of cereal starch. In this work, the capacity of AF4-MALS–RI to monitor the variation in conformation of wheat and barley starch during germination was evaluated. The aim of this work is to study the correlation between conformational properties of strach in wheat and barley and their antidiabetic activities. 2. Materials and methods 2.1. Materials Wheat and barley seeds were obtained from farms in Gongeum and Chuncheon, Korea, respectively. Five week-old male C57BLks/J db/db mice were purchased from Joongang Experimental Animal Co., (Seoul, Korea). Deionized water was obtained from a Milli-Q Plus Ultra-Pure Water system (Millipore, MA, USA). All chemicals were used without further purification. Rat intestinal acetone powder, p-nitrophenyl-␣-d-glucopyranoside, sodium nitrate (NaNO3 ), sodium hydroxide (NaOH), and sodium azide (NaN3 ) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Hydrochloride acid (35–37%) was purchased from Samchun Chemical (Pyeongtaek, Korea). Tea catechin was purchased from General Foods & Flavors Inc. (Seoul, Korea). Chitooligosaccharides was obtained from Kunpoong Bio Co., Ltd. (Seoul, Korea). 2.2. Starch preparation from germinated wheat and barley seeds Wheat and barley seeds were germinated by soaking them in distilled water containing 0.5% (w/w) tea catechin and 1.0% (w/w) chitooligosaccharides at 25 ◦ C for 24 and 48 h, respectively. After soaked germination, seeds were dried for 24 h at 50 ◦ C and pulverized using a grinder (Hibell Co., Ltd., Hwaseong, Korea). Then, 10% (w/w) dispersion of sample was prepared by adding germinated sample into distilled water and autoclaved for 15 min at 121 ◦ C. Finally, the suspension was centrifuged at 7000 rpm for 20 min at 30 ◦ C. Powder samples were obtained by freeze–drying (see Table S1) the supernatants. Wheat powders obtained by 24 h and 48 hgermination were labeled “W-24” and “W-48”, respectively, and the wheat powder obtained without germination was labeled “W0”. Barley powders were labeled likewise “B-0”, “B-24”, and “B-48”, respectively. 2.3. Sample preparation for AF4 analysis of starch Sample solutions for AF4-MALS–RI analysis were prepared by the following procedure: 10 mg of the powder sample was mixed

Table 1 Method used for AF4 analysis of starch. Time (min)

Mode

Vc,initial (mL/min)

Vc,final (mL/min)

Focusing flow rate (mL/min)

3.7 2.0 2.0 2.3 8.0 30 1.0 20

Elution Focusing Focusing + injection Focusing Elution Elution Elution Elution

0.9 – – – 0.9 0.1 0.1 0

0.9 – – – 0.1 0.1 0 0

– 1.0 1.0 1.0 – – – –

with 1 mL of 1 M NaOH in a 20 mL vial, and stirred with a magnetic stirring bar at 400 rpm for 2 min at 70 ◦ C in an oil bath (Daihan Scientific Co., Ltd., Seoul, Korea). Then, 8 mL of the AF4 carrier liquid (deionized water containing 50 mM NaNO3 and 3 mM NaN3 ) was added and stirred at 70 ◦ C. Finally, the solution was neutralized by adding 1 mL of 1 M HCl, and stirred again. The sample solution was filtered through a 0.45 ␮m syringe filter prior to injection into AF4. 2.4. AF4-MALS–RI analysis of starch The AF4 system used in this work was an Eclipse 2 Separations System (Wyatt Technology Europe, Dernbach, Germany). It was connected to a DAWN EOS multiangle light scattering detector (Wyatt Technology, Santa Barbara, CA, USA) operating at the wavelength of 690 nm and a RID-10 differential refractive index detector (Shimadzu, Kyoto, Japan). An Agilent 1100 pump (Agilent Technologies, Waldbronn, Germany) with an in-line vacuum degasser delivered the carrier liquid into the AF4 channel. Between the pump and the AF4 channel was placed a 0.1 ␮m YYLP membrane filter (Millipore Corp. MA, USA) to ensure the carrier liquid entering AF4 channel is particle-free. The channel was assembled with a 350 ␮m-thick Mylar spacer and a regenerated ultrafiltration cellulose membrane with the cut-off of 10 kDa. The actual channel thickness was measured to be 295 ␮m from the elution time of ferritin (from horse spleen) based on the method in [24]. The channel geometry was trapezoidal with the tip-to-tip length of 26.5 cm and breadths at the inlet and the outlet of 2.2 and 0.6 cm, respectively. Injection of the sample into the channel was performed at the flow rate of 0.2 mL/min for 2 min. The concentration of the sample solution was 0.5–1.0 mg/mL, and the sample injection volume was 100 ␮L. In order to avoid excessive retention, a cross-flow programming was employed in this study, where the cross-flow was deceased linearly from 0.9 to 0.1 mL/min for 8 min, and then was maintained at 0.1 mL/min for 30 min as shown in Table 1. The thickness of the sample equilibrium layer increases with decreasing cross-flow rate, resulting in a reduction in retention time, especially for larger ones. The carrier liquid for AF4 was deionized water containing 50 mM NaNO3 and 3 mM NaN3 , which was filtered through a 0.1 ␮m regenerated cellulose membrane filter. 2.5. Determination of in vitro ˛-glucosidase inhibition In order to investigate the inhibition activity of wheat and barley starch, an in vitro ␣-glucosidase inhibition test was performed. 0.3 g of rat intestinal acetone powder was suspended in 9 mL of 0.9% (w/v) saline, and the suspension was sonicated for 30 s 12 times at 37 ◦ C. Then the suspension was centrifuged at 13,000 rpm for 30 min at 4 ◦ C and the supernatant was taken for assaying. Sample extracts were prepared by adding 20 mg of the sample powder into 1 mL of 0.1 M phosphate buffer (pH 6.9) followed by centrifugation at 5600 rpm for 5 min. In order to see whether the starch affect antidiabetic activity, suspensions of entire sample were also prepared by the same manner without centrifugation.

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A mixture of 100 ␮L aforementioned supernatant and 50 ␮L of either the sample extract or sample suspension was preincubated in VS-1203P3N incubator (Vision Scientific Co., Ltd., Bucheon, Korea) for 15 min at 37 ◦ C. After pre-incubation, 50 ␮L of 5 mM p-nitrophenyl-␣-d-glucopyranoside in 0.1 M phosphate buffer (pH = 6.9) was added to the assay mixture. The mixture was incubated for 10 min at 37 ◦ C. Then, absorbance of the mixture was measured using a GENios UV detector (TECAN Ltd., Männedorf, Switzerland) at the wavelength of 405 nm. A solution without sample was used as a control. The ˛-glucosidase inhibitory activity was measured as inhibition % by:

 Inhibition (%) =

Sample 405 AControl 405

AControl −A

 × 100

405

(1)

is the absorbance of the control measured at the where AControl 405 Sample

wavelength of 405 nm, and A405 ple.

is the absorbance of the sam-

2.6. Determination of in vivo antidiabetic activity Effect of starch on hyperglycemia in C57BLks/J db/db mouse was determined by studying the inhibitory action of the B-24 sample and acarbose on postprandial hyperglycemia. Five week-old male C57BLks/J db/db mice were fed solid diet (Joongang Experimental Animal Co., Seoul, Korea) for one week. The mice were housed in a ventilated room at 25 ± 2 ◦ C and 50 ± 7% relative humidity under an alternating 12 h light/dark cycle. After three groups of five male mice were fasted for 24 h, 5–100 mg of inhibitor (B-24 or acarbose) per body weight (kg) was orally administrated. Then the glucose levels of blood taken from tails were measured using CareSens II Blood Glucose Monitor (I-SENS Inc., Seoul, Korea) at 0, 0.5, 1.0, 2.0 and 3.0 h, respectively, based on the glucose oxidase method. Blood samples taken from mice without inhibitor administration were used as control. 2.7. Data treatment In the normal mode of AF4, the hydrodynamic radius (Rh ) can be determined from measured retention time, tr using [25,26]: Rh =

kTV 0 tr Vc w2 t 0

(2)

where k is the Boltzmann constant, T is the absolute temperature, V0 is the void volume,  is the viscosity of the carrier liquid, w is the channel thickness, Vc is the cross-flow rate, and t0 is the void time. It is noted that the use of Eq. (2) requires Vc to be constant (isocratic elution), and is valid to within the relative error of 1% when t0 /tr ≤ 0.029, within 5% when t0 /tr ≤ 0.17, and within 10% when t0 /tr ≤ 0.44 [25]. For ith slice of FFF fractogram, assuming molecules are spherical, the apparent molecular density can be determined from molar mass (Mi ) and molecular volume based on either the radius of gyration (Rg ) or Rh by [27,28]: ∗ g,i =

Mi × q or V (Rg )i × NA

∗ h,i =

Mi ×q V (Rh )i × NA

(3)

where g∗ is the Rg -based density, h∗ is the Rh -based density, NA is the Avogadro’s number, and Vi is the molar volume. q is a constant given by q=

Vsphere (Rg ) Vsphere (Rh )

=

Rg3 Rh3

=

 3 3/2 5

(4)

117

The mass-weighted average apparent density can be obtained by ∗ average

 M × i∗ i =

(5)

Mi

In this study, the molar mass and Rg were obtained from MALS–RI using the Berry method [29,30]:





Kc = R

1 162 + × Rg2 × sin2 Mw 32

   2

(6)

where K is the optical constant, c is the sample concentration, R is the Rayleigh ratio, Mw is the weight average molar mass,  is the wavelength. The reflective index increment (dn/dc) was measured by a batch MALS–RI method. The sample solutions were prepared at various concentrations at 0.01, 0.02, 0.04, 0.06, 0.08, and 0.1 mg/mL. Then the sample solutions were introduced into RI detector by a syringe pump (KD Scientific Inc., FL, USA). The dn/dc values of wheat and barley starch were determined to be 0.153 and 0.150, respectively. MALS data processing was performed using the Astra software (Version 5.3.4.19, Wyatt Technology). 3. Results and discussion 3.1. AF4-MALS–RI analysis of wheat and barley samples In AF4 analysis, no sample overloading was observed at the concentration range of 0.5–1.0 mg/mL used in this study as indicated by the independence of retention times on concentration (see Fig. S1). Also an excellent reproducibility was observed as shown in Fig. S2. Fig. 1 shows AF4-MALS–RI fractograms of three wheat samples (a) and their molar mass and Rg distributions (b). The solid lines are RI detector signals, and the dashed lines are MALS signals observed at the scattering angle of 90◦ (LS90 ). It can be seen in Fig. 1(a) that, as the germination time increases, the intensity and retention of LS90 response gradually decrease, suggesting the amount of high molar mass components was decreased during germination. In the retention time range of about 8 to 20 min, RI responses show the same trends as the LS90 responses. Molar mass distributions in Fig. 1(b) show there are two main populations (probably amylose and amylopectin) having molar masses either lower or higher than around 107 g/mol, respectively. There may also present some non-starch components [31]. It can be seen in Fig. 1(b) that, after 24 h of germination, the molar masses of the two populations were decreased, and the amount of the larger population was greatly reduced. No significant change was observed when the germination time was further increased to 48 h. These results indicate that germination of wheat promotes mobilization of energy-storing compounds having high molar mass (i.e. amylopectin) which are consumed to provide energy needed for metabolisms of wheat seed during germination. In Fig. 1(b), Rg distributions show that wheat samples have broad size distributions ranging from about 30 nm up to about 300 nm. Also it is shown that the slope of Rg vs. molar mass plot gradually increases as the molar mass increases for all three samples, suggesting the molecules become less compact as the molar mass increases. For molecules with molar masses of higher than about 108 g/mol, Rg decreases for the same molar mass as the germination time increases, suggesting molecules become more compact. For molecules with molar mass lower than about 108 g/mol, however, no significant differences in Rg were observed for the same molar mass. This result reveals that the germination affects more significantly the higher molar mass components. In order to study the effect of germination on molecular conformation of wheat starch, hydrodynamic radius Rh and apparent

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(a)

LS90 responses RI responses

0

5

10

15

B-0 B-24 B-48

Detector response

Detector response

W-0 W-24 W-48

20

LS90 responses

RI responses

0

25

5

(b)

350 300

Rg (nm)

250 200

Differential weight fraction

W-0 W-24 W-48 W-0 W-24 W-48

150 100 50

105

106

107 108 Molar mass (g/mol)

109

2.0

W-0 W-24 W-48 W-0 W-24 W-48

1.2

h

1.6

Rg/R

Apparent density (kg/m 3)

25

200 150 100 50

10

0.8 5

0.4

0 106

107

108

109

106

107

108

109

1010

Molar mass (g/mol)

molecular density g∗ were determined according to Eqs. (2) and (3), respectively, and then g∗ and Rg /Rh were plotted against molar mass in Fig. 2. For all three samples, both g∗ and Rg /Rh show similar overall trends. As the molar mass increases, g∗ increases (molecules become more compact), and levels off somewhat, and then decreases (molecules become less compact) until the molar mass reaches around 3.0 × 108 g/mol, after which increases sharply for germinated samples while levels off for the ungerminated sample. For the same molar mass, g∗ tends to increase with increasing germination time. This suggests that germination leads to more compact conformation of wheat starch molecules.

105

B-0 B-24 B-48 B-0 B-24 B-48

105

1010

Fig. 1. AF4-MALS–RI fractograms of wheat samples (a) and distributions of molar mass (solid lines) and Rg (symbols) (b).

15

20

0

0

20

15

300 250

Rg (nm)

(b)

10

Retention time (min)

Retention time (min)

Differential weight fraction

(a)

0.0 1010

Molar mass (g/mol) Fig. 2. Variation of apparent density (triangle) and Rg /Rh (circle) of wheat samples with molar mass.

Fig. 3. AF4-MALS–RI fractograms of barley samples (a) and distributions of molar mass (solid lines) and Rg (symbols) (b).

Additional information on the compactness and the shape of molecules can be obtained from Rg /Rh . Typically the value of Rg /Rh is about 0.78 for a homogeneous hard sphere, and about 2.4 for a stiff rod. And the value of Rg /Rh ranges from 1.5 to 2.1 for random coil conformation [27]. In Fig. 2, for all three samples, Rg /Rh initially decreases rather sharply with increasing molar mass and reaches a minimum, and then increases slowly. Results suggest, during sharp reductions, the molecular conformations change from oblate ellipsoid to swollen micro-gel structures. During slow increases, the values of Rg /Rh remain in a range of 0.3–0.6 (swollen micro-gel structures), suggesting molecules with oblate ellipsoid and swollen micro-gel structures may be joined together [32]. For starch, the values of Rg /Rh are expected to be in the range of 1.0–1.8 [33]. It is noted that the measurement of Rh may suffer from non-ideal retention behaviors (such as secondary relaxation), resulting in a deviation in Rg /Rh [34–36]. It is interesting that the position of the minimum shifts toward higher molar mass after germination. Fig. 3 shows AF4-MALS–RI fractograms of three barley samples (a) and their molar mass and Rg distributions (b). Unlike for the wheat samples, as the germination time increases, the intensity and retention of LS90 response gradually increases, suggesting the amount of component with molar mass higher than about 107 g/mol was increased during germination. Molar mass distributions in Fig. 3(b) show there are also two main populations in terms of molar mass. Up to and including 24 h of germination, no significant changes in the molar mass and the relative content of the two populations were observed. When the germination time was further increased to 48 h, the molar mass of the larger population was increased and the relative content of the smaller population was decreased along with an increase in the

H. Dou et al. / J. Chromatogr. A 1340 (2014) 115–120

25 20

0.8 0.6

Rg/Rh

15

0.4

10

0.2

5 0.0 105

106

107

108

109

1010

Molar mass (g/mol)

80

Rat α-glucosidase inhibition (%)

30

Rg/Rh

Apparent density (kg/m 3)

1.0 B-0 B-24 B-48 B-0 B-24 B-48

Wheat extract Barley extract Barley suspension

70

60

50

40 0

Fig. 4. Variation of apparent density (triangle) and Rg /Rh (circle) of barley samples with molar mass.

3.2. Antidiabetic activity in vitro and in vivo There have been reports on antidiabetic components extracted from various botanical sources. Generally the antidiabetic activity Table 2 Molar mass, Rg , and g∗ of wheat and barley samples determined by AF4-MALS–RI.

W-0 W-24 W-48 B-0 B-24 B-48

Rg (nm) 175 149 144 129 135 159

Mn × 10−5 (g/mol)

Mw × 10−5 (g/mol)

Mw /Mn

23 9 7 8 9 10

473 311 301 160 208 325

21 35 42 20 23 32

∗ ¯ g,average 3

(kg/m ) 5.3 14.7 15.3 15.8 21.5 31.8

24

48

Germination time (h) Fig. 5. Variations of rat ␣-glucosidase inhibition with germination time for three different samples. The error bars represent a standard deviation (n = 3).

of the extracts has been evaluated from lowering of the blood glucose level [37]. Until now, however, there have not been any studies on the antidiabetic activity of starch. Fig. 5 shows changes in the rat ␣-glucosidase inhibition with germination time for three different samples. For both wheat and barley extracts, the ␣-glucosidase inhibition gradually increases with increasing germination time. It is noted that the rat ␣-glucosidase inhibition is always higher for barley than those for wheat, which suggests the potential of germinated barley is higher than germinated wheat as a material for functional foods for reduction of diabetic agents. For barley samples without germination (i.e. B-0), no significant difference in the ␣-glucosidase inhibition was observed between the sample extract and the sample suspension. While for the germinated barley samples (i.e. B-24 and B-48), ␣-glucosidase inhibition of the sample suspensions are higher than those of the sample extracts. One possible explanation is that the macromolecules with high apparent density, i.e. compact conformation induced by germination, would promote an enhancement in ␣-glucosidase inhibition activity [23]. For further investigation on in vitro ␣-glucosidase inhibition activity of germinated barley, the in vivo blood glucose-lowering effect was evaluated with C57BLks/J db/db mouse and the results are shown in Fig. 6. The result shows B-24 has a higher blood glucose-lowering effect than the control. It can be seen that, when acarbose, an antidiabetic drug, was administrated, the blood

800

Blood glucose level (mg/dL)

relative content of the larger population. Results from Figs. 1 and 3 reveal that the metabolism of cereal during germination is strongly dependent on the botanical source of cereal. The Rg distributions in Fig. 3(b) show similar trends as those of wheat samples (Fig. 1(b)). No significant differences were observed among all three samples, except at higher end of the distributions, where Rg of B-48 is higher than those of other two samples for the same molar mass. Fig. 4 shows plots of g∗ and Rg /Rh vs. molar mass, where both g∗ and Rg /Rh show similar trends for all three samples. The plots of g∗ vs. molar mass for all three samples show similar trends to those of the germinated wheat samples, where g∗ initially increases with increasing molar mass, and levels off, and then followed by a sharp increase. In the molar mass range lower than about 8.0 × 107 g/mol, g∗ increases as the germination time increases indicating molecules become more compact by germination. In the entire molar mass range, the values of Rg /Rh are lower than 1.0 again. No significant difference in Rg /Rh among barley samples was observed. Molar mass, Rg , and g∗ of wheat and barley samples determined by AF4-MALS–RI are summarized in Table 2. It is shown that, although Rg and M of W-0 are larger than those of B-0, average ∗ ) of W-0 (5.3 kg/m3 ) is lower than that of B-0 (15.8 kg/m3 ), g,average indicating B-0 has more compact conformation. During germi∗ were gradually nation, all of Rg , Mn ,Mw , Mw /Mn , and g,average increased for the barley samples. While for the wheat samples, ∗ were decreased with increasing all except Mw /Mn , and g,average germination time. These results reveal again that the germination mechanism is dependent on the botanical source of cereal. Detailed conformational characterization of germinated cereal seeds by AF4-MALS–RI may open the way to a better understanding of the germination mechanism.

Samples

119

Acarbose 5 mg/kg B-24 100 mg/kg Control

600

400

200

0 0.0

0.5

1.0

1.5

2.0

2.5

3.0

Time (h) Fig. 6. Effect of B-24 on blood glucose level of mouse after oral administration. The error bars represent a standard deviation (n = 5).

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glucose level decreases lightly with increasing time which is probably due to broad error bars. It has been reported that side effects of diarrhea and flatulence would be caused by oral administration of acarbose [38,39]. Our in vivo test also reveals the presence of the side effects of oral administration of acarbose to a certain extent as indicated in Fig. S3. It is seen from Fig. S3 that the cecums of mice fed with acarbose for two months are significantly larger than those fed with B-24 or control, which probably due to the side effect of flatulence. Thus, the demand for the healthy diet is increasing. 4. Conclusion In this work, we demonstrated that AF4-MALS–RI is a suitable technique for the monitoring of variation in conformation properties of wheat and barley starch during germination process. Our results reveal that, for wheat, the amount of component of ultrahigh molar mass gradually decreased with increasing germination time, whereas for barley, the amount of component of ultrahigh molar mass slightly increased, suggesting the metabolism of cereal seed during germination process is dependent on the botanical source of cereal. Furthermore, it was suggested that germination process induces an enhancement in the apparent density either for wheat or for barley starch, which would contribute to enhance their antidiabetic activity. Detailed conformational characterization of germinated cereal seeds by AF4-MALS–RI can open the way to well understand the germination mechanism. Acknowledgments The authors acknowledge support from Hannam University. This research was also supported by Korea Research Foundation (KRF). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.chroma.2014.03.014. References [1] R. Gilbert, Anal. Bioanal. Chem. 399 (2011) 1425. [2] C.S. Brennan, Mol. Nutr. Food Res. 49 (2005) 716. [3] D.J. Jenkins, C.W. Kendall, L.S. Augustin, S. Franceschi, M. Hamidi, A. Marchie, A.L. Jenkins, M. Axelsen, Am. J. Clin. Nutr. 76 (2002) 266S.

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Study on antidiabetic activity of wheat and barley starch using asymmetrical flow field-flow fractionation coupled with multiangle light scattering.

The ability of asymmetrical flow field-flow fractionation (AF4) coupled online with multiangle light scattering (MALS) and refractive index detector (...
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