Grain Dimension, Nutrition and Nutraceutical Properties of Black and Red Varieties of Rice in India

Abstract

Traditional colored rice varieties in India are the source of carbohydrates, phytochemicals and minerals. They facilitate the growth of probiotics in intestine and protect human from many chronic diseases. The present study investigated the nutritional properties such as total sugars, digestible sugars, resistant sugars, hydrolysis index, glycemic index and total proteins of thirteen colored varieties of rice in India. Nutraceutical properties like anti diabetic and prebiotic activity were investigated by standard methods. Chak hao poreiton and mappillai samba grains were 6.3 mm in length. Lowest length of 5.1 mm was recorded in 60 m Kuruvai. Among the rice varieties, mappillai samba has high concentration of digestible starch of 91% and Chak hao poreiton had low concentration of 62%. Resistant starch was 38% in Chak hao poreiton and 8% in mappillai samba. Lowest glycemic index of 52 and 53 were recorded in karuthakar poha and Chak hao poreiton respectively. Anthocyanin extracted from Chak hao poreiton inhibited 24% of human pancreatic α-amylase activity. It significantly increased the probiotic number from 0.15 CFU/mL to 1.95 CFU/mL. The study revealed that the black rice variety, Chak hao poreiton was rich in resistant starch and exhibited low glycemic index. The anthocyanins from Chak hao poreiton possessed significant antidiabetic and prebiotic activity. Molecular docking studies revealed the interaction of anthocyanin with pancreatic α-amylase, β-glucosidase and GLUT1.

Full text

Grain Dimension, Nutrition and Nutraceutical Properties of
Black and Red Varieties of Rice in India
MALA RAJENDRAN*and KEERTHANA RAVI CHANDRAN
Department of Biotechnology, Mepco Schlenk Engineering College, Sivakasi, Tamil Nadu, India.
Abstract
Traditional colored rice varieties in India are the source of carbohydrates,
phytochemicals and minerals. They facilitate the growth of probiotics in
intestine and protect human from many chronic diseases. The present
study investigated the nutritional properties such as total sugars, digestible
sugars, resistant sugars, hydrolysis index, glycemic index and total proteins
of thirteen colored varieties of rice in India. Nutraceutical properties like anti
diabetic and prebiotic activity were investigated by standard methods. Chak
hao poreiton and mappillai samba grains were 6.3 mm in length. Lowest
length of 5.1 mm was recorded in 60 m kuruvai. Among the rice varieties,
mappillai samba has high concentration of digestible starch of 91% and
chak hao poreiton had low concentration of 62%. Resistant starch was 38%
in chak hao poreiton and 8% in mappillai samba. Lowest glycemic index
of 52 and 53 were recorded in karuthakar poha and Chak hao poreiton
respectively. Anthocyanin extracted from Chak hao poreiton inhibited
24% of human pancreatic α-amylase activity. It signicantly increased the
probiotic number from 0.15 CFU/mL to 1.95 CFU/mL. The study revealed
that the black rice variety, Chak hao poreiton was rich in resistant starch
and exhibited low glycemic index. The anthocyanins from Chak hao poreiton
possessed signicant antidiabetic and prebiotic activity. Molecular docking
studies revealed the interaction of anthocyanin with pancreatic α-amylase,
β-glucosidase and GLUT1.
Current Research in Nutrition and Food Science
www.foodandnutritionjournal.org
ISSN: 2347-467X, Vol. 08, No. (3) 2020, Pg. 903-923
CONTACT Mala Rajendran maalsindia@mepcoeng.ac.in Department of Biotechnology, Mepco Schlenk Engineering College,
Sivakasi, Tamil Nadu, India.
© 2020 The Author(s). Published by Enviro Research Publishers.
This is an Open Access article licensed under a Creative Commons license: Attribution 4.0 International (CC-BY).
Doi: 10.12944/CRNFSJ.8.3.20
Article History
Received: 30 March 2020
Accepted: 05 November
2020
Keywords
Amylase;
Anthocyanin;
Docking;
Glycemic Index;
Hydrolysis Index;
Probiotics.
Introduction
India is the global capital of diabetes with
approximately 69 million people suffering from
diabetes. Pre-diabetic people are in danger of
developing insulin resistant diabetes. Uncontrolled
diabetes leads to many other chronic diseases like
heart, liver, kidney, stroke, nervous disorders etc.1
The major driving force for the rapid emergence of

904RAJENDRAN & CHANDRAN, Curr. Res. Nutr Food Sci Jour., Vol. 8(3) 903-923 (2020)
diabetes is the consumption of energy dense food,
less intake of fruits, vegetables and omega 3 fatty
acids. Rice is the staple food in many countries.2
Asia produces the 95% needs of rice globally.3
About 60% of caloric needs of Asian people are met
by rice. Some rice varieties have low concentration
of readily digestible sugars and high concentration
of resistant sugars. The resistant sugars are not
digested by the human gastrointestinal tract. The
probiotic population of intestine digests them. Rice
varieties rich in resistant starch have low glycemic
index (GI). GI is an indicator of the rate at which
postprandial blood sugar level is increased within
90 minutes of digestion. The GI of rice varies widely
from 40 to121.4 GI of rice is classied into low (<55),
medium (55) and high (>70). Understanding the
hydrolysis index (HI) and GI of rice varieties are
of prime importance to prevent diabetes mellitus.
Rice with higher GI values induce diabetes.5,6 and
those with low GI value does not impose stress on
pancreas to secrete insulin. One powerful strategy
to control diabetes is to limit the release of glucose
from food by inhibiting the activity of α-amylase
and glucosidase.7 One of the promising strategies
to reduce the complications and counteract the
metabolic alterations of diabetes is to use inhibitors
of α-amylase and glucosidase. Synthetic inhibitors
of amylase and glucosidase such as acarbose and
miglitol induce gastrointestinal complications and
hepatic injury. Side e󰀨ects associated with these
drugs limit their success.8 Hence, research towards
the identication of natural inhibitors becomes prime
importance.
Red, purple and black rice were used since ancient
time in India.9 They are good sources of resistant
starch, minerals and nutraceuticals.10 Colored rice
varieties available in India includes Mattai, Kiarali,
Black Kavuni, Chak hao poreiton, Thavala kanna
matta, Onamatta, Mapillai samba, Navara, Red
Kavuni, Kuruvaikar, Poonkar, Kuzhiyadichan etc.
Recently these rice varieties were evaluated for
their nutraceutical properties such as antioxidants,
antidiabetic etc.11 The color and aroma of rice plays
a key role in consumer preference.12 The color of the
endosperm distinguishes rice into black red, brown
and white.13 Di󰀨erent anthocyanins contribute to
the color of rice. Red varieties are unique with high
mineral concentration while the black varieties are
rich in minerals, protein and crude ber.
Changing the food practice based on the consumption
of functional foods and foods with pharmaceutical
value are complimentary approaches to mitigate
diabetes and chronic diseases mapped with
diabetes.14-16 Nutritional therapy aims to use
foods with nutraceutical value. Nutraceuticals are
products which provide nutrition and pharmaceutical
value to the consumer. Many nutraceuticals play
a vital role in protection against diabetes,17-18
cardiovascular diseases,19 neurological and
nephrological disorders.20 They block the absorption
of cholesterol and decrease the harmful e󰀨ects
of atherosclerosis.21 They function as potent
antioxidants and anti-inammatory agents.22 Due
to increasing awareness among people about the
side e󰀨ects of synthetic drugs used to increase
the glucose uptake in tissues23 and the benets of
consuming functional foods, research is focused
on natural compounds as therapeutic agents.
The anthocyanin in rice are chemically avonoids
which serves as an antioxidant, antidiabetic,
antihyperlipidemic and anti-ageing agents.24-25
Recently the correlation between disease and gut
microbiome is unraveled. Establishing the probiotics
in gut is essential for maintaining health.
There is only scarce scientic information on the
grain properties, resistant starch and nutraceutical
value of di󰀨erent colored varieties of rice available
in India. Hence, the present study was aimed to
analyze the grain characteristics, GI, resistant
starch, total proteins, anti-diabetic and prebiotic
activity of few colored rice varieties available in India.
Materials and Methods
Collection of Rice Varieties
Thirteen unique traditional rice varieties originated
in di󰀨erent states of India like Tamil Nadu, Manipur
and Kerala, were gifted by Spirit of earth, Chennai,
Tamil Nadu, India.
Grain Dimensions
Length, Width, Thickness and Aspect ratio
Length (L) and width (W) of the rice samples were
measured. Thickness (T) was measured with Vernier
caliper of accuracy 0.01mm. Aspect ratio (Ra) and
sphericity (Sp) of the rice were determined using the
following formula,

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Rα= W x100/L
Sp= (LWT) 1/3/L x 100
Based on the FAO standards, the grains are
classied as long, medium and short as per the
following criteria:
Long: Kernel length > 6 mm and length/width ratio
3 and above
Medium: Kernel length between 5.2 mm and 6 mm,
L/W ratio < 3
Short: Kernel length <5.2 mm and L/W ratio <2
Equivalent Diameter
The equivalent diameter of the rice was calculated
using the following formula followed by Haq et.al.26
De = (LBT) 1/3
Thousand Kernel Weight
Thousand kernels were counted and weighed in
digital balance with an accuracy of 0.001g. The
measurement was replicated thrice and the mean
value was recorded.27
Nutritional Properties
Estimation of Total Starch, Readily Digestible
Starch and Resistant Starch
Total sugars, readily digestible sugars and resistant
sugars were estimated by Glucose Oxidase-
Peroxidase (GOD-POD) method.
Estimation of Total Starch
The concentration of total starch in the rice
samples were estimated using porcine pancreatic
amylase.28 Rice samples were powdered, sieved
and homogenized with 5 mL of distilled water. To
the sample, 1 mL of 100 U amylase (pancreatin)
was added and incubated at 370C for 15 min.
The volume was made up to 25 mL with distilled
water. To 1 mL of the digested sample, 10 µL of
(14 U) amyloglucosidase and 2 mL of phosphate
bu󰀨er (pH 4.75) were added and incubated in shaker
for 60 min. It was diluted to 5 mL and the glucose was
estimated using GOD-POD. The available starch
was calculated using the formula,
Available Starch (%) = (µg of glucose*0.9*25)/
sample weight (mg, dry basis)
Estimation of Readily Digestible and Slowly
Digestible Starch
Rice sample (1g) was homogenized with 50 mL of
phosphate bu󰀨er (pH 6.9). 1 mL of 100 U amylase
(Pancreatin) was added and incubated at 370C in
shaker. The glucose concentration was estimated
at a regular interval of 30 min up to 90 min using
GOD-POD.29
Resistant Starch
The concentration of resistant starch in rice
varieties was determined after complete digestion of
digestible starch.19 Powdered sample (100 mg) was
solubilized with 10 mL of 0.2M phosphate bu󰀨er and
incubated with 1 mL of 100 U amylase (Pancreatin)
for 16 h to completely hydrolyze the digestible starch.
The sample was centrifuged at 10,000 rpm for
10 min and the residue was solubilized with 2.0M
KOH. Glucose was estimated using GOD-POD
method. The obtained value of glucose was
multiplied by a factor of 0.9 to calculate the resistant
starch.
Digestible Starch
The digestible starch was calculated using the
following formula,
Digestible starch = Total Starch – Resistant Starch
Hydrolysis Index (HI)
HI Index was determined from the ratio of area under
the hydrolysis curve (AUC) of test sample and bread
as the reference sample.19 AUC was constructed
using the concentration of starch hydrolyzed until
90 min as described in the readily digestible starch.
HI = (AUC of test sample/AUC of bread) *100
Glycemic Index (GI)
The GI of rice samples was calculated from the
formula given by Goni et al.29
GI = 39.71 + 0.549*HI

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Estimation of Total Proteins
The concentration of total proteins in the rice
samples were estimated by Biuret method.30
500 mg of powdered rice samples were extracted
with 10 mL of NaOH at 600C for 90 min. The samples
were cooled to room temperature and centrifuged
at 4000 g at 4°C for 30 min. To 100µL of the
supernatant, 1 mL of Biuret reagent, was added and
incubated at room temperature for 5 min. The optical
density was recorded at 546 nm.
Nutraceutical Properties
Extraction of Anthocyanins from Rice Samples
Anthocyanin was extracted using methanol acidied
with 1.5N HCl (70:30) as per the protocol of Sun
et al.31 The procedure consisted of dissolving
1mg/mL of the rice powder in two di󰀨erent bu󰀨er
solutions of hydrochloric acid-potassium chloride
bu󰀨er (pH 1.5) and sodium acetate bu󰀨er (pH 4.5).
The extract was dried in a rotary evaporator at 400C.
Antidiabetic Activity
Antidiabetic activity of rice extract was quantied by
their ability to inhibit porcine pancreatic amylase that
releases reducing sugars from starch.32 500 µL of
amylase (16 U/mg) and 500µL of the anthocyanin
extract of rice varieties (1mg/mL) were incubated
at 250C for 10 min. To the mixture, 500 µL of starch
(0.05% in phosphate bu󰀨ered saline of pH 6.9)
was added and incubated at room temperature for
30 min. 500µL of the reaction mixture was withdrawn
for the determination of reducing sugars released by
amylase. A control was maintained simultaneously
with enzyme and substrate without the rice extract.
To the sample 1000 µL of dinitro salicylic acid was
added and heated in a boiling water bath for 10 min.
The tubes were cooled to room temperature and the
color was read at 540 nm. Inhibition of amylase was
calculated using the formula,
Inhibition of Amylase (%) = (Absorbance of control-
Absorbance of sample / Absorbance of control)*100
Probiotic Activity of Anthocyanin Extracted
from Rice
The prebiotic activity of anthocyanins was evaluated
by viable plate count method. The probiotic
organisms were purchased from Bilac (300 million
cells/100 mg powder). The bacilli were activated in
MRS broth for 24 hr. The To 50 mL of MRS medium
1mL of activated probiotics (1x108 CFU/mL) and
anthocyanin powder (1 mg/mL) were added and
incubated for 36 hr. A tube without anthocyanin
extract was used as control. The sample was diluted
and plated in MRS agar plate to count the viable
colonies.33
Statistical Analysis
All experiments were replicated thrice, and the data
represented are the mean of three replicates. Graphs
were plotted with standard error of the mean using
Graph Pad Prism 5.
Molecular Docking of Anthocyanin
Preparation of Receptor
Crystal structure of human pancreatic α-amylase
(2QMK), human cytosolic β-glucosidase (2JFE) and
human glucose transporter-GLUT1(4PYP), were
retrieved from protein data bank. The bound ligands,
cofactors and solvent molecules associated with the
protein structures were removed and saved in .pdb
format as an input format for PyRx and Discovery
Studio Visualizing software.
Preparation of the Ligands
As the black rice variety was superior to other
varieties in its resistant sugar concentration
and nutraceutical properties, docking study was
restricted to bioactive compounds in black rice
only. The major anthocyanin pigments from black
rice such as cyanidin, cyanidin-3-glucoside,
cyanidin-3-rutinoside, peonidin-3-glucoside and
peonidin-3-rutinoside were selected to evaluate their
interaction with amylase, glucosidase and GLUT1.
To compare the e󰀩ciency of anthocyanins, three
clinically available drugs such as acarbose, miglitol
and voglibose were used. The ligands were retrieved
from PubChem database in .sdf format and then
converted to .pdb format using Open Babel GUI tool.
The ligands were loaded into the PyRx software.
Molecular Docking
The ligands and proteins were docked using
Autodock Vina incorporated in Pyrx software and
their binding a󰀩nities were obtained. The docked
results were compiled, visualized and the receptor-
ligand interactions were analyzed using Discovery
Studio Visualizer.

907RAJENDRAN & CHANDRAN, Curr. Res. Nutr Food Sci Jour., Vol. 8(3) 903-923 (2020)
Results and Discussion
Grain Dimension
There is very less scientific information about
the grain dimensions of the colored rice varieties
available in India. The rice varieties collected in the
present study is represented in Figure 1. The gure
clearly depicts the di󰀨erence in the color between
the rice varieties. The di󰀨erence is attributed to
the variation in the composition of phytochemicals.
The color of the rice is due to the avonoid group of
compounds called anthocyanins, which are potent
antioxidants.31, 34 The dimensions of rice grains
are represented in Table 1. Physical dimensions
of rice grains are critical in screening, grading and
discriminating the quality. Size based classication
is essential for further processing of agro products
in machines.34 Length of the rice ranged from
5.1 mm to 6.3 mm. Width of the rice varieties varied
between 2.5 mm and 3 mm. Lower most length
(5.1 mm) and width (2.6 mm) were recorded in 60m
kuruvai. Mappillai samba variety showed highest
length (6.3 mm) and width (2. 9 mm). The results
agree with the observations of Rui et al.35 and Yang
et al.36 Difference in the grain size among the
di󰀨erent varieties of rice is due to the di󰀨erence
in the genetic composition of the seed and soil
quality. Similar results were reported in the
previous studies.37 Positive correlation between
the morphological parameters like area, perimeter,
length, diameter width, roundness, thickness and
grain weight are reported earlier.38 Grain morphology
is related to milling quality. The overall appearance
of rice is dependent on the uniformity in size, shape
and thickness.39
Table 1: Grain Dimensions of Rice
S.No Rice Variety Length Width Thickness L/W De(mm) Aspect Sp(%)
(mm) (mm) (mm) ratio (%)
1 Chak hao poreiton 6.3 2.5 1.9 2.52 3.1 39.68 49.20
2 Mapillai samba (raw) 6 3 2.1 2 3.36 50 56
3 Mapillai samba (boiled) 6.3 2.9 1.8 2.17 3.2 46.03 50.79
4 60m kuruvai 5.1 2.6 1.9 1.96 2.93 50.9 57.45
5 Karuthakar poha 5.7 2.8 1.8 2.03 3.06 49.12 53.68
6 Kothamalli samba (raw) 5.5 2.7 2 2.03 3.09 49.09 56.18
7 Kothamalli samba (boiled) 5.1 2.7 1.8 1.88 2.92 52.94 57.25
8 Kuzhiyadichan 5.8 2.7 2.1 2.14 3.2 46.55 55.17
9 Navara boiled 5.5 2.5 1.7 2.2 2.86 45.45 52
10 Perungar 6.2 3 2 2.06 3.34 48.38 53.87
11 Kullakar (raw) 5.3 2.6 1.7 2.03 2.86 49.05 53.96
12 Onamatta 6.1 3 1.9 2.03 3.26 49.18 53.4
13 Thavalakannamatta 5.5 2.5 1.6 2.2 2.8 45.45 50.90
Signicant variation was observed in the aspect
ratio and roundedness among rice varieties. Lower
roundedness indicates the cylindrical nature of rice.40
Chak hao poreiton variety has less aspect ratio of
39.68 and kothamalli samba (boiled) has the highest
aspect ratio of 52.94. Wide variations in the grain
dimension was reported by Correa et al.41 and Shittu
et al.42 Lower roundedness indicates the cylindrical
nature of rice.40 The results corroborate well with
the total starch concentration presented in Figure 3.
The total starch in kothamalli samba was higher than
chak hao poreiton. The results clearly conrm that
large grain favors dry matter accumulation than small
grain relative to their hull weight. Grain dimension is
useful in deciding the sieve during milling process.25
Size, shape, sphericity, aspect ratio and color are
some of the grain parameters that govern the quality
of rice. Grain weight increases with grain size and
the duration of grain lling which in turn depend on
the panicles.43

908RAJENDRAN & CHANDRAN, Curr. Res. Nutr Food Sci Jour., Vol. 8(3) 903-923 (2020)
Weight of thousand kernels is represented in
Figure 2. Thousand kernel weight was 18g in chak
hao poreiton and 28g in perungar. Weight of grain
was less in boiled variety of mappillai samba than
raw variety. It is an indicator of seed quality44 and it
is due to the milling process.45 Consumer preference
and pricing of rice is based on the grain dimension
and quality.
Fig.1: Rice varieties
Fig.2: Thousand kernal weight of rice

909RAJENDRAN & CHANDRAN, Curr. Res. Nutr Food Sci Jour., Vol. 8(3) 903-923 (2020)
Nutritional Prole of Rice
Total Starch
Starch in cereals is composed of repeated glucose
units arranged as straight chain in amylose and
branched chain in amylopectin. Total starch
concentration present in the rice varieties was
assessed and the results are shown in Figure 3.
The concentration of total starch in rice varied from
61% (kullakar -raw) to 85 % (mapillai samba-
boiled). The concentration of total starch was 78% in
kothamalli samba (raw) and it was slightly higher in
processed sample (81%). Similarly, a slight increase
in total starch was observed in boiled variety of
mapillai samba than raw variety. During processing
conditions, the starch in the rice varieties were
solubilized which leads to increased starch content.
The present results corroborate with the reports
of Omar et.al.46 which documented the variation
in starch concentration between 81-92% of total
constituents. Starch from rice is di󰀨erent from other
plant sources by their small size and the presence
of hypoallergenic protein.47 The concentration
of amylose in the starch determines the rate of
digestion of starch. Compactness of amylose
reduces its exposed surface area for digestion by
salivary and pancreatic amylase.48
Fig.3: The concentration of total starch in rice
Fig.4: The concentration of digestible starch in rice
Digestible Starch
The concentration of digestible starch present
in the rice varieties are shown in Figure 4. The
concentration of digestible starch in Chak hao
poreiton, Kullakar (raw) and bread were 62%, 90%
and 97% respectively. The digestibility of the starch

910RAJENDRAN & CHANDRAN, Curr. Res. Nutr Food Sci Jour., Vol. 8(3) 903-923 (2020)
varies widely due to processing conditions, genetic
modication in the seeds and storage conditions.
Bread is easily digestible because of the exposure
of amylose and amylopectin to hydrolyzing enzymes.
Many colored varieties have low digestibility
because of the complex structure of amylose and
amylopectin, which are not easily accessible to
the enzymes. Signicant portion of starch was not
digestible in chak hao poreiton. The concentration of
readily digestible starch increases the burst release
of glucose into blood stream.
Fig.5: The concentration of resistant starch in rice
Resistant Starch
Resistant starch is dened as “the fraction of starch
that escapes digestion in the small intestine and that
is fermented in the large intestine”.49 It is an example
of complex carbohydrate that resists digestion in
the stomach, and they function as soluble ber.
The concentration of resistant starch present in
the rice is depicted in Figure 5. It varied widely
between varieties with 38 % in chak hao poreiton
and 8% in mapillai samba. The resistant starch is
fermented by the microora in the large intestine.49
Among the rice varieties used, chak hao poreiton,
has the highest concentration of resistant starch,
which reduces its GI.50 60m kuruvai has second
highest concentration of 19 %. Boiled mapillai samba
(14%) showed high concentration than raw variety
(8%). Richness of colored varieties of rice with
resistant starch was supported by the observations
of Deepa et.al.51 Resistant starch decrease the post
prandial glucose and improves insulin sensitivity.52, 53
Rice rich in amylose content are resistant to digestion
by amylase.4 Resistant starch increases the growth
of benecial bacteria in colon by supplying the free
fatty acids, acetate, propionic acid and butyric acid.
These short chain fatty acids increase the absorption
of minerals in the bowel and prevent bowel related
disorders.54
Hydrolysis Index
Hydrolysis of starch determines the body’s response
to metabolic conditions such as hyperglycemia and
hyperlipidemia.55 The hydrolysis of starch at di󰀨erent
time intervals is represented in Figure 6. Starch
hydrolysis was very rapid in bread where more than
90% of starch was hydrolyzed within 90 min. In chak
hao poreiton only 20% of starch was hydrolyzed in
90 min. Kothamalli samba variety rapidly digested
90% of starch within 90 min. Other varieties of rice
were intermediate between bread and Chak hao
poreiton. The Hydrolysis Index of the food sample
is calculated from the starch hydrolysis curve with
bread as the reference food and the results are
shown in Figure 7. The HI of bread was 100.56
Karuthakar poha and chak hao poreiton have low
HI of 22 and 25 respectively. This low HI may be
due to the fact that the rice samples may contain
non-starch polysaccharides.56, 57 Boiled mapillai
samba has lower hydrolysis index than raw variety.
The results are in accordance with the observations
of Perera.58 Variation in the hydrolysis of starch
among the di󰀨erent sources of plant and within the
varieties were supported by the observations of
starch digestibility both among plant sources and
within a single variety.59, 60

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Glycemic Index
GI determines the rate at which the body converts
starch into a reducing sugar. It is directly proportional
to HI value. The GI values of rice varieties are
presented in Figure 8. GI of rice varieties varied
signicantly between 83 and 52. Higher glycemic
index of bread is attributed to the solubility of
starch and its susceptibility to amylase action which
Fig.6: Hydrolysis of starch
Fig.7: Hydrolysis Index of rice

912RAJENDRAN & CHANDRAN, Curr. Res. Nutr Food Sci Jour., Vol. 8(3) 903-923 (2020)
releases glucose rapidly. Studies by Miller et.al.4
reported the wide variation in GI value of rice from
62 to 93. Raw Mapillai samba has the highest GI
(83) compared to boiled variety (52). Still higher
GI of 102 was reported by Frei et.al.59 Karuthakar
Poha and chak hao poreiton have low GI of 52 and
53 respectively. The observed variation in the GI
among the rice varieties is due to the di󰀨erence
in the composition of starch, particle size, the
concentration of other nutrients such as lipids, ber
and proteins.13 Varieties like mapillai samba and
kullakar rice with higher GI values release high
concentration of glucose.60 Therefore, it leads to
hyper insulinemia and culminates insulin receptor
down regulation.61 Moreover, foods with higher
glycemic index triggers hunger rapidly and induce
over eating and obesity.62 American Diabetes
Association describes the signicance of controlling
blood sugar to prevent the worst the consequence
of diabetes mellitus.31,63 Rice with low GI value is
preferred recently due to increased awareness about
the relationship between the GI and diabetes. Low
glycemic index diets enhance insulin sensitivity and
improve metabolic and cardiovascular risk factors.
Foods with low glycemic index protect people from
obesity, type II diabetes,64-66 breast cancer, gall
bladder disease67 and cardiovascular disease.68, 69
Low glycemic food reduces the absorption of glucose
and imposes no stress to pancreas to secrete insulin,
gastric inhibitory peptide and glucagon like peptide.
Low GI food reduces obesity and diseases linked
with obesity.70
Fig.8: Glycemic Index of rice
Total Proteins
The total proteins present in the rice varieties is
shown in Figure 9. Rice is an important source of
protein. The protein concentration of almost all the
rice samples is less than 20%. High concentration
of protein (16%) was observed in chak hao poreiton
followed by mapillai samba raw (11%). The results
agree with the previous records.71 Rice varieties
with high concentration of proteins decrease the
access of enzyme for starch hydrolysis.71 As the
concentration of protein was high in Chak hao
poreiton, the hydrolysis index and GI are also low
in the same variety.
Antidiabetic Activity of Anthocyanin
Inhibition of amylase by anthocyanins is evident from
Figure10. Inhibition of amylase by anthocyanin of
chak hao poreiton was highest with 24% followed
by kothamalli samba (14%). Lower most inhibition
of 4% was exhibited by mapillai samba variety. The
results are similar to the observation of Stephen
et al.72 The signicant di󰀨erence in the amylase
activity was correlated to the difference in the
concentration of anthocyanins in the rice varieties.
Anthocyanins from different food sources were
reported to possess inhibition towards amylase
and glucosidase.73-75 Inhibition of amylase activity

913RAJENDRAN & CHANDRAN, Curr. Res. Nutr Food Sci Jour., Vol. 8(3) 903-923 (2020)
reduces the release of sugars into the blood thus
reducing the burden of diabetes.73 Anthocyanins
compete for the catalytic sites of amylase and
glucosidase by mimicking the structure of starch.76
Anthocyanin glucosides mimic the transition state
complex of enzyme substrate complex and block
the enzyme activity. Other studies supported that
the bulky structure of anthocyanin masks the
active site like a cap and prevent the binding of
carbohydrates to the hydrolyzing enzyme.77 Few
other studies described the inhibition of amylase
and glucosidase by noncompetitive and mixed
inhibition.78-81 Anthocyanins exhibit antidiabetic
activity, not only by inhibiting the hydrolytic
enzymes but also inhibiting the glucose transporters
that are essential for the transport of glucose.
So, anthocyanins are potent in regulating the
glucose level by multiple ways.81 Hence, the
traditional colored varieties of rice are nutritionally
and pharmaceutically signicant.
Fig.9: Total proteins in rice
Fig.10: Antidiabetic activity of rice varieties
Prebiotic Activity of Anthocyanin
Gut microbiome plays a vital role in the health and
diseased status of human being. The biological e󰀨ect
of any active ingredient of food or pharmaceutical
formulation depends on the colonic microbiome.
Hence in the present study, the e󰀨ect of anthocyanin
in maintaining the gut microbiome was assessed and
the results are presented in Figure 11. The number

914RAJENDRAN & CHANDRAN, Curr. Res. Nutr Food Sci Jour., Vol. 8(3) 903-923 (2020)
of viable probiotic bacteria increased signicantly
in the medium supplemented with anthocyanin.
In control without anthocyanin, the number of
colonies was log 0.15CFU/mL. It was signicantly
increased to log 1.95 CFU/mL in Chak hao
poreiton. Growth promoting e󰀨ect of anthocyanin
was supported by the observations of Sun et.al31
and Díaz-Rizzolo et.al82 The anthocyanin in Chak
hao poreiton is primarily glycosylated derivatives.
These glycosyl units are hydrolyzed by glucosidases
synthesized by probiotics. The released glucose
is used for the growth of probiotics.83 The growth
promoting activity of Karuthakar poha increase the
bacterial count to log 1.0 CFU/mL. Growth inducing
e󰀨ect of anthocyanin on Bifidobacterium bifidum,
Bifidobacterium adolescentis, Bifidobacterium
infantis and Lactobacillus acidophilus were supported
in previous studies.
Fig.11: Prebiotic activity of anthocyanins
Firmicutes/bacteroidetes which are abundant in
the gut of hyperlipedemic and obese human were
reversed by the supplementation of anthocyanin
extract from black rice. Supplementation of
anthocyanin increased the bacteria from phylum such
as Lactobacillus, Bifidobacterium, Parabacteroides,
Oscillospira, Akkermansia, Ruminococcus, and
Butyricimonas and simultaneously decreased
Clostridium and Desulfovibrio.84-87
Table 2: Binding a󰀩nity of anthocyanins to carbohydrate
regulating enzymes and glucose transporter
Ligand Binding a󰀩nity (kcal/mol)
Amylase Glucosidase GLUT 1
Acarbose - 7.5 - 3.0 - 8.9
Miglitol - 5.7 - 3.5 - 5.8
Voglibose - 8.1 - 3.8 - 8.5
Cyanidin - 7.9 - 4.3 - 8.5
Cyanidin-3-glucoside - 9.9 -10.0 -10.4
Cyanidin-3-rutinoside -10.8 -10.8 -10.9
Peonidin-3-glucoside - 6.8 - 3.6 - 9.3
Peonidin-3-rutinoside - 6.8 -10.0 - 9.6

915RAJENDRAN & CHANDRAN, Curr. Res. Nutr Food Sci Jour., Vol. 8(3) 903-923 (2020)
Molecular Docking of Anthocyanin
The interaction of major anthocyanins of black colored
rice with pancreatic α-amylase, β-glucosidase and
GLUT1 was analyzed by molecular docking. The
results were compared with clinically available
drugs such as acarbose, miglitol and voglibose.
The free energy of binding of the ligands and
controls with the target proteins are shown in
Table 2. The table reveals the strong a󰀩nity between
the anthocyanins and proteins than the control
drugs. Druggability analysis of cyanidin-3-glucoside,
cyanidin-3-rutinoside, peonidin-3-glucoside and
peonidin-3-rutinoside showed more than 2 violations
in Lipinski rule. Cyanidin has good drug likeliness
with the binding a󰀩nity ofc-7.9 kcal/mol, -4.3kcal/mol
and -8.5kcal/mol against amylase, glucosidase and
GLUT 1 respectively.The binding a󰀩nity of cyanidin
was greater than acarbose and miglitol against
α-amylase Druggability β- glucosidase. A󰀩nity to
GLUT1 was equal to voglibose.
Fig.12: 2D interaction view of the ligands against pancreatic α-amylase

916RAJENDRAN & CHANDRAN, Curr. Res. Nutr Food Sci Jour., Vol. 8(3) 903-923 (2020)
Fig.13: 2D interaction view of the ligands against α-glucosidase
Based on the results of drug likeliness, only
cyanidine was used for further analysis.Interaction
of acarbose, miglitol, voglibose and cyanidin with
amylase, glucosidase and GLUT 1 are shown in
Figure 12,13, and 14 respectively. Voglibose binds
with Asp197, Glu 300 and Asp 233 which are the
active site residues of pancreatic α- amylase.88
Similarly, cyanidin also binds with Glu 233 and
Asp 300 conrming the competitive inhibition. The
binding of cyanidin with the active site residues are
supported by the observations of Xu et.al.89 Acarbose
and miglitol binds with α-amylase in many sites other
than the active sites. Oudjeriouat, et.al.90 reported
the uncompetitive inhibition of barely α-amylase by
acarbose.

917RAJENDRAN & CHANDRAN, Curr. Res. Nutr Food Sci Jour., Vol. 8(3) 903-923 (2020)
Fig.14: 2D interaction view of the ligands against GLUT 1
Active site of glucosidase is comprised of Glu 165,
Glu 273 and Gln 307. Hydrophobic amino acids such
as Phe179, Phe 121, Phe 225, Phe 433, Tyr 308,
Tyr 309, Trp 345 and Trp 425 covers the substrate.91
Active site binding was not observed in controls
and cyanidin conrming the absence of competitive
inhibition. Cyanidin binds with glucosidase through
vanderwaals forces and hydrogen bond. Strongest
a󰀩nity of cyanidin to glucosidase than acarbose
corroborates with the results of Chen et.al.92
GLUT1 mobilizes glucose to tissues and reduces
the concentration of postprandial glucose. Hence,
in the present study, the binding of cyanidin with

918RAJENDRAN & CHANDRAN, Curr. Res. Nutr Food Sci Jour., Vol. 8(3) 903-923 (2020)
GLUT1 was evaluated. Control drugs and cyaniding
revealed similar bindig sites as represented in
Figure 14.Trp 388 and Trp 412 are absolutely
essential for transport of glucose through GLUT1.
Acarbose and cyanidine interact with both residues.
Trp 388 was bound by all controls and cyanidin.
Similar interaction was documented by Son and
Lee.93 In addition to the essential sites, the interaction
involved many residues which contributed to higher
binding a󰀩nity. The results conrm the interaction of
cyanidin with α-amylase, β-glucosidase and glucose
transporter to reduce the postprandial glucose level.
Conclusion
Among the rice varieties used, chak hao poreiton has
low concentration of total starch, high concentration
of resistant starch, lowermost, GI and high
concentration of proteins. The variety is the richest
source of anthocyanins that possess antidiabetic
and probiotic activity. Molecular docking revealed
the binding of cyanidin from Chak hao poreiton with
pancreatic α-amylase, α-glucosidase and GLUT 1
with key residues essential for their function.
Acknowledgements
The authors thank “Spirit of Earth”, Chennai,
Tamil Nadu, and India for providing the unique
rice varieties. The authors thank Ms. Gazana
Iraivan, M.Tech, Department of Biotechnology,
Mepco Schlenk Engineering College for molecular
docking analysis. The authors thank the Head of
the department, Principal and Management board,
Mepco Schlenk Engineering College (Autonomous),
Sivakasi, Tamil Nadu, India for all support.
Author’s contributions
Mala Rajendran conceived the concept and
designed the experimental plan and Keerthana Ravi
Chandran executed the experiments.
Funding
The author(s) received no nancial support for the
research, authorship, and/or publication of this
article.
Conict of Interest
The authors do not have any conict of interest.
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