JOURNAL OF MEDICINAL FOOD

J Med Food 12 (4) 2009, 704–713

Full Communication
# Mary Ann Liebert, Inc. and Korean Society of Food Science and Nutrition
DOI: 10.1089=jmf.2008.0122

Evaluation of Indigenous Grains from the Peruvian Andean Region

for Antidiabetes and Antihypertension Potential Using In Vitro Methods
Lena Galvez Ranilla,1 Emmanouil Apostolidis,2 Maria Ines Genovese,1
Franco Maria Lajolo,1 and Kalidas Shetty2
1
Laboratório de Quı´mica, Bioquı´mica e Biologia Molecular de Alimentos, Departamento de Alimentos e Nutrição
Experimental, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, Brazil;
and 2Department of Food Science, Chenoweth Laboratory, University of Massachusetts, Amherst, Massachusetts, USA

ABSTRACT The health-relevant functionality of 10 thermally processed Peruvian Andean grains (five cereals, three
pseudocereals, and two legumes) was evaluated for potential type 2 diabetes-relevant antihyperglycemia and antihypertension
activity using in vitro enzyme assays. Inhibition of enzymes relevant for managing early stages of type 2 diabetes such as
hyperglycemia-relevant a-glucosidase and a-amylase and hypertension-relevant angiotensin I-converting enzyme (ACE) were
assayed along with the total phenolic content, phenolic profiles, and antioxidant activity based on the 1,1-diphenyl-2-
picrylhydrazyl radical assay. Purple corn (Zea mays L.) (cereal) exhibited high free radical scavenging-linked antioxidant
activity (77%) and had the highest total phenolic content (8
1 mg of gallic acid equivalents=g of sample weight) and
a-glucosidase inhibitory activity (51% at 5 mg of sample weight). The major phenolic compound in this cereal was proto-
catechuic acid (287
15 mg=g of sample weight). Pseudocereals such as Quinoa (Chenopodium quinoa Willd) and Kañiwa
(Chenopodium pallidicaule Aellen) were rich in quercetin derivatives (1,131
56 and 943
35 mg [expressed as quercetin
aglycone]=g of sample weight, respectively) and had the highest antioxidant activity (86% and 75%, respectively). Andean
legumes (Lupinus mutabilis cultivars SLP-1 and H-6) inhibited significantly the hypertension-relevant ACE (52% at 5 mg
of sample weight). No a-amylase inhibitory activity was found in any of the evaluated Andean grains. This in vitro
study indicates the potential of combination of Andean whole grain cereals, pseudocereals, and legumes to develop effec-
tive dietary strategies for managing type 2 diabetes and associated hypertension and provides the rationale for animal and
clinical studies.

KEY WORDS:  a-amylase inhibitory activity  Andean grains  angiotensin I-converting enzyme  antioxidant activity
 a-glucosidase inhibitory activity  hypertension  phenolic phytochemicals  type 2 diabetes

INTRODUCTION Andean grains have been cultivated for thousands of

years since pre-Colombian times and include the ‘‘pseudo-
T he Andean region forms a long chain of moun-
tains over 7,000 km long that stretch from Southern
Venezuela to the South of Chile. The longest north–south
cereals’’ Quinoa (Chenopodium quinoa Willd.), Kañiwa
(Chenopodium pallidicaule Aellen), and Amaranth (Amar-
anthus caudatus L.), the Andean lupin or ‘‘Tarwi’’ (Lupinus
mountain range in the world, the Andes encompass a tre-
mutabilis Sweet), and several pigmented varieties of corns
mendous range of ecosystems and are home to a rich variety
(Zea mays L.). The purple corn is the most representative
of plant and animal species and human communities.1 For
and consumed among these cereals. Nutritionally, Andean
example, in Peru, there are 25,000 species of plants, which
pseudocereals are often closer to the ideal protein balance
corresponds to 7–10% of the total species existing in the
than any other common cereal grains, being at least equal to
world. Of this high number, 38 species are domesticated,
milk in protein quality. In particular, they have high lysine
including tubers, roots, grains, fruits, and vegetables,
content. Therefore, in order to obtain a suitable amino acid
whereas a large number of medicinal and ornamental plants
profile to cover the needs of the human diet, it is not nec-
still have not been domesticated.2
essary to combine them with other crops such as legumes,
rich in lysine but lacking in methionine and cysteine, as
Manuscript received 7 May 2008. Revision accepted 10 December 2008. happens for other cereals.3 Further, the Andean legume
‘‘Tarwi’’ (L. mutabilis Sweet) has the highest protein and
Address correspondence to: Kalidas Shetty, Department of Food Science, Chenoweth
Laboratory, University of Massachusetts, Amherst, MA 01003, USA, E-mail: kalidas
lipid content of all the domesticated species of lupin, and its
@foodsci.umass.edu protein value is comparable to that present in soybeans.4

704
 ANDEAN GRAINS AND TYPE 2 DIABETES POTENTIAL
Andean crops have shown a high level of resistance to
drought, frost, salinity, pests, and diseases.2 Therefore, An-
dean grains may be promising with regard to their diversity
of phenolic secondary metabolites (phytochemicals) syn-
thesized to counteract such adverse climatic and growing
conditions. Phytochemicals and in particular phenolic me-
tabolites are bioactive non-nutrient plant compounds found
in fruits, vegetables, grains, and other plant foods. These
metabolites not only are involved in various stress and de-
fense responses in plants (primary function)5 but also are
currently being linked to the reduction of the risk of major
chronic diseases when consumed via diet.6 For example,
plant polyphenols, a large group of natural antioxidants, are
important candidates linked to the protective effects of
vegetables and fruits against cancer and cardiovascular
diseases.7
Among Andean grains, flavonoids such as kaempferol
and quercetin glycosides were found in the ‘‘Kancolla’’
variety of C. quinoa seeds.8 Similarly, Rastrelli et al.9
identified new flavonol triglycosides in seeds of C. pallidi-
caule. Further, the Andean purple corn had shown inter-
esting health-related properties such as antioxidant,10
antimicrobial,11 and prevention of obesity and amelioration
of hyperglycemia in mice,12 which were mainly related to its
rich content of anthocyanins. Further, other phenolic com-
pounds such as phenolic acids and flavonols, also identified
in purple corn, showed potent antimutagenic properties
in vitro.13 These have potential for managing chronic dis-
ease emergence resulting from globalization and narrower
choices of the food supply that are high in refined and sol-
uble carbohydrate sources.
The effects of globalization and urbanization on the food
supply have influenced dietary patterns and lifestyle be-
haviors among traditional population groups throughout the
world.14 In a recent study carried out in Peru, the migration
of Peruvian Amerindian women from the rural Cuzco region
(Andes) to urban areas in Lima (coast), resulting in different
degrees of physical activity (high in Cuzco, low in Lima)
and altitudes of living (highland in Cuzco, lowland in Lima),
was associated with the increased risk for obesity and car-
diovascular diseases.15 In Latin America, as elsewhere,
traditional food patterns rich in complex carbohydrates,
micronutrients, fiber, and phytochemicals are being replaced
with diets high in animal products and refined carbohydrates
Table 1. Pseudocereals, Cereals, and Legumes from the Andean Region
Code Common name
1 Kañiwa
2 Red Quinoa
3 Kiwicha
4 Corn 1 (purple pigmented)
5 Corn 2 (yellow, purple-red mottled)
6 Corn 3 (yellow, red blotched)
7 Corn 4 (half yellow-half red)
8 Corn 5 (purple mottled)
9 Tarwi SLP-1
10 Tarwi H-6
705
and oils, situations that are leading to the rapid increase of
obesity and chronic diseases such as type 2 diabetes, hy-
pertension, and cardiovascular diseases.14
Type 2 diabetes is a condition associated with elevated
glycemic index, insulin resistance, and eventually declining
pancreatic function that results in absolute or relative insulin
deficiency. The importance of poor glycemic index man-
agement leading to insulin resistance as part of the natural
history of diabetes is its association with metabolic syn-
drome: the human condition characterized by the presence of
coexisting traditional risk factors for cardiovascular disease,
such as hypertension, dyslipidemia, glucose intolerance, and
obesity, in addition to nontraditional cardiovascular disease
risk factors, such as inflammatory processes and abnormal-
ities of the blood coagulation system.16 Managing hyper-
glycemia and hypertension through a whole grain diet could
be effective on early-stage management of type 2 diabetes.
Andean grains provide a potential resource for South
America and other regions of the developing world because
their exceptional nutritional characteristics with the poten-
tial for alleviating malnutrition. Specific research on phe-
nolic bioactive compound-linked health benefits from these
grains may also show their potential for managing hyper-
glycemia and hypertension linked to type 2 diabetes and
related cardiovascular complications.
Based on the above rationale, the objective of this study
was to investigate the health-relevant functionality of phe-
nolic phytochemicals related to type 2 diabetes-relevant
antihyperglycemia and antihypertension potential of An-
dean grains using in vitro enzyme models. Therefore, phe-
nolic profiles, antioxidant activity, and in vitro inhibition of
enzymes relevant for managing early stages of type 2 dia-
betes such as hyperglycemia-relevant a-glucosidase and a-
amylase and hypertension-relevant angiotensin I-converting
enzyme (ACE) were investigated.
MATERIALS AND METHODS
Materials
Dry grains including pseudocereals and cereals (corn)
were obtained from an Andean local market in Arequipa,
Peru. Seeds of L. mutabilis Sweet H-6 and SLP-1 were
provided by the Legume and Cereal Program of Agraria
University (Lima, Peru) (Table 1).
Scientific name Type
C. pallidicaule Aellen Pseudocereal
C. quinoa Willd Pseudocereal
A. caudatus L. Pseudocereal
Z. mays L. Cereal
Z. mays L. Cereal
Z. mays L. Cereal
Z. mays L. Cereal
Z. mays L. Cereal
L. mutabilis Sweet Legume
L. mutabilis Sweet Legume
706
Porcine pancreatic a-amylase (EC 3.2.1.1), baker’s yeast
a-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
purchased from Sigma.
Extract preparation
Dry grains (10 g) were added to 15 mL of distilled water
and soaked overnight at 258C in order to facilitate the sub-
sequent thermal processing. Pseudocereals and legumes
were thermally processed by autoclaving at 1208C for 10
minutes. A longer autoclaving time was required (20 min-
utes) in the case of corn samples. After the thermal treat-
ment, 85 mL of distilled water was added to the cooked
grains, and the mixture was homogenized for 1 minute using
a Waring
laboratory blender (Waring Laboratory, Tor-
rington, CT) set on ‘‘high’’ speed. The homogenate was
centrifuged at 9,300 g for 30 minutes, and the pH of the
supernatant was corrected to 6–8. An aliquot of the final
extract was recentrifuged at 1,157 g (centrifuge model
5415D, rotor F45-24-11, Eppendorf, Westbury, NY) for 10
minutes before each in vitro assay. Extractions were per-
formed in duplicate.
Total phenolics assay
The total phenolics were determined by the Folin-
Ciocalteu method as modified by Shetty et al.17 In brief,
1 mL of the grain 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% Na2CO3 was added to the reaction mixture and
allowed to stand for 60 minutes. The absorbance was read at
725 nm. The standard curve was established using various
concentrations of gallic acid in 95% ethanol, and results
were expressed as mg of gallic acid equivalents=g of sample
weight.
Antioxidant activity by 1,1-diphenyl-2-picrylhydrazyl
radical (DPPH) inhibition assay
The DPPH scavenging activity was determined by an
assay modified by Kwon et al.18 To 1.25 mL of 60 mM
DPPH in 95% ethanol, 250 mL of each grain extract was
added, and the decrease in absorbance was monitored after 1
minute at 517 nm. The percentage of inhibition was calcu-
lated by:
A517 control  A517 extract
Inhibition ¼ · 100
A517 control
a-Amylase inhibition assay
The a-amylase inhibitory activity was determined by an
assay modified from the Worthington Enzyme Manual.19 A
total of 500 mL of each grain extract and 500 mL of 0.02 M
sodium phosphate buffer (pH 6.9 with 0.006 M NaCl) con-
taining a-amylase solution (0.5 mg=mL) were incubated at
RANILLA ET AL.
258C for 10 minutes. After preincubation, 500 mL of a 1%
starch solution in 0.02 M sodium phosphate buffer (pH 6.9
with 0.006 M NaCl) was added to each tube at timed inter-
vals. The reaction mixtures were then incubated at 258C for
10 minutes. The reaction was stopped with 1.0 mL of dini-
trosalicylic acid color reagent. The test tubes were then in-
cubated in a boiling water bath for 5 minutes and cooled to
room temperature. The reaction mixture was then diluted
after adding 15 mL of distilled water, and absorbance was
measured at 540 nm. Sample blanks (buffer instead of en-
zyme solution) and a control (buffer in place of sample
extract) were included as well. The a-glucosidase inhibitory
activity was calculated according to the equation below:
A540 control  A540 extract
Inhibition ¼ · 100
A540 control
a-Glucosidase inhibition assay
A modified version of the assay described by the Wor-
thington Enzyme Manual20 was followed.21 A volume of
50 mL of grain extract diluted with 50 mL of 0.1 M potassium
phosphate buffer (pH 6.9) and 100 mL of 0.1 M potassium
phosphate buffer (pH 6.9) containing a-glucosidase solution
(1.0 U=mL) was incubated in 96-well plates at 258C for 10
minutes. After preincubation, 50 mL of 5 mM p-nitrophenyl-
a-d-glucopyranoside solution in 0.1 M potassium phosphate
buffer (pH 6.9) was added to each well at timed intervals.
The reaction mixtures were incubated at 258C for 5 minutes.
Before and after incubation, absorbance readings were re-
corded at 405 nm by a microplate reader (Thermomax;
Molecular Devices Co., Sunnyvale, CA) and compared to a
control that had 50 mL of buffer solution in place of the
extract. The a-glucosidase inhibitory activity was expressed
as percentage of inhibition and was calculated as follows:
DA405 control  DA405 extract
Inhibition ¼ · 100
DA405 control
ACE inhibition assay
ACE inhibition was performed by a method modified by
Kwon et al.18 A volume of 50 mL of grain extract was in-
cubated with 200 mL of 0.1 M NaCl-borate buffer (0.3 M
NaCl, pH 8.3) containing 2 mU of ACE solution at 258C for
10 minutes. After preincubation, 100 mL of a 5.0 mM sub-
strate (hippuryl-histidyl-leucine) solution was added to the
reaction mixture. Test solutions were incubated at 378C for
1 hour. The reaction was stopped with 150 mL of 0.5 N HCl.
The hippuric acid formed was detected and quantified by
high-performance liquid chromatography (HPLC). A vol-
ume of 5 mL of sample was injected using an Agilent ALS
1100 autosampler into an Agilent 1100 series HPLC appa-
ratus (Agilent Technologies, Palo Alto, CA) equipped with a
DAD 1100 diode array detector. The solvents used for the
gradient were (A) 10 mM phosphoric 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,
 ANDEAN GRAINS AND TYPE 2 DIABETES POTENTIAL 707

100
18 minutes). The analytical column used was Agilent 10
Total Phenolics
Zorbax SB-C18 (2504.6 mm inner diameter) with packing 9 DPPH scavenging activity
a
material of 5 mm particle size at a flow rate of 1 mL=minute
8 80
at room temperature. During each run the absorbance was

Total phenolics (mg Gallic acid/g)

DPPH Scavenging Activity (%)

recorded at 228 nm, and the chromatogram was integrated 7

using the Agilent Chemstation enhanced integrator for de- 6 60

tection of liberated hippuric acid. Pure hippuric acid was
5
used to calibrate the standard curve and retention time. The b b

percentage of inhibition was calculated considering the area 4 40

of the hippuric acid peak according to the equation below: c
3 d

Areacontrol  (Areasample  Areasample blank ) 2 20

Inhibition ¼ · 100 e f e
f ef
Areacontrol  Areablank 1

0 0
HPLC analysis of phenolic profiles 1 2 3 4 5 6 7 8 9 10

The grain extracts (2 mL) were filtered (pore size,
0.2 mm). A volume of 5 mL of sample was injected using an
Agilent ALS 1100 autosampler into an Agilent 1100 series
HPLC apparatus equipped with a DAD 1100 diode array
detector. The solvents used for gradient elution were (A)
10 mM phosphoric acid (pH 2.5) and (B) 100% methanol.
The methanol concentration was increased to 60% for the
first 8 minutes and to 100% over the next 7 minutes, then
decreased to 0% for the next 3 minutes, and was main-
tained for the next 7 minutes (total run time, 25 minutes).
The analytical column used was Agilent Zorbax SB-C18
(2504.6 mm inner diameter) with packing material of 5 mm
particle size at a flow rate of 1 mL=minute at room tem-
perature. During each run the absorbance was recorded at
306, 333, and 525 nm, and the chromatogram was integrated
using the Agilent Chemstation enhanced integrator. Pure
standards of quercetin aglycone and protocatechuic and
p-coumaric acids in 100% methanol were used to calibrate
the standard curves and retention times.
Statistical analysis
Two extractions were performed for each sample, and all
in vitro analysis were carried out six times (n ¼ 12). In the
case of HPLC analysis, the experiments were performed
at least in triplicates. Results were expressed as mean

standard deviation values. Data were subjected to one-way
analysis of variance, with means compared using Tukey’s
test (P < 05), and Pearson correlations were calculated ac-
cording to the Statistica software package version 5.0
(StatSoft, Tulsa, OK).
RESULTS
Total phenolics, antioxidant activity,
and HPLC phenolic profiles
Figure 1 shows the total phenolic contents from Andean
pseudocereals, cereals, and legumes correlated to their
DPPH radical scavenging-linked antioxidant activity.
The total phenolic content in water extracts of cooked
Andean grains varied from 0.80
0.07 to 8
1 mg=g.
Sample 4 (purple corn) exhibited the highest total phenolic
FIG. 1. Total phenolics and DPPH radical scavenging activity of
Andean grains. Bars with different letters are significantly different
(P < .05).
content (8
1 mg of gallic acid=g), followed by the two
legumes (samples 9 and 10) (L. mutabilis Sweet) (4
0.2
and 4.1
0.3 mg=g for SLP-1 and H-6 cultivars, respec-
tively). Both Kañiwa and Quinoa (samples 1 and 2, re-
spectively), which belong to the same botanical genus
(Chenopodium), showed similar total phenolic contents
(2.3
0.1 and 2.8
0.1 mg=g, respectively). On the other
hand, the other partially pigmented corn seeds (samples
5–8) had lower phenolic levels (from 0.9
0.1 to 1.1

0.1 mg=g).
The antioxidant activity based on the DPPH radical in-
hibition assay ranged from 6% to 86% and had a moderate
but statistically significant correlation with the total phe-
nolic contents (r ¼ 0.53, P < .05) (Table 2). Although Qui-
noa (sample 2) did not have the highest total phenolic levels,
it exhibited the highest antioxidant activity among all An-
dean grains (86%). Kañiwa and purple corn were the second
best grains with high antioxidant activity (75% and 77%,
respectively).
Except for purple corn, the antioxidant activity among
corn samples was moderate (from 24% to 36%). Lupin
cultivars (sample 9 and 10) showed similar trends in their
total phenolic contents. However, the ability to inhibit the
Table 2. Pearson Correlation Coefficients for Antioxidant
Activity (AA), a-Glucosidase Inhibitory Activity (GLUC),
and ACE Inhibition of Andean Pseudocereals,
Cereals, and Legumes
AA GLUCa ACEb
TP 0.53* 0.60* 0.20
AA 0.50* 0.13
GLUC 0.09
TP, total phenolics.
*P < .05.
a
For a 5-mg sample weight (n ¼ 120).
b
For a 5-mg sample weight (n ¼ 102).
708 RANILLA ET AL.

Table 3. Phenolic Compounds Detected
by HPLC in Andean Grains
Phenolic compound (mg=g of sample weight)
Common Quercetin
Code name derivativesa
1 Kañiwa 943
35
2 Quinoa 1,131
56
4 Corn 1 ND
6 Corn 3 ND
7 Corn 4 ND
Data are mean
SD values. ND, not detected.
a
Expressed as quercetin aglycone.
DPPH free radical was higher in the H-6 cultivar (42%) than
in the SLP-1 lupin cultivar (24%). The pseudocereal known
as ‘‘Kiwicha’’ (A. caudatus L.) had the lowest antioxidant
activity (8%), which was proportional to its low total phe-
nolic content.
Individual phenolic compounds detected by HPLC-diode
array detector in water extracts of autoclaved Andean grains
are shown in Table 3. Chenopodium species (samples 1 and
2) were characteristic for their content of quercetin deriva-
tives (943–1,132 mg=g, expressed as quercetin aglycone).
Only protocatechuic acid was detected in purple corn,
whereas low levels of p-coumaric acid were found in corns
3 and 4 (samples 6 and 7, respectively).
a-Glucosidase, a-amylase, and ACE-I
inhibitory activities
All Andean grains inhibited the yeast a-glucosidase in a
dose-dependent manner (Fig. 2). The a-glucosidase inhibi-
tory activities ranged from 11% to 51% at 5 mg of sample
weight. Andean purple corn exhibited the highest inhibition
against a-glucosidase (51%), followed by corn 3 (sample 6)
(40%) and Quinoa (sample 2) (30%). Kiwicha (A. caudatus
L.), the pseudocereal with the lowest total phenolic contents
and antioxidant activity, also had the lowest a-glucosidase
inhibitory activity (11%). Legumes showed moderate a-
Protocatechuic p-Coumaric glucosidase inhibitory activities. However, the H-6 lupin
acid acid cultivar (sample 10) was more effective against the a-
ND ND glucosidase enzyme than the SLP-1 lupin cultivar (sample 9)
ND ND (26% and 19%, respectively). According to Table 2, statis-
287
15 ND tical correlations between total phenolic contents and a-
ND 22
1 glucosidase inhibitory activities and between the antioxidant
ND 34
5 activity and a-glucosidase inhibitory activities were signif-
icant (r ¼ 0.60 and r ¼ 0.50, respectively, P < .05). How-
ever, comparing both Pearson correlation coefficients, the
ability of Andean grain water extracts for inhibiting a-
glucosidase in vitro was better correlated to their total phe-
nolic contents than to their free radical scavenging-linked
antioxidant activity.
The a-amylase inhibitory activity was also screened
among Andean grains. However, no a-amylase inhibitory
activity was detected in any of the grains.
Only legumes (lupin cultivars) exhibited a significant
inhibitory effect on ACE at a dose of 5 mg of sample weight
(Fig. 3), but no ACE inhibition was detected when lower
doses of sample were assayed. Likewise, no significant
differences (P > .05) between the ACE inhibitory activities
of both lupin cultivars were observed (52% for SLP-1 and
H-6 cultivars). According to Pearson correlations (Table 2),
neither the total phenolic contents nor the antioxidant ac-
tivities were correlated with the ACE inhibitory activities of
lupin cultivars.
DISCUSSION
Total phenolic content, antioxidant activity,
and HPLC phenolic profiles
Overall, the total phenolic contents in Andean grains
were proportional to their antioxidant activity, and this fact

100 10
Glucosidase Inhibitory Activity (%): 1 mg 100 10
90 Glucosidase Inhibitory Activity (%): 2.5 mg
Glucosidase Inhibitory Activity (%): 5 mg ACE-I Inhibitory Activity (%): 5 mg
Total Phenolics Total Phenolics
80 8
Total Phenolics (mg Gallic acid/g)

80 8
70
Total Phenolics (mg Gallic acid/g)
ACE-I Inhibitory Activity (%)

60 6
% Inhibition

a
60 6
a a
50
b
40 4
c
d 40 4
d
30 e
f
20 g 2
h
20 2
10

0 0
1 2 3 4 5 6 7 8 9 10
0 0
1 2 3 4 5 6 7 8 9 10
FIG. 2. Dose-dependent changes in a-glucosidase inhibitory ac-
tivity of Andean grains (mg of sample weight) and comparison with FIG. 3. ACE-I inhibitory activity (%) of Andean grains (5 mg of
the total phenolic content. Bars with different letters are significantly sample weight) and comparison with the total phenolic content. Bars
different (P < .05). with different letters are significantly different (P < .05).
 ANDEAN GRAINS AND TYPE 2 DIABETES POTENTIAL
appeared to be correlated to the presence of specific phe-
nolic compounds detected by HPLC-diode array detector.
Among analyzed Andean grains, purple corn (Z. mays L.)
was the most relevant grain because of its highest total
phenolic contents and DPPH radical scavenging-linked an-
tioxidant activity, followed by both legumes (L. mutabilis
Sweet) and the pseudocereals Kañiwa (C. pallidicaule
Aellen) and Quinoa (C. quinoa Willd).
Purple corn, rich in anthocyanins, has been cultivated for
centuries in the Andean region. In Peru, people consume a
typical drink made by boiling purple corn called ‘‘Chicha
Morada,’’ which is believed by folklore use to improve
health.22 The average anthocyanin content of whole purple
corn from Peru was 1,640 mg=100 g of fresh weight,23
higher than fresh blueberries (73–430 mg=100 g of fresh
weight).24 Anthocyanins such as cyanidin-3-glucoside,
pelargonidin-3-glucoside, and peonidine-3-glucoside have
been identified in a purple corn extract from Peru. However,
other non-anthocyanin phenolic compounds such as quer-
cetin derivatives and ferulic and p-coumaric acid derivatives
were also detected.25 The high free radical scavenging
activity of purple corn has been associated to its high con-
tent of anthocyanins.13,26 In the current study, only proto-
catechuic acid was identified, and no anthocyanins were
detected when the autoclaved purple corn water extract was
analyzed by HPLC-diode array detector at 525 nm.
It is well known that anthocyanins are sensitive to heat
and can easily convert to the colorless chalcone form
during heating.27 Sadilova et al.28 indicated that successive
deglycosylation reactions represent the initial steps of deg-
radation of anthocyanins following thermal treatment at
pH 1, yielding the corresponding aglycones. The latter
would then be cleaved into a phenolic acid and a phenolic
aldehyde. Further, Jing and Giusti29 observed a consistent
decrease of protein at 1008C in a purple corn water extract
indicating a possible protein denaturation at high temper-
atures, which could result in anthocyanin complexation and
precipitation leading to a declining total anthocyanin con-
tents.
Cyanidin 3-glucoside was identified as the major antho-
cyanin present in purple corn together with its acylated
equivalents.25,26,30 Under neutral conditions and high tem-
peratures, cyanidin glycosides are consecutively hydro-
lyzed mainly to protocatechuic acid. However, other minor
compounds such as 2,4-dihydroxybenzoic acid and 2,4,6-
trihydroxybenzoic acid are also formed.31 Similarly, Sadi-
lova et al.28 showed that thermal treatment lead to the
formation of protocatechuic acid and phloroglucinaldehyde
as a result of cyanidin-3-glucoside deglycosylation and fur-
ther cleavage of the respective aglycone. This rationale
would explain the detection of protocatechuic acid as the
main individual phenolic compound in water extracts of
thermally processed purple corn analyzed in this study.
Currently, a purple corn extract from powders obtained by
extraction of ground purple corn in a 60% aqueous ethanol
solution, filtering, and further spray-drying is being com-
mercially sold for the nutraceutical market. This powder has
been shown to contain high levels of anthocyanins with
709
potential health benefits attributed to those compounds.13,25
However, these powder preparations failed to consider that
purple corn is still traditionally consumed as a beverage
prepared by boiling purple corn in water. According to re-
sults in this study, the high total phenolic levels and DPPH
radical scavenging-linked antioxidant activity of water ex-
tracts from thermally processed purple corn seeds could be
correlated with the presence of protocatechuic acid formed
as result of anthocyanin degradation by the autoclaving
process.
L. mutabilis Sweet, also known as ‘‘pearl lupin’’ or
‘‘Tarwi,’’ is a valuable legume protein that has been found
in the Andean region since ancient times. With regard to its
phenolic contents and antioxidant activities assayed in this
study, both cultivars (SLP-1 and H-6) showed high total
phenolic contents linked to moderate antioxidant activities,
although no specific phenolics using current methods were
detected by HPLC-diode array detector in water extracts
from thermally processed lupin seeds. No reports on the
antioxidant activity of L. mutabilis have been published to
date. However, results obtained for other lupin species in-
dicated that the antioxidant activity (measured by the
photochemiluminescence assay) had no correlation with the
total phenolic contents in seeds of Lupinus angustifolius.32
On the other hand, Tsaliki et al.33 observed a high corre-
lation between total phenolic contents and phospholipid
levels with the antioxidant activity of Lupinus albus ssp.
graecus, using the method based on the coupled oxidation
of b-carotene and linoleic acid. In this study, lupin anti-
oxidant activity based on the DPPH radical inhibition was
moderately proportional to their total phenolic contents,
suggesting the contribution of other non-phenolic com-
pounds, probably carotenoids, most likely degraded and
solubilized after thermal treatment. Carotenoids such as
zeoxanthin and b-carotene have been found in L. mutabilis
and Lupinus luteus in higher concentrations than in L.
angustifolius and L. albus.34 Further, the ability of carot-
enoids to scavenge the DPPH free radical has been previ-
ously reported.35,36
Quinoa (C. quinoa Willd) is one of the oldest crops of the
Andes, being cultivated for at least 7,000 years.2 Quinoa
produces high-protein grains under ecologically extreme
conditions, and this makes it important for the diversifica-
tion of future agricultural systems, such as in high-altitude
areas of the Himalayas and North Indian Plains.37 Kañiwa
(C. pallidicaule Aellen) provides exceptional nutritional
qualities with high iron content in leaves and seeds. Also, its
flour, like Quinoa, can be used by people with celiac disease,
who cannot eat the gluten present in wheat, rye, barley, and
oat. Kañiwa cultivation is actually limited to the harshest
and coldest areas of the Andes, above 3,800 m above sea
level.2 Dini et al.8 reported the presence of kaempferol,
quercetin glycosides, and a glycoside of vanillic acid in raw
whole flour of the Quinoa seed variety Kancolla. Similarly,
10 flavonol glycosides, mainly quercetin, isorhamnetin, and
kaempferol glycosides, have been identified in the raw whole
flour from the seeds of C. pallidicaule.9 In the current study,
only Chenopodium species exhibited high concentrations
710 RANILLA ET AL.
of quercetin derivatives, and this likely was correlated with
their high radical scavenging-linked antioxidant activities.
The strong free radical-scavenging properties of quercetin
have been attributed to its special structural arrangement
that includes an o-30 ,40 -dihydroxy moiety in the B ring and
the presence of a 2,3-double bond in combination with both
the 4-keto group and the 3-hydroxyl group in the C ring, for
electron delocalization.38 Since these grains are commonly
consumed after a cooking process, results from this study
indicate for the first time the presence of quercetin deriva-
tives in cooked grains of Quinoa and Kañiwa and their
potential free radical scavenging-linked antioxidant activity.
Less colored corn seeds have been shown to contain free
and esterified phenolic acids. Del Pozo-Insfran et al.26
identified six derivatives of ferulic acid (88.8–816 mg=g)
along with the free form (2480 mg=g), p-coumaric acid
(6.6 mg=g), two protocatechuic acid derivatives (4.2 and
14.2 mg=g), and gallic acid (3.9 mg=g) in a white corn
genotype seed after its processing into nixtamal (cooked
kernels). In addition, low concentrations of vanillic acid,
p-hydroxybenzoic acid, and protocatechuic acid have been
found in yellow corn (3.7, 1.3, and 3.0 mg=g, respectively).39
In this study, only p-coumaric acid was identified in two
partially pigmented corn seeds (yellow-red blotched and
half yellow-half red, 22 and 34 mg=g of sample weight, re-
spectively) after thermal processing by autoclaving.
a-Glucosidase and a-amylase inhibitory activity
Adult-onset type 2 diabetes is a metabolic disorder
characterized by hyperglycemia leading subsequently to
defects in insulin secretion, insulin action, or combinations.
The long-term manifestation of type 2 diabetes can result in
the development of microvascular or macrovascular com-
plications.40
Early intervention in type 2 diabetes by using carbo-
hydrate utilization inhibitors can delay the digestion of
complex carbohydrates and intestinal uptake of glucose,
thus significantly reducing postprandial glycemic and in-
sulinemic excursions.41 Potential compounds are competi-
tive inhibitors of pancreatic a-amylase for delaying ingested
carbohydrate breakdown and of intestinal brush border
a-glucosidases with the subsequent inhibition of uptake of
glucose and oligosaccharides, resulting in lower glycemic
index.42 However, because of their strong a-amylase inhi-
bition, synthetic a-glucosidase inhibitors have undesirable
side effects, such as flatulence, diarrhea, and abdominal
cramping. In addition, some of them may increase the in-
cidence of renal tumors and serious hepatic injury and acute
hepatitis.43–46 Therefore, consumption of natural dietary
inhibitors from constituents in food could be effective
therapy at reduced cost for managing postprandial hyper-
glycemia with minimal adverse reactions as compared to
modern synthetic pharmaceuticals.40
Results from the current study indicate that cooked An-
dean grains, especially purple corn, the yellow-red blotched
corn (sample 6), and Quinoa (C. quinoa Willd), have po-
tential for in vitro a-glucosidase inhibition with no effect on
pancreatic a-amylase. Such profile has interesting func-
tionality for potentially controlling intestinal glucose ab-
sorption and likely not generating side effects linked to high
pancreatic a-amylase inhibitory activity.47
The significant correlation observed between the a-
glucosidase inhibitory activity of Andean grains and their
total phenolic contents (r ¼ 0.60, P < .05) likely indicates
the potential of Andean grain-linked phenolic compounds
for inhibiting the hyperglycemia-relevant a-glucosidase
enzyme. Thus, in purple corn and Quinoa, the a-glucosidase
inhibitory activity may probably be associated with the
contents of protocatechuic acid and quercetin derivatives,
respectively. Previous reports with clonal herbal extracts
reported high a-glucosidase inhibitory activity in vitro with
specific standard phenolics. Pure protocatechuic acid on a
constant weight and constant pH basis showed 56% inhi-
bition, followed by quercetin (37%). Further, these standard
phenolics did not have any inhibitory activity against por-
cine pancreatic a-amylase.18
The potential of purple corn for the amelioration of hy-
perglycemia and prevention of obesity and cholesterol has
been correlated mainly with the presence of cyanidin-3-
glycosides.12,48 Similarly, cholesterol reduction and in-
crease of antioxidant capacity by continuous consumption
of the anthocyanin-rich Andean purple corn have been re-
cently demonstrated in hypercholesterolemic rats.49 Insights
from the present study suggest that protocatechuic acid, the
major degradation product of cyanidin-glycosides from
purple corn following thermal treatment, could have po-
tential in type 2 diabetes prevention as part of a dietary
strategy. Recently, protocatechuic acid has been shown to
exhibit other therapeutic effects such as induction of cell
death in HepG2 hepatocellular carcinoma and a neuropro-
tective effect on rotenone-induced apoptosis of PC12 (a
neuroendocrine cell line) via ameliorating the mitochondrial
dysfunction.50,51
Kiwicha (A. caudatus L.), a highly nutritious Andean
grain crop,52 was not interesting with regard to its potential
to inhibit key enzymes relevant for type 2 diabetes man-
agement. Further, cooked grains of Kiwicha showed low
total phenolic contents and the lowest antioxidant activity.
A previous study reported the a-amylase inhibitory activity
in vitro of two varieties of Kiwicha (Oscar blanco and
Victor red; 51% and 28%, respectively, at 25 mg=mL).53
However, analyses in this previous investigation were
based on methanolic extracts of ground raw seeds.53 This
would explain differences in results obtained in this study,
where a thermal processing was applied prior to analysis of
water extracts from Kiwicha grains to reflect consumption
practices.
ACE-I inhibitory activity
Hypertension is a risk factor of cardiovascular disease and
is also associated with long-term diabetes. ACE, a key en-
zyme involved in maintaining vascular tension, converts
angiotensin I to angiotensin II, a potent vasoconstrictor and
stimulator of aldosterone secretion by the adrenal gland.
 ANDEAN GRAINS AND TYPE 2 DIABETES POTENTIAL
Inhibition of ACE is considered a useful therapeutic ap-
proach in the treatment of high blood pressure in patients
both with and without diabetes.54,55 Control of hypertension
via modulation of ACE by dietary bioactive factors could be
an important strategy to manage this risk factor.56
Among Andean grains, only lupin cultivars inhibited
significantly the ACE enzyme, indicating their potential
antihypertension activity, and therefore these legumes can
be targeted for potentially combating this macrovascular
complication of hyperglycemia.
Lupin species are of fundamental nutritional importance
because their higher contents of protein comparable to that
of soybean. L. mutabilis seeds have 48% dry weight of
protein and 27% fat content, which is similar to soybeans
but higher than other legumes.57 In general, legumes that
contain a higher quantity of proteins produce higher levels
of peptides by peptic digestion or by food processing, and
such legumes show higher ACE inhibitory activities than
cereals.58 Lupin protein isolates (L. albus) and its hydro-
lysates were found to inhibit in vitro the ACE to a degree
of 0.7–43.4% (150 mg=mL of sample).59 However, the
same authors reported later that the ACE inhibitory activ-
ities of lupin protein isolates (L. albus) decreased to
0–2.72% (150 mg=mL of sample) after high temperature
treatments at 1258C.60 Results from this study indicate that
cooked Andean lupin seeds (L. mutabilis) exhibit in vitro
ACE inhibitory activity that was likely not associated with
phenolics but more likely with its protein content. The role
of protein in ACE inhibition has to be confirmed with
further studies.
CONCLUSIONS
Type 2 diabetes has reached epidemic proportions, with
the greatest increases in the developing countries of Africa,
Asia, and South America.61 In Peru, the prevalence of type
2 diabetes varies from 1% to 8%, and the regions with the
highest prevalence are Piura and Lima.62 Metabolic syn-
drome associated with type 2 diabetes is highly prevalent
among Peruvian Andean Hispanics, particularly in older
women.63 Further, type 2 diabetes was found in 17% of
Peruvian women, with the highest incidence in the poorest
areas.64 In Amerindian populations, one explanation may
be the rapid transition from hunter-gatherer societies to an
agricultural lifestyle, the transition to urbanization, and
changes of traditional unrefined healthy diets towards re-
fined high calorie diets devoid of protective phytochemi-
cals.15,65 Paradoxically, Peru is also one of the countries
with a wide diversity of plants and Andean crops poten-
tially rich in health-relevant bioactive phenolic compounds.
Research focused on functional benefits of local Andean
crops for managing the above noted diet-linked chronic
diseases and return to diversity of traditional whole food
dietary patterns is urgently needed.
The current study using in vitro assays provides insights
about the antihyperglycemia and antihypertension potential
of thermally processed Andean pseudocereals, cereals, and
legumes from Peru. Purple corn (Z. mays L.) (cereal) had
711
the highest total phenolic content, free radical scavenging
linked-antioxidant activity, and a-glucosidase inhibitory
activity, characteristics likely correlated with its content of
protocatechuic acid. Pseudocereals such as Quinoa (C.
quinoa Willd) and Kañiwa (C. pallidicaule Aellen), rich in
quercetin derivatives, exhibited high antioxidant activities
and moderate a-glucosidase inhibitory activities, whereas
only legumes (L. mutabilis SLP-1 and H-6) had high ACE
inhibition, indicating their potential antihypertension ac-
tivity. Cooked Andean grains did not have a-amylase in-
hibitory activity. Therefore, the potential for managing
glucose level, hypertension, and cellular redox status for
prevention of type 2 diabetes and its complications without
side effects linked to high a-amylase inhibition may be
achieved by combining Andean cereals, pseudocereals, and
legumes as a part of the traditional overall diet and food
diversity. Such combinations of diet designs need to be
further validated by animal and clinical studies.
AUTHOR DISCLOSURE STATEMENT
No competing financial interests exist.
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