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JOURNAL OF MEDICINAL FOOD
J Med Food 10 (2) 2007, 266–275
© Mary Ann Liebert, Inc. and Korean Society of Food Science and Nutrition
DOI: 10.1089/jmf.2006.234
Health Benefits of Traditional Corn, Beans, and Pumpkin: In Vitro Studies for
Hyperglycemia and Hypertension Management
Y.-I. Kwon,
1
E. Apostolidis,
1
Y.-C. Kim,
2
and K. Shetty
1
Departments of
1
Food Science and
2
Nutrition, University of Massachusetts, Amherst, Massachusetts
ABSTRACT Levels of obesity-linked non–insulin-dependent diabetes mellitus (NIDDM) and hypertension are highest among
indigenous communities in North America. This is linked to changes in dietary pattern towards high calorie foods such as
sugar, refined grain flour, and sweetened beverages. Therefore, a return to traditional dietary patterns may help to reduce these
disease problems because of better balance of calories and beneficial nutrients. Further protective non-nutrient phenolic phy-
tochemicals against NIDDM and hypertension are potentially high in these foods but less understood. In this study antidia-
betic- and antihypertension-relevant potentials of phenolic phytochemicals were confirmed in select important traditional plant
foods of indigenous communities such as pumpkin, beans, and maize using in vitro enzyme assays for -glucosidase, -amy-
lase, and angiotensin I-converting enzyme (ACE) inhibitory activities. In vitro inhibitory activities of these enzymes provide
a strong biochemical rationale for further in vivo studies and dietary management strategy for NIDDM through the control of
glucose absorption and reduction of associated hypertension. These enzyme inhibitory activities were further compared to to-
tal soluble phenolic content and antioxidant activity of the above-targeted plant foods. Pumpkin showed the best overall po-
tential. Among the varieties of pumpkin extracts P5 (round orange) and P6 (spotted orange green) had high content of total
phenolics and moderate antioxidant activity coupled to moderate to high -glucosidase and ACE inhibitory activities. There-
fore this phenolic antioxidant-enriched dietary strategy using specific traditional plant food combinations can generate a whole
food profile that has the potential to reduce hyperglycemia-induced pathogenesis and also associated complications linked to
cellular oxidation stress and hypertension.
KEY WORDS: •-amylase inhibitor •angiotensin I-converting enzyme inhibitor •beans •corn •-glucosidase in-
hibitor •hypertension •non–insulin-dependent diabetes mellitus •phenolic phytochemicals •pumpkin •traditional diet
INTRODUCTION
C
HRONIC DISEASES
such as diabetes mellitus (DM) and
cardiovascular disease are among the leading causes of
death globally. These diseases are strongly associated with
diets high in calories and reduced physical activity. In North
America these diseases disproportionately affect certain in-
digenous communities, who also have high prevalence of
obesity in all age groups and in both sexes.
1–3
The intro-
duction of new high calorie foods such as sugar, refined
grain flour, bread, and sweetened tea took place rapidly and
has resulted in the increase of chronic disease among this
population.
4
Therefore, a return to traditional dietary patterns may help
to reduce these disease problems because of better balance
of calories and beneficial nutrients. Traditional diet is used
to identify all food within a particular culture available from
local natural resources.
3
The major qualities of traditional
diet in certain North American indigenous communities that
require attention are the various species and species diver-
sity that are accepted as food from the natural environment
in diverse climates and latitude. A plant food-based tradi-
tional diet comprising corn, bean, and pumpkin was com-
mon among select indigenous communities of North Amer-
ica and has potential for preventing chronic disease
challenges such as DM and cardiovascular disease.
DM is a serious chronic metabolic disorder that has a sig-
nificant impact on health, quality of life, and life expectancy.
Two types of DM are currently known: insulin-dependent
DM and non–insulin-dependent DM (NIDDM).
5
The most
common acute complications of NIDDM due to the hyper-
glycemia-induced pathogenesis are metabolic problems and
infection.
6
Hyperglycemia, a condition characterized by an
abnormal postprandial increase of blood glucose level, has
been linked to the onset of NIDDM and associated oxida-
tion-linked vascular complications.
5,6
Recent studies have
shown that the glucose-induced increased levels of mito-
chondrial reactive oxygen species (ROS) produced by the
mitochondrial electron transport chain seem to be the causal
link between elevated levels of glucose and the pathways
Manuscript received 16 October 2006. Revision accepted 5 February 2007.
Address reprint requests to: K. Shetty, Department of Food Science, University of Mass-
achusetts, Chenoweth Laboratory, 100 Holdsworth Way, Amherst, MA 01003, E-mail:
kalidas@foodsci.umass.edu
266
responsible for hyperglycemia-induced vascular complica-
tions.
7
Studies indicate that hyperglycemia triggers the gen-
eration of free radicals and oxidative stress in capillary en-
dothelial cells in the retina, mesangial cells in the renal
glomerulus, and neuron cells in the peripheral nerves.
8,9
Therefore, it is essential to regenerate critical cellular an-
tioxidant responses to manage cellular redox status for pre-
venting these NIDDM complications resulting from hyper-
glycemia.
10,11
Effective dietary strategies can contribute to
solutions for managing both hyperglycemia and proper cel-
lular redox status.
A sudden rise in blood glucose levels, causing hyper-
glycemia, in NIDDM patients is due to hydrolysis of starch
by pancreatic -amylase and absorption of glucose in the
small intestine by -glucosidase. Inhibition of these en-
zymes involved in the breakdown of starch and uptake of
glucose can significantly decrease the postprandial increase
of blood glucose after a mixed carbohydrate diet and there-
fore can be an important strategy in the management of
NIDDM.
12
However, previous reports have indicated that
excessive inhibition of pancreatic -amylase could result in
the abnormal bacterial fermentation of undigested starch in
the colon, and therefore mild -amylase inhibitory activity
is useful.
13,14
High -amylase inhibition is the main draw-
back of currently used therapeutic -glucosidase inhibitors
such as the drug acarbose, resulting in side effects such as
abdominal distention, flatulence, meteorism, and possibly
diarrhea.
15,16
Therefore, natural inhibitors from dietary
plants are useful as they have lower inhibitory activity
against -amylase and a stronger inhibitory activity against
-glucosidase and can be used as effective therapy for post-
prandial hyperglycemia with minimal side effects.
17
One of the long-term complications of NIDDM is hyper-
tension, or high blood pressure. Angiotensin I-converting
enzyme (ACE) is an important enzyme involved in main-
taining vascular tension. ACE converts angiotensin I to an-
giotensin II, a potent vasoconstrictor and stimulator of al-
dosterone secretion by the adrenal gland.
18
Inhibition of
ACE is considered a useful therapeutic approach in the treat-
ment of high blood pressure in both diabetic and nondia-
betic patients,
19–21
and dietary phenolic phytochemicals
have promising potential. Previous in vitro and in vivo ani-
mal and clinical studies have also indicated the potential of
specific phenolic phytochemicals in hypertension manage-
ment with direct absorption into the blood.
17,22,23
Phenolic compounds or phenolic phytochemicals are sec-
ondary metabolites of plant origin and are important parts
of the diet,
24–26
providing potential antioxidant benefits for
managing oxidation stress-related chronic diseases such as
diabetes and cardiovascular disease.
27–32
Important plant
foods in select traditional diet of indigenous communities
such as maize (corn), pumpkin, and beans have moderate to
high phenolic phytochemicals. A combination of these plant
foods can be targeted for management of hyperglycemia and
hypertension associated with NIDDM.
Our hypothesis is that the biochemical rationale for di-
etary management of hyperglycemia and hypertension lies
in the phenolic-enriched antioxidant activity and -glucosi-
dase and ACE inhibitory potential of traditional diet. Any
dietary management of hyperglycemia linked to NIDDM
and related complications from oxidative dysfunction can
benefit from specific enzyme inhibitory activities combined
with antioxidant activity in the same whole food profile from
combinations of plant foods. This has potential for high com-
pliance and fewer side effects. Traditional plant-based foods
such as pumpkin, bean, and maize have good nutrient ben-
efits with balanced calories, and their general nutrient pro-
file has been outlined by U.S. Department of Agriculture
and the American Diabetes Association studies.
33,34
How-
ever, phenolic protective factors linked to antidiabetic and
antihypertension potential have not been understood. There-
fore, the objective of this research was to investigate sev-
eral types of commonly available plant foods typical of tra-
ditional diet in select indigenous communities such as
pumpkins, beans, and maize for in vitro -amylase, -glu-
cosidase, and ACE inhibitory activities using mammalian
enzyme sources such as rat intestinal -glucosidase, porcine
pancreatic -amylase, and rabbit lung ACE. These in-
hibitory activities were compared to total phenolic content
and antioxidant activity in the water extracts of various types
of the above-mentioned plant foods. These in vitro studies
provide a stronger biochemical rationale for future animal
and clinical studies.
MATERIALS AND METHODS
Materials
Pumpkin (Cucurbita pepo) [round yellow (P1), striped
yellow green (P2), striped round yellow green (P3), elon-
gated brown (P4), round orange (P5), spotted orange green
(P6), and round green (P7)] and maize (Zea mays) [yellow
strained (M1), small yellow (M2), black pigmented (M3),
white blotched (M4), light red (M5), small red (M6), and
dark red (M7)] were purchased from a local market in
Hadley, MA. Maize seed samples were harvested from cob
by color and shape from several traditional varieties. Bean
varieties [Glycine max black (B1), Vigna angularis (B2),
Canavalia spp. (B3), Cicer arietinum (B4), G. max yellow
(B5), and Canavalia ensiformis (B6)] were purchased from
a local market in Hadley, MA. Commonly consumed G. max
and C. arietinum were included for comparisons to the tra-
ditional Canavalia genus that has been domesticated from
Arizona to Peru
35
and V. angularis common in Americas.
Porcine pancreatic -amylase (EC 3.2.1.1), rat intestinal
-glucosidase (EC 3.2.1.20), and rabbit lung ACE (EC
3.4.15.1) were purchased from Sigma Chemical Co. (St.
Louis, MO). Unless noted, all chemicals also were pur-
chased from Sigma.
Extract preparation
To reflect cooked food, for each type of pumpkin, bean,
and maize, a 10-g sample was added to 10 mL of distilled
DIET CONTROL FOR DIABETES AND HYPERTENSION 267
water and autoclaved for 10 minutes, and then 40 mL of dis-
tilled water was added and homogenized for 1 minute using
a Waring
®
laboratory blender (Waring Laboratory, Tor-
rington, CT) set on “HIGH.” The homogenate was cen-
trifuged at 9,300 gfor 10 minutes. The supernatant was vac-
uum-filtered through a Whatman #2 filter and then used as
the crude extract for in vitro assays. Nonautoclaved samples
were also prepared similarly for comparison and to observe
trends of loss following autoclaving.
Total phenolics assay
The total phenolics were determined by an assay modi-
fied from Shetty et al.
36
Briefly, 1 mL of pumpkin, bean, or
maize extract was transferred into a test tube and mixed with
1 mL of 95% ethanol and 5 mL of distilled water. To each
sample 0.5 mL of 50% (vol/vol) Folin-Ciocalteu reagent was
added and mixed. After 5 minutes, 1 mL of 5% Na
2
CO
3
was
added to the reaction mixture and allowed to stand for 60
minutes. The absorbance was read at 725 nm. The ab-
sorbance values were converted to total phenolics and were
expressed in micrograms of gallic acid equivalents per mil-
liliter of the sample extract. Standard curves were estab-
lished using various concentrations of gallic acid in 95%
ethanol.
Antioxidant activity by 1,1-diphenyl-2-picrylhydrazyl
radical (DPPH) inhibition assay
The DPPH scavenging activity was determined by an as-
say modified from Kwon et al.
17
To 3 mL of 60 MDPPH
in ethanol, 250 L of each pumpkin, bean, or maize extract
was added, and the decrease in absorbance was monitored
every 1 minute for 3 minutes at 517 nm. The readings were
compared with the controls, which contained 250 L of 95%
ethanol instead of the extract. The percentage inhibition was
calculated by:
% inhibition 100
-Amylase inhibition assay
The -amylase inhibitory activity was determined by an
assay modified from the Worthington Enzyme Manual.
37
Porcine pancreatic -amylase (EC 3.2.1.1) was purchased
from Sigma. A total of 500 L of pumpkin, bean, or maize
extract and 500 L of 0.02 Msodium phosphate buffer (pH
6.9 with 0.006 MNaCl) containing -amylase solution (0.5
mg/mL) were incubated at 25°C for 10 minutes. After prein-
cubation, 500 L of a 1% starch solution in 0.02 Msodium
phosphate buffer (pH 6.9 with 0.006 MNaCl) was added to
each tube at timed intervals. The reaction mixtures were then
incubated at 25°C for 10 minutes. The reaction was stopped
with 1.0 mL of dinitrosalicylic acid color reagent. The test
tubes were then incubated in a boiling water bath for 5 min-
utes and cooled to room temperature. The reaction mixture
A
517
Control
A
517
Extract
A
517
Control
was then diluted after adding 10 mL of distilled water, and
absorbance was measured at 540 nm:
% inhibition 100
-Glucosidase inhibition assay
A modified version of the assay described by Worthington
Enzyme Manual was followed.
38,39
Rat intestine acetone pow-
der as a crude enzyme extract was purchased from Sigma. A
volume of 50 L of sample extract and 100 L of 0.1 M
phosphate buffer (pH 6.9) containing crude -glucosidase so-
lution (25 mg/mL) was incubated in 96-well plates at 25°C
for 10 minutes. After preincubation, 50 L of 5 mMp-nitro-
phenyl--
D
-glucopyranoside solution in 0.1 Mphosphate
buffer (pH 6.9) was added to each well at timed intervals. The
reaction mixtures were incubated at 37°C for 30 minutes. Be-
fore and after incubation, absorbance readings were recorded
at 405 nm by a micro-array reader (Thermomax, Molecular
Device Corp., Sunnyvale, CA) and compared to a control that
had 50 L of buffer solution in place of the extract. The -
glucosidase inhibitory activity was expressed as percentage
inhibition and was calculated as follows:
% inhibition 100
ACE inhibition assay
ACE inhibition was assayed by a method modified by
Kwon et al.
17
The substrate (hippuryl-histidyl-leucine) and
ACE from rabbit lung (EC 3.4.15.1) were purchased from
Sigma. Fifty microliters of extracts was incubated with 100
L of 1.0 MNaCl-borate buffer (pH 8.3) containing 2.0 mU
of ACE solution at 37°C for 10 minutes. After preincuba-
tion, 100 L of a 5.0 mMsubstrate (hippuryl-histidyl-
leucine) solution was added to the reaction mixture. Test so-
lutions were incubated at 37°C for 1 hour. The reaction was
stopped with 150 L of 0.5 NHCl. The hippuric acid formed
was detected and quantified by the high performance liquid
chromatography method. A volume of 5 L of sample was
injected using an Agilent ALS 1100 autosampler into an Ag-
ilent 1100 series high performance liquid chromatograph
(Agilent Technologies, Palo Alto, CA) equipped with a
DAD 1100 diode array detector. The solvents used for the
gradient were (A) 10 mMphosphoric acid (pH 2.5) and (B)
100% methanol. The methanol concentration was increased
to 60% for the first 8 minutes and to 100% for 5 minutes
and then decreased to 0% for the next 5 minutes (total run
time, 18 minutes). The analytical column used was Nucle-
osil 100-5C
18
, 250 4.6 mm i.d., with packing material of
5 m particle size at a flow rate of 1 mL/minute at ambi-
ent temperature. During each run the chromatogram was
recorded at 228 nm and integrated using the Agilent Chem-
station enhanced integrator for detection of liberated hip-
puric acid. Pure hippuric acid (purchased from Sigma) was
A
405
Control
A
405
Extract
A
405
Control
A
540
Control
A
540
Extract
A
540
Control
268 KWON ET AL.
used to calibrate the standard curve and retention time. The
percentage inhibition was calculated by:
% inhibition 100
Statistical analysis
All experiments were performed at least in triplicates.
Analysis at every time point from each experiment was car-
ried out in triplicates. Means and SD values were calculated
from replicates within the experiments, and analyses were
done using Microsoft (Redmond, WA) Excel 2003. The re-
sults were statistically analyzed by analysis of variance and
Duncan’s multiple range tests. Statistical significance was
accepted at a level of P.05.
RESULTS
Total soluble phenolics and antioxidant activity
Water extracts of pumpkin (C. pepo). The total soluble phe-
nolics were determined by an assay modified from Shetty et
al.
36
Water extracts of P5 (round orange) had an average phe-
nolic content of 172 g/g fresh weight (FW), which was high-
est among all the extracts evaluated (Fig. 1). P1 (round yel-
low), P6 (spotted orange green), P4 (elongated brown), and
P3 (striped round yellow green stripes) extracts had 146 g/g
FW, 107 g/g FW, 93 g/g FW, and 86 g/g FW of soluble
phenolics, respectively (Fig. 1). Cooking through autoclaving
enhanced phenolic content only in the P1 and P5 varieties.
The ability of phenolics to inhibit the DPPH radical for-
mation was measured in both autoclaved (to reflect cook-
ing) and nonautoclaved pumpkin extracts. The nonauto-
claved water extract of P4 had the highest DPPH radical
inhibition capacity (33%), followed by autoclaved extract of
P6 (30%), autoclaved extract of P5 (26%), and nonauto-
claved extract of P2 (striped yellow green, 23%) (Fig. 1).
DPPH radical inhibition was not proportional to the con-
centration of total soluble phenolics in all extract samples
(Fig. 1). The Pearson’s correlation coefficients between
DPPH scavenging activity and total phenolic content (auto-
claved and nonautoclaved samples) were 0.21 and 0.2098,
respectively.
Water extracts of bean. The nonautoclaved B5 (G. max
yellow) had 1.3 mg/g FW of phenolics, which was highest
among all the beans evaluated (Fig. 2). The nonautoclaved
B3 (Canavalia spp.), B4 (C. arietinum), and autoclaved B4
had 1.1 mg/g FW, 0.9 mg/g FW, and 0.9 mg/g FW of sol-
uble phenolics, respectively (Fig. 2). The nonautoclaved ex-
tracts of B2 (V. angularis), B3, B5, and B6 (C. ensiformis)
had higher total soluble phenolics than autoclaved extracts.
There was variation of total phenolic content in various bean
types, with nonautoclaved generally having higher pheno-
lics than autoclaved extracts and therefore indicating some
loss following cooking.
E
Control
E
Sample
E
Control
E
Blank
The ability of phenolics to inhibit the DPPH radical for-
mation was measured in both autoclaved and nonautoclaved
bean samples and reflected the free radical scavenging-
linked antioxidant activity. Autoclaved B4 had the highest
antioxidant activity (23%), followed by autoclaved B5
(22%), nonautoclaved B2 (20%), and nonautoclaved B5
(19%) (Fig. 2). The free radical scavenging-linked antioxi-
dant activity of all autoclaved bean extracts was generally
low and was proportional to the concentration of soluble to-
tal phenolics compared to other species evaluated in this
study (Fig. 2) (the Pearson’s correlation coefficient of au-
toclaved beans between DPPH scavenging activity and to-
tal phenolic content was 0.7601). The phenolic content of
beans (legumes) in general was 7- to 10-fold higher than
pumpkin but with a similar range of antioxidant activity.
Water extracts of maize (Z. mays). Nonautoclaved water
extracts of M7 (dark red) had 0.5 mg/g FW of phenolics,
which was highest among all the extracts tested (Fig. 3). The
nonautoclaved M6 (small red) extract and autoclaved M2
(small yellow) extract had close to 0.4 mg/g FW soluble
DIET CONTROL FOR DIABETES AND HYPERTENSION 269
FIG. 1. Total soluble phenolics and DPPH radical scavenging ac-
tivity in autoclaved and nonautoclaved extracts of pumpkin: round
yellow (P1), striped yellow green (P2), striped round yellow green
(P3), elongated brown (P4), round orange (P5), spotted orange green
(P6), and round green (P7). Total soluble phenolic content (in g/mL)
and DPPH radical scavenging activity (%) are plotted.
A–G
Data are
mean SD values of total soluble phenolic content (nonautoclaved)
of three replicated samples.
a–g
Data are mean SD values of total
soluble phenolic content (autoclaved) of three replicated samples.
Columns with different letters indicate statistically significant differ-
ences among groups at P.05. The Pearson’s correlation coefficient
of total phenolic content between nonautoclaved and autoclaved sam-
ples is 0.8137. The Pearson’s correlation coefficient of DPPH scav-
enging activity between nonautoclaved and autoclaved samples is
0.0398. The Pearson’s correlation coefficients between DPPH scav-
enging activity and total phenolic content (nonautoclaved and auto-
claved samples) are 0.2098 and 0.21, respectively.
phenolics (Fig. 3). Overall, phenolic content of maize was
generally lower than beans but higher than pumpkin.
The free radical scavenging-linked antioxidant activity of
the extracts was monitored using the DPPH radical inhibi-
tion assay. The autoclaved water extracts of M5 had the
highest DPPH radical inhibition activity (38%), followed by
M4 (white blotched, 29%), and nonautoclaved M7 (28%)
(Fig. 3). The Pearson’s correlation coefficients between
DPPH scavenging activity and total phenolic content (auto-
claved and nonautoclaved samples) were 0.02 and 0.7601,
respectively.
Amylase/glucosidase inhibition
Water extracts of pumpkin. -Amylase and -glucosidase
inhibitory activities and antioxidant activity were compared
with autoclaved extracts of pumpkin reflecting the cooking
process. All samples were prepared on a constant volume
basis (200 mg FW/mL). Figure 4 shows -amlyase and
-glucosidase inhibitory activities of various autoclaved ex-
tracts of pumpkin, and the trends were compared to DPPH
scavenging-linked antioxidant activity. The -amylase in-
hibitory activity of autoclaved extracts of pumpkin was di-
rectly proportional to the rat intestinal -glucosidase in-
hibitory activity and DPPH scavenging activity (Fig. 4). The
Pearson’s correlation coefficient between -amylase and -
glucosidase inhibitory activity was 0.91. The Pearson’s cor-
relation coefficients between DPPH scavenging activity and
the inhibitory activity of the two enzymes (-amylase and
-glucosidase) were 0.8429 and 0.9123, respectively.
All the samples of pumpkin had slightly higher -amy-
lase inhibitory potential than -glucosidase (Fig. 4). Extracts
P3, P5, and P7 had high -glucosidase inhibitory activity,
which was similar in range to -amylase inhibitory activity.
However, P4 and P6 extracts had much higher -amylase
inhibitory activity than -glucosidase inhibitory activity
(Fig. 4).
Water extracts of bean. Figure 5 shows -amylase and
-glucosidase inhibitory activities of various autoclaved ex-
tracts of bean in comparison to antioxidant activity. All the
270 KWON ET AL.
FIG. 2. Total soluble phenolics and DPPH radical scavenging ac-
tivity in autoclaved and nonautoclaved extracts of bean: G. max black
(B1), V. angularis (B2), Canavalia spp. (B3), C. arietinum (B4), G.
max yellow (B5), and C. ensiformis (B6). Total soluble phenolic con-
tent (in g/mL) and DPPH radical scavenging activity (%) are plot-
ted.
A–F
Data are mean SD values of total soluble phenolic content
(nonautoclaved) of three replicated samples.
a–f
Data are mean SD
values of total soluble phenolic content (autoclaved) of three repli-
cated samples. Columns with different letters indicate statistically sig-
nificant differences among groups at P.05. The Pearson’s correla-
tion coefficient of total phenolic content between nonautoclaved and
autoclaved samples is 0.8353. The Pearson’s correlation coefficient of
DPPH scavenging activity between nonautoclaved and autoclaved
samples is 0.4349. The Pearson’s correlation coefficients between
DPPH scavenging activity and total phenolic content (nonautoclaved
and autoclaved samples) are 0.2196 and 0.73, respectively.
FIG. 3. Total soluble phenolics and DPPH radical scavenging ac-
tivity in autoclaved and nonautoclaved extracts of maize: yellow
strained (M1), small yellow (M2), black pigmented (M3), white
blotched (M4), light red (M5), small red (M6), and dark red (M7). To-
tal soluble phenolic content (in g/mL) and DPPH radical scaveng-
ing activity (%) are plotted.
A–G
Data are mean SD values of total
soluble phenolic content (nonautoclaved) of three replicated samples.
a–d
Data are mean SD values of total soluble phenolic content (au-
toclaved) of three replicated samples. Columns with different letters
indicate statistically significant differences among groups at P.05.
The Pearson’s correlation coefficient of total phenolic content between
nonautoclaved and autoclaved samples is 0.2126. The Pearson’s cor-
relation coefficient of DPPH scavenging activity between nonauto-
claved and autoclaved samples is 0.1716. The Pearson’s correlation
coefficients between DPPH scavenging activity and total phenolic con-
tent (nonautoclaved and autoclaved samples) are 0.7601 and 0.02, re-
spectively.
extracts showed a comparable inhibition of the -amylase
but did not have high inhibitory activity against -glucosi-
dase. The -glucosidase and -amylase inhibitory activities
of autoclaved extracts of bean were not proportional to the
DPPH scavenging activity (Fig. 5). The Pearson’s correla-
tion coefficient between -amylase and -glucosidase in-
hibitory activity was 0.25. The Pearson’s correlation coef-
ficients between DPPH scavenging activity and the
inhibitory activities of the two enzymes (-amylase and -
glucosidase) were 0.3341 and 0.2568, respectively.
Water extracts of maize. The -amylase inhibitory activ-
ity of maize extracts was not proportional to the antioxidant
activity (Fig. 6) (the Pearson’s correlation coefficient was
0.2289). M2 had the highest -amlyase inhibitory activ-
ity (89%), followed by M5 (56%) and M1 (40%) (Fig. 6).
-Glucosidase inhibitory activity in autoclaved extracts of
maize was also evaluated. Samples M1, M2, and M5 had
higher -amylase inhibitory activity compared to -glu-
cosidase inhibitory activity. Sample M5 had also moderate
antioxidant activity potential. Samples M4 and M6 had
higher -glucosidase inhibitory activity than -amylase in-
hibitory activity. The range of -glucosidase inhibitory ac-
tivity was 28–48%, which can be considered moderate po-
tential.
ACE inhibition and total soluble phenolic content
Water extracts of pumpkin. The ability of the autoclaved
extracts of pumpkin to inhibit the activity of ACE was in-
vestigated. ACE inhibition was assayed by a method mod-
ified by Kwon et al.
17
P5 (80%), P1 (76%), and P6 (61%)
had the highest ACE inhibitory activity (Fig. 7). ACE in-
hibitory activity of the pumpkin types correlate well with
the total soluble phenolic content (Fig. 7) (the Pearson’s cor-
relation coefficient was 0.8934). The P5 and P6 extracts had
high ACE-I inhibitory activity combined with moderate to
high -amylase/-glucosidase inhibitory activities and mod-
erate antioxidant activity (Figs. 4 and 7).
Water extracts of bean. The ACE inhibitory activity of
autoclaved bean extracts was not proportional to the con-
centration of the total soluble phenolics (Fig. 8) (the Pear-
son’s correlation coefficient was 0.7015). Samples B1, B2,
DIET CONTROL FOR DIABETES AND HYPERTENSION 271
FIG. 4. Comparison of porcine pancreatic -amylase and rat in-
testinal -glucosidase inhibitory activity and DPPH radical scaveng-
ing activity of autoclaved extracts of pumpkin: round yellow (P1),
striped yellow green (P2), striped round yellow green (P3), elongated
brown (P4), round orange (P5), spotted orange green (P6), and round
green (P7). Each assay was carried out with 200 mg FW sample/mL
concentration.
A–D
Data are mean SD values of -amylase inhibitory
activity (autoclaved) of three replicated samples.
a–g
Data are mean
SD values of -glucosidase inhibitory activity (autoclaved) of three
replicated samples. Columns with different letters indicate statistically
significant differences among groups at P.05. The Pearson’s cor-
relation coefficient between -amylase and -glucosidase inhibitory
activity is 0.91. The Pearson’s correlation coefficients between DPPH
scavenging activity and inhibitory activity against the two enzymes
(-amylase and -glucosidase) are 0.8429 and 0.9123, respectively.
FIG. 5. Comparison of porcine pancreatic -amylase and rat in-
testinal -glucosidase inhibitory activity and DPPH radical scaveng-
ing activity of autoclaved extracts of bean: G. max black (B1), V. an-
gularis (B2), Canavalia spp. (B3), C. arietinum (B4), G. max yellow
(B5), and C. ensiformis (B6). Each assay was carried out with 200 mg
FW sample/mL concentration.
A
Data are mean SD values of -
amylase inhibitory activity (autoclaved) of three replicated samples.
a–c
Values are mean SD values of -glucosidase inhibitory activity
(autoclaved) of three replicated samples. Columns with different let-
ters indicate statistically significant differences among groups at P
.05. The Pearson’s correlation coefficient between -amylase and -
glucosidase inhibitory activity is 0.25. The Pearson’s correlation co-
efficient between DPPH scavenging activity and inhibitory activity
against the two enzymes (-amylase and -glucosidase) are 0.3341
and 0.2568, respectively.
and B6 had the highest ACE inhibitory activity (87%), fol-
lowed by B3 (83%), B5 (63%), and B4 (28%) (Fig. 8). All
bean types (except B4) had high ACE inhibitory activity.
Water extracts of maize. Samples M2, M5, and M6 had
the highest ACE inhibitory activity, but overall activity was
uniformly lower than that observed for several beans and
pumpkin (Fig. 9). ACE inhibitory activity of the maize sam-
ples was moderately correlated with the total soluble phe-
nolic content (Fig. 9) (the Pearson’s correlation coefficient
was 0.5258) and was also correlated well with the -glu-
cosidase inhibitory activity (Figs. 6 and 9) (the Pearson’s
correlation coefficient was 0.8795).
DISCUSSION
Total phenolics and antioxidant activity
Inhibition of DPPH radical formation by pumpkin extract
samples was not proportional to the concentration of total
soluble phenolics (Fig. 1). The Pearson’s correlation coef-
ficients between DPPH scavenging activity and total phe-
nolic content (autoclaved and nonautoclaved samples) were
0.21 and 0.2098, respectively. As reported previously, this
could be due to variation in content of specific high water-
soluble phenolics such as protocatechuic acid.
17
It is likely
that the profile of individual phenolics and nonphenolic an-
tioxidants such as carotenoids or xanthophylls in the extracts
may also be important in contributing to the antioxidant ac-
tivity rather than only the total phenolics content. In the case
of bean samples the nonautoclaved extracts such as B2 (V. an-
gularis), B3 (C. arietinum), B5 (G. max), and B6 (C. ensi-
formis) had higher total soluble phenolics than autoclaved ex-
tracts. The antioxidant activity of all autoclaved bean extracts
was generally more proportional to the concentration of solu-
ble total phenolics than other species evaluated in this study
(Fig. 2) (the Pearson’s correlation coefficient was 0.73). In-
terestingly, in the case of maize samples, except for M7 ex-
tract, antioxidant activity of all autoclaved maize extracts was
much higher than that of the nonautoclaved extracts evaluated.
Generally, pigmented maize samples had slightly higher total
soluble phenolics and antioxidant activity (Fig. 3). This result
indicates that thermal processing such as autoclaving/cooking
and pigmentation are important factors to enhance antioxidant
activity of maize-based foods. This may be due to the com-
position and profile modification of individual soluble pheno-
lics in the extracts due to likely polymerization from thermal
treatments. Furthermore, other components of pigments may
contribute to antioxidant activity.
272 KWON ET AL.
FIG. 6. Comparison of porcine pancreatic -amylase, rat intestinal
-glucosidase inhibitory activity, and DPPH radical scavenging ac-
tivity of autoclaved extracts of maize: yellow strained (M1), small yel-
low (M2), black pigmented (M3), white blotched (M4), light red (M5),
small red (M6), and dark red (M7). Each assay was carried out with
200 mg FW sample/mL concentration.
A–D
Data are mean SD val-
ues of -amylase inhibitory activity (autoclaved) of three replicated
samples.
a–f
Data are mean SD values of -glucosidase inhibitory
activity (autoclaved) of three replicated samples. Columns with dif-
ferent letters indicate statistically significant differences among groups
at P.05. The Pearson’s correlation coefficient between -amylase
and -glucosidase inhibitory activity is 0.69. The Pearson’s correla-
tion coefficients between DPPH scavenging activity and inhibitory ac-
tivity against the two enzymes (-amylase and -glucosidase) are
0.2289 and 0.0364, respectively.
FIG. 7. Comparison of rabbit lung ACE and total soluble phenolic
content of pumpkin extracts: round yellow (P1), striped yellow green
(P2), striped round yellow green (P3), elongated brown (P4), round
orange (P5), spotted orange green (P6), and round green (P7). Each
assay was carried out with 200 mg FW sample/mL concentration.
a–d
Data are mean SD values of three replicated samples. Columns
with different letters indicate statistically significant differences
among groups at P.05. The Pearson’s correlation coefficient be-
tween ACE inhibitory activity and total phenolic content is 0.8934.
Amylase/glucosidase inhibition
Each pumpkin type had different enzyme inhibitory ac-
tivity (Fig. 4), and these different responses to mammalian
enzyme sources will be an important factor for designing in
vivo studies using animal models such as the rat and mouse,
where the mammalian in vitro enzyme response will be more
preferable. Among various autoclaved pumpkin extracts P3,
P5, and P7 would be most preferable because of moderate
-glucosidase inhibitory activity with moderate -amylase
inhibitory activity that can potentially reduce side effects of
undigested starch linked to high -amylase inhibitory ac-
tivity. Most bean types have high -amylase inhibitory ac-
tivity and low -glucosidase inhibitory activity (Fig. 5) and
therefore should be carefully used for managing glycemic
response because of the potential side effects of undigested
starch. Too much of an unbalanced bean diet would likely
have abdominal side effects such as flatulence and diarrhea
arising from excessive inhibition of pancreatic -amylase.
This also can lead to abnormal bacterial fermentation of
undigested starch in the colon, enhancing the formation of
gases and butyrate.
40,41
In the case of maize, M1, M2, and
M5 showed comparable inhibition of -glucosidase but also
had much higher inhibitory activity against -amylase,
which also indicates the potential for side effects from undi-
gested starch. However, M3, M4, M6, and M7 extracts of
maize showed comparable inhibition of -glucosidase but
have less inhibitory activity against porcine pancreatic -
amylase. These extracts also had moderate antioxidant ac-
tivity (Fig. 6). Therefore, M3, M4, M6, and M7 extracts of
maize, which have moderate antioxidant activity, have the
potential for the management of glycemic response in DM
patients and warrant further animal studies. This strategy
would likely have lower abdominal side effects arising from
excessive inhibition of pancreatic -amylase.
Based on these results the -glucosidase inhibitory activity
and moderate antioxidant activity in select pumpkin (P3, P5,
and P7) and maize (M3, M4, M6, and M7) types would be
helpful to manage glucose uptake and the glucose-induced in-
creased levels of mitochondrial ROS linked to hyperglycemia.
Therefore, it is not surprising that select pumpkin types when
used in a diverse diet on a consistent basis may have poten-
tial benefits for improving NIDDM and the pumpkin has been
recommended by the U.S. Department of Agriculture Food
Guide Pyramid called “My Pyramid”
33
and the Diabetes Food
Pyramid of the American Diabetes Association.
34
ACE inhibition
Two pumpkin extracts, P5 and P6, had high ACE in-
hibitory activity and also had a high content of total phe-
nolics along with moderate to high -amylase/-glucosidase
inhibitory activity and moderate antioxidant activity (Figs.
1, 4, and 7). These in vitro results indicate that P5 and P6
with high ACE inhibitory activity and -glucosidase in-
hibitory activity and moderate antioxidant activity have the
potential to manage hyperglycemia-induced hypertension
and oxidation-linked vascular complications and provide a
strong biochemical rationale for further animal and clinical
DIET CONTROL FOR DIABETES AND HYPERTENSION 273
FIG. 8. Comparison of rabbit lung ACE and total soluble phenolic
content of bean extracts: G. max black (B1), V. angularis (B2),
Canavalia spp. (B3), C. arietinum (B4), G. max yellow (B5), and C.
ensiformis (B6). Each assay was carried out with 200 mg FW sam-
ple/mL concentration.
a–c
Data are mean SD values of ACE in-
hibitory activity of three replicated samples. Columns with different
letters indicate statistically significant differences among groups at
P0.05.
FIG. 9. Comparison of rabbit lung ACE and total soluble phenolic
content of maize extracts: yellow strained (M1), small yellow (M2),
black pigmented (M3), white blotched (M4), light red (M5), small red
(M6), and dark red (M7). Each assay was carried out with 200 mg
FW sample/mL concentration.
a–c
Data are mean SD values of three
replicated samples. Columns with different letters indicate statistically
significant differences among groups at P.05.
studies. All bean types [except B4 (G. max, yellow)] had
high ACE inhibitory activity. The ACE inhibitory activity
of autoclaved bean extracts was not proportional to the con-
centration of the total soluble phenolics (Fig. 8) (the Pear-
son’s correlation coefficient was 0.7015). The lack of cor-
relation of the ACE inhibitory activity with phenolic content
may suggest nonphenolic small peptide compounds may the
bioactive factors contributing to the total ACE inhibitory ac-
tivity.
42
ACE inhibitory activity of the maize extracts cor-
related well with the -glucosidase inhibitory activity (Figs.
6 and 9) (the Pearson’s correlation coefficient was 0.8795).
Based on these results, correlation between ACE inhibitory
activity and -glucosidase inhibitory activity of maize ex-
tracts showed that specific types of maize are potentially
useful to manage hyperglycemia and associated macrovas-
cular complication such as hypertension.
IMPLICATIONS
Hyperglycemia, a condition characterized by an abnormal
postprandial increase of blood glucose level, has been linked
to the onset of NIDDM and associated vascular complications.
This study provides insights that phenolics and associated free
radical scavenging-linked antioxidant activity of select plant
foods of traditional diet of indigenous communities in North
America such as specific varieties of pumpkin, bean, and maize
have the potential for in vitro -glucosidase inhibition. This
indicates the potential to reduce glucose absorption in the in-
testine. Additionally, several pumpkin and maize varieties in-
dicate high -glucosidase inhibitory activity with moderate -
amylase inhibitory activity. Such combinations found in a
diversity of traditional plant foods have the potential to con-
trol glucose absorption and not generate undigested starch-
linked side effects. Undigested starch-linked complications
from drug therapy result in the abnormal bacterial fermenta-
tion of undigested carbohydrates in the colon.
15,16
A further
benefit from this study is the indication that some select types
of bean and pumpkin have high ACE inhibitory potential,
which indicates potential antihypertension activity, and there-
fore these foods can be targeted for combating this macrovas-
cular complication of hyperglycemia.
Hyperglycemia-induced microvascular complications are
likely from oxidative dysfunction from mitochondrial ROS.
Insights from this study indicate that select types of pump-
kin, bean, and maize varieties have moderate phenolic phy-
tochemical content with moderate free radical scavenging-
linked antioxidant activity and therefore have the potential
to contribute to the reduction of hyperglycemia-induced mi-
crovascular complications. The above benefits taken to-
gether indicate the potential of traditional diet combinations
of maize, beans, and pumpkin to reduce hyperglycemia and
associated macro- and microvascular complications and sup-
port the evidence that diets rich in fruits and vegetables are
associated with lower incidences of oxidation-linked dis-
eases such as diabetes and cardiovascular disease.
27–32
Based on these in vitro results, traditional plant foods of
select indigenous communities of North America have the
potential to contribute as a useful dietary strategy for con-
trolling postprandial hyperglycemia and hypertension linked
to NIDDM and to reduce associated microvascular compli-
cations from oxidative stress. This in vitro study therefore
further provides the strong biochemical rationale for dietary
recommendations of the diabetes food guide pyramid sug-
gested by the U.S. Department of Agriculture
33
and the
American Diabetes Association.
34
This also provides the sci-
entific basis for further in vivo rat and human clinical stud-
ies with low toxicity risk.
REFERENCES
1. Jackson Y: Nutrition in American Indian health: past, present and
future. J Am Diet Assoc 1986;86:1561–1565.
2. Young TK: Diabetes mellitus among native Americans in Canada
and the United States. An epidemiological review. Am J Hum Biol
1993;35:399–413.
3. Story M, Evans M, Fabsitz RR, Clay TE, Rock BH, Broussard B:
The epidemic of obesity in American Indian communities and the
need for childhood obesity-prevention programs. Am J Clin Nutr
1999;69(Suppl):747S–754S.
4. Harriet VK, Olivier P: Dietary change and traditional food sys-
tems of indigenous peoples. Annu Rev Nutr 1996;16:417–442.
5. Harris MI, Zimmer P: Classification of diabetes mellitus and other
categories of glucose intolerance. In: International Textbook of
Diabetes Mellitus (Alberti KGGM, De Fronzo RA, Keen H, Zim-
mer P, eds.), John Wiley, London, 1992, pp. 3–18.
6. Nishikawa T, Edelstein D, Du XL, Yamagishi S, Matsumura T,
Kaneda Y, Yorek MA, Beebe D, Oates PJ, Hammes HP, Giardino
I, Brownlee M: Normalizing mitochondrial superoxide produc-
tion blocks three pathways of hyperglycaemic damage. Nature
2000;404:787–790.
7. Kawamura M, Heinecke JW, Chait A: Pathophysiological con-
centrations of glucose promote oxidative modification of low den-
sity lipoprotein by a superoxide dependent pathway. J Clin Invest
1994;94:771–778.
8. Brownlee M: The pathobiology of diabetic complications. Dia-
betes 2005;54:1615–1625.
9. Kaiser N, Sasson S, Feener EP, Boukobza N, Higashi S, Moller
DE: Differential regulation of glucose transport and transporters
by glucose in vascular endothelial and smooth muscle cells. Di-
abetes 1993;42:80–89.
10. Heilig CW, Concepcion LA, Riser BL, Freytag SO, Zhu M, Cortes
P: Overexpression of glucose transporters in rat mesangial cells
cultured in a normal glucose milieu mimics the diabetic pheno-
type. J Clin Invest 1995;96:1802–1814.
11. Lee AY, Chung SS: Contributions of polyol pathway to oxida-
tive stress in diabetic cataract. FASEB J 1999;13:23–30.
12. Puls W, Keup U, Krause H, Thomas PG, Hoffmeister F: Glu-
cosidase inhibition: a new approach to the treatment of diabetes,
obesity and hyperlipoproteinemia. Naturwissen Schaften 1977;64:
536–537.
13. Horii S, Fukasse K, Matsuo T, Kameda K, Asano N, Masui Y:
Synthesis and -
D
-glucosidase inhibitory activity of N-substituted
valiolamine derivatives as potent oral antidiabetic agents. J Med
Chem 1987;29:1038–1046.
274 KWON ET AL.
14. Bischoff H, Puls W, Krause HP, Schutt H, Thomas G: Pharma-
cological properties of the novel glucosidase inhibitors BAY m
1099 (miglitol) and BAY o 1248. Diabetes Res Clin Pract 1985;
1:53–57.
15. Puls W, Keup U: Metabolic studies with an amylase. In: Recent
Advances in Obesity Research, Vol. 3 (Howard A, ed.), Newman,
London, 1975, p. 391.
16. Bischoff H: Pharmacology of glucosidase inhibitor. Eur J Clin
Invest 1994;24:3–10.
17. Kwon Y-I, Vattem DV, Shetty K: Evaluation of clonal herbs of
Lamiaceae species for management of diabetes and hypertension.
Asia Pac J Clin Nutr 2006;15:107–118.
18. Skeggs LT, Khan JR, Shumway NP: The preparation and func-
tion of the hypertension-converting enzyme. J Exp Med 1956;
103:295–299.
19. Lieberman J: Elevation of serum angiotensin I-converting enzyme
(ACE) level in sarcoidiosis. Am J Med 1975;59:365–372.
20. Erdos EG, Skidgel RA: The angiotensin I-converting enzyme. Lab
Invest 1987;56:345–348.
21. Johnston JI, Franz V: Renin-angiotensin system: a dual tissue and
hormonal system for cardiovascular control. J Hypertens 1992;10:
13–26.
22. Suda I, Oki T, Masuda M, Nishiba Y, Furuta S, Matsugano K,
Sugita K, Tarahara N: Direct absorption of acylated anthocyanin
in purple-fleshed sweet potato into rats. J Agric Food Chem
2002;50:1672–1676.
23. Suda I, Oki T, Masuda M, Kobayashi M, Nishiba Y, Furuta S:
Physiological functionality of purple-fleshed sweet potato con-
taining anthocyanins and their utilization in foods. Jpn Agric Res
Q2003;37:167–173.
24. Mann J: Secondary metabolism. In: Oxford Chemistry Series,
Clarendon Press, Oxford, 1978, p. 116.
25. Bravo L: Phenolic phytochemicals: chemistry, dietary sources, me-
tabolism, and nutritional significance. Nutr Rev 1998;56:317–333.
26. Crozier A, Burns J, Aziz AA, Stewart AJ, Rabiaz HS, Jenkins GI,
Edwards CA, Lean MEJ: Antioxidant flavonols from fruits, veg-
etables and beverages: measurements and bioavailability. Biol Res
2000;33:79–88.
27. Shetty K: Biotechnology to harness the benefits of dietary phe-
nolics; focus on Lamiaceae. Asia Pac J Clin Nutr 1997;6:162–171.
28. Shetty K: Phytochemicals: biotechnology of phenolic phyto-
chemicals for food preservatives and functional food applications.
In: Encyclopedia of Food Science and Technology, 2nd ed. (Fran-
cis FJ, ed.), Wiley, New York, 1999, pp. 1901–1909.
29. Shetty K: Biosynthesis of rosmarinic acid and applications in med-
icine. J Herbs Species Med Plants 2001;8:161–181.
30. Shetty K, Labbe RL: Foodborne pathogens, health and
role dietary phytochemicals. Asia Pac J Clin Nutr 1998;7:
270–276.
31. Block G, Patterson B, Subar A: Fruit, vegetables, and cancer pre-
vention: a review of the epidemiological evidence. Nutr Cancer
1992;18:1–29.
32. Serdula MK, Byers MH, Simoes E, Mendlein ML, Coates RJ: The
association between fruit and vegetable intake and chronic dis-
ease risk factors. Epidemiology 1996;7:161–165.
33. U.S. Department of Agriculture: Mypyramid, inside the pyramid.
http://www.mypyramid.gov/pyramid/grains.html (accessed Au-
gust 2006).
34. American Diabetes Association: Using the diabetes pyramid.
http://www.diabetes.org/nutrition-and-recipes/nutrition/food
pyramid.jsp (accessed August 2006).
35. Sauer J, Kaplan L: Canavalia beans American prehistory. Am An-
tiquity 1969;34:417–424.
36. Shetty K, Curtis OF, Levin RE, Wikowsky R, Ang W: Preven-
tion of verification associated with in vitro shoot culture of
oregano (Origanum vulgare) by Pseudomonas spp. J Plant Phys-
iol 1995;147:447–451.
37. Worthington V, ed.: Alpha amylase. In: Worthington Enzyme Man-
ual, Worthington Biochemical Corp., Freehold, NJ, 1993, pp. 36–41.
38. Worthington V, ed.: Maltase--glucosidase. In: Worthington En-
zyme Manual, Worthington Biochemical Corp., Freehold, NJ,
1993, p. 261.
39. McCue P, Kwon Y-I, Shetty K: Anti-amylase, anti-glucosidase
and anti-angiotensin I-converting enzyme potential of selected
foods. J Food Biochem 2005;29:278–294.
40. Englyst HN, Hay S, Macfarkane GT: Polysaccharide break-down
by mixed populations of human faecal bacteria. FEMS Microbiol
Ecol 1987;95:163–17l.
41. Weaver GA, Tangel CT, Krause JA, Parfitt MM, Jenkins PL,
Rader JM, Lewis BA, Miller TL, Wolin MJ: Acarbose enhances
human colonic butyrate. J Nutr 1997;127:717–723.
42. Lo WM, Li-Chan EC: Angiotensin converting enzyme inhibitory
peptides from in vitro pepsin-pancreatin digestion of soy protein.
J Agric Food Chem 2005;53:3369–3376.
DIET CONTROL FOR DIABETES AND HYPERTENSION 275
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