Journal of Dietary Supplements

ISSN: 1939-0211 (Print) 1939-022X (Online) Journal homepage: http://www.tandfonline.com/loi/ijds20

Antidiabetic and Antioxidant Potentials of

Amaranthus hybridus in Streptozotocin-Induced
Diabetic Rats

T. Balasubramanian MPharm, PhD, M. Karthikeyan MPharm, K. P.

Muhammed Anees BPharm, C. P. Kadeeja BPharm & K. Jaseela BPharm

To cite this article: T. Balasubramanian MPharm, PhD, M. Karthikeyan MPharm, K. P.

Muhammed Anees BPharm, C. P. Kadeeja BPharm & K. Jaseela BPharm (2017): Antidiabetic
and Antioxidant Potentials of Amaranthus hybridus in Streptozotocin-Induced Diabetic Rats,
Journal of Dietary Supplements, DOI: 10.1080/19390211.2016.1265037

To link to this article: http://dx.doi.org/10.1080/19390211.2016.1265037

Published online: 27 Jan 2017.

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JOURNAL OF DIETARY SUPPLEMENTS
http://dx.doi.org/./..

ARTICLE

Antidiabetic and Antioxidant Potentials of Amaranthus hybridus

in Streptozotocin-Induced Diabetic Rats
T. Balasubramanian, MPharm, PhD, M. Karthikeyan, MPharm, K. P. Muhammed Anees,
BPharm, C. P. Kadeeja, BPharm, and K. Jaseela, BPharm
Department of Pharmacology, Al Shifa College of Pharmacy, Kerala, India

ABSTRACT KEYWORDS
Context: Amaranthus hybridus (Amaranthaceae) has been used as a folk anti-hyperglycemic; ethanol
medicine in southern parts of India for the treatment of diabetes. Objec- extract; malondialdehyde;
tive: This research evaluates the antidiabetic and antioxidant effects maranthaceae; superoxide
dismutase; serum
of Amaranthus hybridus ethanol leaf extract (AHELE) in streptozotocin-
biochemical parameters
induced diabetic rats. Methods: Blood glucose levels of diabetic rats were
measured on days 1, 4, 7, and 15 after oral administration of AHELE at
doses of 200 and 400 mg/kg for 14 days. The effects of extract were
observed on serum glutamate oxaloacetate transaminase, serum glu-
tamate pyruvate transaminase, serum alkaline phosphatase, choles-
terol, high- and low-density lipoprotein, antioxidant potential, and
histopathological changes. Result: AHELE (200 and 400 mg/kg) caused
a significant (p < 0.001) reduction in blood glucose levels on day 15
(152.2 and 181.2 mg/dL, respectively versus diabetic control 287.0 mg/dL).
The extract also improved serum biochemical parameters associated
with diabetes. A significant (p < 0.001) decrease in malondialdehyde
protein (liver: 15.92, 12.29 nmol/mg, and kidney: 13.92, 10.29 nmol/mg
vs. diabetic control 25.49, 24.49 nmol/mg), increase in superoxide dis-
mutase protein (14.01; 17.47 IU/mg, and 25.01; 37.47 IU/mg vs. diabetic
control 9.65; 15.65 IU/mg), catalase protein (35.80, 44.49, and 39.80,
49.69 nmol/min/mg vs. diabetic control 18.45, 20.85 nmol/min/mg) and
reduced glutathione protein (44.77, 55.08 and 40.77, 51.08 μM/gm vs. dia-
betic control 29.81, 26.50 μM/gm) were observed. Conclusions: The study
reveals that treatment of diabetic rats with AHELE significantly reduced
hyperglycemia-associated oxidative damage. This could provide a ratio-
nale for the use of the plant to treat diabetes in folk medicine.

Introduction
Diabetes mellitus is a chronic metabolic disorder characterized by hyperglycemia, hyper-
lipemia, azoturia, and sometimes ketonemia, resulting from an absolute or relative deficiency
of insulin secretion or relative lack of insulin or malfunctioning of insulin action (Kumar,
Kumar, & Prakash, 2011). In recent times, many antidiabetic agents have been introduced,
still diabetes and the related complications continue to be a major medical problem not only
in developed countries but also in developing countries. Plant use presents a new area of
interest for the discovery of new antidiabetic drugs. A number of clinical studies suggest

CONTACT T. Balasubramanian tbaluanandhi@gmail.com Department of Pharmacology, Al Shifa College of Pharmacy,

Poonthavanam Post, Kizhattur Village, Perinthalmanna , Malappuram District, Kerala, India.
Color versions of one or more figures in the article can be found online at www.tanfonline.com/ijds.
©  Taylor & Francis Group, LLC
2 T. BALASUBRAMANIAN ET AL.

that the antioxidants in medicinal plants are key factors in reducing the incidence of diabetic
complications (Balasubramanian, Senthilkumar, & Karthikeyan, 2013). Finding new natural
sources of antidiabetics with antioxidant potential can be useful as future therapy for diabetic
patients.
Amaranthus hybridus L. (family Amaranthaceae), commonly known as “Cheera,” in
Malayalam, is an erect and branched annual herb distributed throughout tropical and tem-
perate regions of India as a common weed in agricultural fields and wastelands. In the tradi-
tional medicinal system, different parts of the plant Amaranthus hybridus (A. hybridus) have
been mentioned to be useful in treating a variety of diseases. Traditionally, the plant has been
used in treating dysentery, diarrhea, ulcers, and hemorrhage of the bowel due to its astringent
property (He, Corke, & Cai, 2003; Krochmal & Krochmal, 1973; Sumitra & Sandeep, 2011).
In southern India, the leaves are used in folk medicine for the treatment of diabetes. Leaves
possess antibacterial effect, cleansing effect, and also help to reduce tissue swelling (Sumitra &
Sandeep, 2011). In Nigeria, A. hybridus leaves combined with condiments are used to prepare
soup (Maiyol et al., 2010; Mepha, Eboh, & Banigbo, 2007). In the Congo, their leaves are eaten
as spinach or green vegetable (Dhellot, Matouba, & Maloumbi, 2006). These leaves boiled and
mixed with groundnut sauce are eaten as salad in Mozambique and West Africa (Oliveria &
DeCarvalho, 1975). The Amaranthus species contain amaranthine, quercetin, and kaempferol
glycosides (Zeashan, Amresh, & Singh, 2008). A. hybridus leaves are used as an antidote for
snake and scorpion bite (Chopra, 1958; Shinwari, Khan, & Nakaike, 2003).
Amaranthus species were of great importance in pre-Colombian American people’s diets
(González et al., 2007), as A. cruentus and A. hybridus have a high nutritional value (Fer-
nand et al., 2012). The consumption of A. cruentus products is advised for patients with celiac
disease as well as for diabetic patients (Guerra-Matias & Areas, 2005). A. hybridus has been
used traditionally for the treatment of liver infections and knee pain as well as for its laxative,
diuretic, and cicatrization properties (Fernand et al., 2012).
Furthermore, recent studies have established the anti-hyperglycemic activities of other
species of Amaranthus genus as A. spinosus (Sangameswaran & Ramadas, 2010) and A. viridis
(Ramdas et al.,2012a; Jayaveera et al., 2012). However, based on literature, there is no scien-
tific report proving the anti-hyperglycemic efficacy of this particular species. Based on this,
the present investigation was to study the anti-hyperglycemic activity of A. hybridus in various
experimental animal models. It can be claimed that the present study is the first to investigate
and demonstrate the anti-hyperglycemic effects of A. hybridus.

Materials and methods

Drugs and chemicals

Streptozotocin (STZ) was purchased from SISCO Research Laboratory, Mumbai, India.
Glibenclamide was obtained from Prudence Pharma Chem, Ankeshwara, Gujarat, India. All
other chemicals, including solvents used in the experiments, were purchased locally (Merck
and SD Fine Chemicals, Mumbai, India), and were of analytical grade. Standard kits were
obtained from Span Diagnostics, Mumbai, India.

Plant material
The leaves of A. hybridus L. were collected during the month of June 2013 from agricul-
tural and fallow fields of Kulukkallur, Palakkad district, Kerala. The plant was taxonomically
 JOURNAL OF DIETARY SUPPLEMENTS 3

identified and authenticated by Dr. Prabhu Kumar, Scientist, Plant Systematics and Genetic
Resources Division, Centre for Medicinal Plant Research (CMPR), Department of AYUSH,
Government of India, Kottakal, Malappuram district, Kerala, and a voucher specimen
(ACPPCTB-1) is preserved in our laboratory for further reference. The leaves of the plant were
washed, shade-dried, and powdered with a mechanical grinder. The powdered plant material
was then passed through a 60-mesh sieve and stored in an air-tight container for future use.

Preparation of plant extract

The shade-dried coarse powdered leaves of A. hybridus (500 g) were packed in the Soxhlet
apparatus and extracted with 1.5 L of 95% ethanol at a temperature of 40–50°C for 72 hr. The
solvent was completely removed in a rotary evaporator under reduced pressure at a temper-
ature of 40°C and a semisolid mass was obtained (Amaranthus hybridus ethanol leaf extract
[AHELE], yield 14% w/w). The dried AHELE was suspended in 5% tween 80 in normal saline
and used for the present study.

Preliminary phytochemical screening

The extract was screened for the presence of various phytoconstituents employing the fol-
lowing standard screening test (Horbone, 1998; Kokate, Purohit, & Gokhale, 1998; Trease &
Evans, 2002):
Test for steroids: Libermann–Burchard test and Salkowski test.
Test for triterpenoids: Noller test.
Test for alkaloids: Mayer’s test, Dragendroff ’s test, Wagner’s test, and Hager’s test.
Test for flavonoids: Lead acetate test and Shinoda’s test.
Test for glycosides: Legal’s test and A. Tollen’s test.
Test for tannins: Ferric chloride test.
Test for saponins: Foam test and blood hemolysis test.
Test for proteins: Millon’s test, Ninhydrin test, and Biurette test.
Test for carbohydrate: Molisch’s test, Fehling’s test, and Benedict’s test.

Experimental animals
Studies were conducted using Wistar albino rats of either sex weighing 150–200 g. They were
purchased from Small Animal Breeding Station (SABS), Government Veterinary Medical
College, Mannuthy, Thrissur (district), Kerala, India. The animals were randomly grouped
(n = 6) and housed in polyacrylic cages (38 × 23 × 10 cm) and maintained under standard
laboratory conditions (temperature 25 ± 2°C; relative humidity 55 ± 10%) with dark and
light cycle (14/10 hr). They were fed with a standard dry pellet diet (Small Animal Breeding
Unit, Government Veterinary College, Mannuthy, Thrissur District, Kerala, India) and water
ad libitum. The rats were acclimatized to laboratory conditions for one week before com-
mencement of the experiment and were maintained in a well-ventilated animal house. Ani-
mals described as fasting were deprived of food for at least 12 hr but were allowed free access to
drinking water. All procedures described were reviewed and approved by the Al Shifa College
of Pharmacy Animal Ethical Committee (Reg. No.: 1195/ac/08/CPCSEA, August 21, 2013).

Acute oral toxicity study

An acute oral toxicity study was performed as per Organization for Economic Co-operation
and Development (OECD)-423 guidelines (Ecobichon, 1997). Wistar albino rats (150–200 g)
4 T. BALASUBRAMANIAN ET AL.

were randomly distributed into six groups of three animals each. The animals were fasted
overnight and AHELE was administered orally at a dose of up to 2,000 mg/kg body weight.
Mortality and general behavior, such as grooming, sedation, hyperactivity, loss of righting
reflex, respiratory rate, and convulsions in the animals, were observed periodically for 72 hr.
The animals were observed continuously for the initial 2 hr and intermittently for the next
6 hr, and then again at 24, 48, and 72 hr following drug administration.

Oral glucose tolerance test

The oral glucose tolerance test (OGTT) (Aybar et al., 2001) was performed in overnight fasted
(18 hr) normal rats. Rats were divided into four groups of six animals each. Group I served
as control and received normal saline (5 mL/kg). Groups II and III were treated with AHELE
orally at doses of 200 and 400 mg/kg respectively. Group IV received the reference drug gliben-
clamide orally at a dose of 0.5 mg/kg. After 30 min of drug administration, the rats of all the
groups were treated orally with 2 g/kg glucose. Blood samples were taken from the tail vein at
0 min (just before administration of glucose) and at 30, 60, 90, 120, and 240 min after glucose
loading, and blood glucose levels were measured by a reflective glucometer (Eusure Blood
glucometer, Eumed Biotechnology Co. Ltd., Taiwan) using the glucose oxidase method.

Experimental

Induction of experimental diabetes

Rats were fasted for 16 hr before the induction of diabetes with STZ. A freshly prepared solu-
tion of STZ (40 mg/kg) in 0.1 M cold citrate buffer, pH 4.5, was injected intraperitoneally in a
volume of 1 mL/kg (Siddique et al., 1989), and the control rats were injected with the citrate
buffer alone. In order to control the hypoglycemia during the first day after STZ adminis-
tration, diabetic rats were given 5% glucose solution orally. Hyperglycemia was confirmed
by the elevated fasting glucose levels in blood, determined at 48 hr, and then on day 6 after
injection. Rats with moderate diabetes exhibiting fasting blood glucose levels in the range of
250–280 mg/100 mL were selected for the studies.

Sub-acute anti-hyperglycemic study (14 Days)

Rats were fasted for 16 hr and classified into five groups of six animals each (Nagappa et al.,
2003). Group I, normal control, animals were given normal saline orally at a dose of 5 mL/kg.
Group II, STZ-diabetic control, received normal saline at a dose of 5 mL/kg orally. Groups
III and IV, STZ-diabetic, rats were treated with AHELE orally at a dose of 200 and 400 mg/kg
respectively. Group V, STZ-diabetic, rats were administered with glibenclamide at a dose of
0.5 mg/kg orally. The treatment was continued once daily for 14 days. Fasting blood glucose
level of each animal was determined on days 1, 4, 7, 10, and 15 after the initiation of treat-
ment. The body weights of animals were also monitored on days 1, 4, 7, 10, and 15. Blood
was collected on the 15th day from the overnight-fasted rats by retro-orbital bleeding using
micro-capillary technique.

Estimation of serum biochemical parameters

Serum was separated and used for the determination of biochemical parameters
such as serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate
 JOURNAL OF DIETARY SUPPLEMENTS 5

transaminase (SGPT), serum alkaline phosphatase (SALP), bilirubin, creatinine, urea, total
cholesterol, triglycerides, and total proteins (using Automated Span Diagnostic Reagents,
Mumbai, India).

Estimation of liver glycogen

The glycogen content was determined from liver by Plummer’s method (Plummer, 1978), and
the glycogen content was expressed as milligram/gram of liver tissue (Kanai, 1998).
The rats were sacrificed and their organs (liver and kidney) were excised, rinsed in ice-
cold normal saline (pH 7.4), blotted dry, and weighed. A 10% w/v of homogenate was pre-
pared in 1.15% KCl and processed for the estimation of lipid peroxidation (Ohkawa, Ohishi,
& Yagi, 1979), and reduced gluthathione content (Ellman, 1959), superoxide dismutase (SOD)
(Kakkar, Das, & Viswanathan, 1984), catalase (CAT) (Aebi, 1974), and total proteins (Lowry
et al., 1951) were estimated.

Histopathological study
The rats were sacrificed on the 15th day after collection of blood samples, and liver and pan-
creas tissues were harvested. The fragments from the tissues were fixed in 10% neutral forma-
lin solution, embedded in paraffin, and stained with hematoxylin and eosin (H&E).

Statistical analysis
The experimental data were expressed as mean ± standard error mean (SEM). The data were
analyzed using ANOVA and Dunett’s test. The results were considered statistically significant
at p < 0.05. All statistical analyses were performed using GRAPH PAD software.

Results

Preliminary phytochemical analysis

The qualitative phytochemical screening of AHELE revealed the presence of flavonoids, gly-
cosides, terpenoids, saponins, alkaloids, tannins, and steroids.

Acute oral toxicity study

Administration of AHELE to Wistar rats up to 2,000 mg/kg did not show any mortality and
gross behavioral changes. Further dosing was not performed to estimate the LD50 (lethal
dose) value. According to the OECD-423 guidelines for acute toxicity, an LD50 dose of
2,000 mg/kg and above is categorized as unclassified, and hence the drug is found to be safe.
Based on acute toxicity studies, the dose 200 mg/kg (low dose, i.e., 10%) and 400 mg/kg (high
dose, i.e., 20%) of AHELE was selected as the therapeutic dose.

Oral glucose tolerance test

At 30 min, the blood glucose level was found to be rapidly increased from the fasting value in
glucose-fed control rats. This increased glucose level was significantly (p < 0.05 and <0.001)
tolerated in glucose-fed rats treated with AHELE (200 and 400 mg/kg) and glibenclamide
6 T. BALASUBRAMANIAN ET AL.

Table . Effect of oral administration of Amaranthus hybridus ethanol leaf extract on blood glucose level of
glucose-loaded rats.
Blood glucose level (mg/dL) at different time intervals
Groups  min  min  min  min  min  min

Control (% tween . ± . . ± . . ± .  ± . . ± . . ± .
 in .% NaCl,
 mL/kg)
AHELE  mg/kg . ± . . ± .∗ . ± .∗ . ± .∗ . ± .∗ . ± .∗
AHELE  mg/kg . ± . . ± .∗∗ . ± .∗∗ . ± .∗∗ . ± .∗∗ . ± .∗∗
GLIB . mg/kg . ± . . ± .∗∗ . ± .∗∗ . ± .∗∗ . ± .∗∗ . ± .∗∗

Control: Glucose-loaded rats; AHELE : Glucose-loaded rats treated with -mg/kg Amaranthus hybridus ethanol leaf
extract; AHELE : Glucose-loaded rats treated with -mg/kg Amaranthus hybridus ethanol leaf extract; GLIB .:
Glucose-loaded rats treated with .-mg/kg glibenclamide.
Values are given as mean ± SEM,  rats in each group;
∗ p < .
∗∗ p < . as compared with the control group.

(0.5 mg/kg) within 30 min. AHELE (200 and 400 mg/kg) produced a dose-dependent reduc-
tion in blood glucose levels, which were significantly (p < 0.001) lower than those of control
groups. Glibenclamide (0.5 mg/kg) showed a significant decrease in fasted blood sugar lev-
els compared with the control group and produced significant hypoglycemia at 240 min after
glucose administration (Table 1).

Sub-acute anti-hyperglycemic study

Significant (p < 0.001) increase in fasting blood glucose levels was observed in STZ-induced
diabetic rats. Repeated oral administration of AHELE at a dose of 200 and 400 mg/kg in STZ-
induced diabetic rats for 14 days significantly (p < 0.05 and <0.001) reduced the elevated
fasting blood glucose levels on days 1, 4, 7, 10, and 15 after initiation of treatment when com-
pared with diabetic control rats. The effect of AHELE is comparable with that of glibenclamide
(Table 2).

Table . Effect of oral administration of Amaranthus hybridus ethanol leaf extract on blood glucose level by
sub-acute treatment in STZ-induced diabetic rats.
Fasting blood glucose level (mg/dL)
Groups st day th day th day th day th day

Normal control (% tween . ± . . ± . . ± . . ± . . ± .
 in .% NaCl,
 mL/kg)
DM control (% tween  . ± . . ± .∗∗ . ± .∗∗ . ± .∗∗ . ± .∗∗
in .% NaCl,  mL/kg)
DM + AHELE  mg/kg . ± . . ± .a . ± .a . ± .a . ± .a
DM + AHELE  mg/kg . ± . . ± .b . ± .b . ± .b . ± .b
DM+ GLIB . mg/kg . ± . . ± .b . ± .b . ± .b . ± .b

DM control: Diabetic mellitus rats; DM + AHELE : Diabetic mellitus rats treated with -mg/kg Amaranthus hybridus
ethanol leaf extract; DM + AHELE : Diabetic rats treated with -mg/kg Amaranthus hybridus ethanol leaf extract; DM +
GLIB .: Diabetic rats treated with .-mg/kg glibenclamide.
Values are given as mean ± SEM,  rats in each group
∗∗ p < . as compared with the normal control group.
a p < ..
b p <. as compared with the diabetic control group.
 JOURNAL OF DIETARY SUPPLEMENTS 7

Table . Effect of oral administration of Amaranthus hybridus ethanol leaf extract on body weight in STZ-
induced diabetic rats.
Body weight (g)
Groups st day th day th day th day th day

Normal control (% tween . ± . . ± . . ± . . ± . . ± .
 in .% NaCl,
 mL/kg)
DM control (% tween  . ± . . ± .∗∗ . ± .∗∗ . ± .∗∗ . ± .∗∗
in .% NaCl,  mL/kg)
DM + AHELE  mg/kg . ± . . ± .a . ± .b . ± .b . ± .b
DM + AHELE  mg/kg . ± . . ± .a . ± .b . ± .b . ± .b
DM + GLIB . mg/kg . ± . . ± .a . ± .b . ± .b . ± .b

DM control: Diabetic mellitus rats; DM + AHELE : Diabetic mellitus rats treated with -mg/kg Amaranthus hybridus
ethanol leaf extract; DM + AHELE : Diabetic rats treated with -mg/kg Amaranthus hybridus ethanol leaf extract; DM +
GLIB .: Diabetic rats treated with .-mg/kg glibenclamide.
Values are given as mean ± SEM,  rats in each group;
∗∗ p < . as compared with the normal control group.
a p < .
b p < . as compared with the diabetic control group.

Effect on body weight

The body weight was slightly increased in normal control rats (in 14 days period) compared
with initial body weight, whereas in the diabetic control rats, there was a significant reduction
in average body weight over the same period. AHELE-treated diabetic rats gained significant
weight but the increase remained lesser than normal controls (Table 3).

Effect on biochemical parameters in serum

In STZ-induced diabetic control rats, there was a significant (p < 0.001) increase in SGOT,
SGPT, SALP, creatinine, urea, bilirubin, triglycerides, cholesterol, and LDL levels but were
reduced by both AHELE (200 and 400 mg/kg) and glibenclamide. In addition, there was a
significant (p < 0.001) decrease in serum total proteins and HDL levels in diabetic control
rats compared with normal control. Oral administration of AHELE increased the serum total
protein and HDL levels when compared with diabetic control rats (Tables 4 and 5).

Effect on hepatic glycogen content

The liver glycogen content was significantly decreased in STZ-induced diabetic rats as com-
pared with normal rats. On the other hand, in AHELE-treated diabetic rats, the glycogen
content in liver increased significantly (p < 0.001) when compared with diabetic control rats
(Table 6).

Effects on hepatic and renal in vivo antioxidant activities

Lipid peroxidation
As depicted in Tables 6 and 7, the amount of malondialdehyde (MDA), an end product of
lipid peroxidation, in the rats’ liver and kidney tissues, increased significantly in STZ-induced
diabetic control rats compared with the normal control rats. The treatment of rats with
AHELE (200 and 400 mg/kg) and glibenclamide resulted in a significant decrease in the
concentration of MDA than in diabetic control rats.
 8

Table . Effect of oral administration of Amaranthus hybridus ethanol leaf extract on serum biochemical parameters level by sub-acute treatment in STZ-induced diabetic
T. BALASUBRAMANIAN ET AL.

rats.
Total bilirubin Serum creatinine Serum urea
Groups AST (IU/dL) ALT (IU/dL) ALP (IU/dL) (mg/dL) Total protein (g/dL) (mg/dL) (mg/dL)

Normal control (% tween  in .% NaCl, . ± . . ± . . ± . . ± . . ± . . ± . . ± .
 mL/kg)
DM control (% tween  in .% NaCl, . ± .∗∗ . ± .∗∗ . ± .∗∗ . ± .∗∗ . ± .∗∗ . ± .∗∗ . ± .∗∗
 mL/kg)
DM + AHELE  mg/kg . ± .b . ± .b . ± .b . ± .b . ± .b . ± .b . ± .b
DM + AHELE  mg/kg . ± .b . ± .b . ± .b . ± .b . ± .b . ± . . ± .b
DM + GLIB . mg/kg . ± .b . ± .b . ± .b . ± .b . ± .b . ± .b . ± .b

DM control: Diabetic mellitus rats; DM + AHELE : Diabetic mellitus rats treated with -mg/kg Amaranthus hybridus ethanol leaf extract; DM + AHELE : Diabetic rats treated with -mg/kg
Amaranthus hybridus ethanol leaf extract;DM + GLIB .: Diabetic rats treated with .-mg/kg glibenclamide.
Values are given as mean ± SEM,  rats in each group;
∗∗ p < . as compared with the normal control group.
b p < . as compared with the diabetic control group.
 JOURNAL OF DIETARY SUPPLEMENTS 9

Table . Effect of oral administration of Amaranthus hybridus ethanol leaf extract on serum lipid profiles in
STZ-induced diabetic rats.
Cholesterol Triglycerides
Groups (mg/dL)) (mg/dL) LDL (mg/dL) HDL (mg/dL)

Normal control (% tween . ± . . ± . . ± . . ± .
 in .% NaCl,
 mL/kg)
DM control (% tween  . ± .∗∗ . ± .∗∗ . ± .∗∗ . ± .∗∗
in .% NaCl,  mL/kg)
DM + AHELE  mg/kg . ± .b . ± .b . ± .b . ± .b
DM + AHELE  mg/kg . ± .b . ± .b . ± .b . ± .b
DM + GLIB . mg/kg . ± .b . ± .b . ± .b . ± .b

DM control: Diabetic mellitus rats; DM + AHELE : Diabetic mellitus rats treated with -mg/kg Amaranthus hybridus
ethanol leaf extract; DM + AHELE : Diabetic rats treated with -mg/kg Amaranthus hybridus ethanol leaf extract; DM +
GLIB .: Diabetic rats treated with .-mg/kg glibenclamide.
Values are given as mean ± SEM,  rats in each group;
∗∗ p < . as compared with the normal control group.
b p < . as compared with the diabetic control group.

Reduced glutathione content superoxide dismutase and catalase

The total glutathione (GSH) content, SOD, and CAT activities in the STZ-induced diabetic
rats were significantly (p < 0.001) decreased in liver and kidneys. However, the hepatic and
renal SOD, CAT activities, and GSH levels were significantly elevated in the diabetic rats
treated with AHELE (200 and 400 mg/kg) and glibenclamide when compared with the dia-
betic control rats (Tables 6 and 7).

Histopathological studies of pancreas

Figure 1(a) presents the section of normal control pancreas showing normal exocrine acni
cells and langerhans islets with clusters of purple-stained β-cells. Figure 1(b) with diabetic
control pancreas shows severe congestion in pancreatic parenchyma, damaged islets shrunken

Table . Effect of oral administration of Amaranthus hybridus ethanol leaf extract on liver in vivo antioxidant
system and glycogen by sub-acute treatment in STZ-induced diabetic rats.
Lipid
peroxidation
(nmol of Glutathione Superoxide Catalase
MDA/mg (μM/gm dismutase (nmol/min/mg Liver glycogen
Groups protein) protein) (IU/mg protein) protein) (mg/gm of liver)

Normal control (% tween . ± . . ± . . ± . . ± . . ± .
 in .% NaCl,
 mL/kg)
DM control (% tween  . ± .∗∗ . ± .∗∗ . ± .∗∗ . ± .∗∗ . ± .∗∗
in .% NaCl,  mL/kg)
DM + AHELE  mg/kg . ± .b . ± .b . ± .b . ± .b . ± .b
DM + AHELE  mg/kg . ± .b . ± .b . ± .b . ± .b . ± .b
DM + GLIB . mg/kg . ± .b . ± .b . ± .b . ± .b . ± .b

DM control: Diabetic mellitus rats; DM + AHELE : Diabetic mellitus rats treated with -mg/kg Amaranthus hybridus
ethanol leaf extract; DM + AHELE : Diabetic rats treated with -mg/kg Amaranthus hybridus ethanol leaf extract; DM +
GLIB .: Diabetic rats treated with .-mg/kg glibenclamide; MDA: Malondialdehyde.
Values are given as mean ± SEM,  rats in each group;
∗∗ p < . as compared with the normal control group.
b p < . as compared with the diabetic control group.
10 T. BALASUBRAMANIAN ET AL.

Table . Effect of oral administration of Amaranthus hybridus ethanol leaf extract on in vivo antioxidant sys-
tem of kidney by sub-acute treatment in STZ-induced diabetic rats.
Lipid
peroxidation
(nmol of Glutathione Superoxide Catalase
MDA/mg (μM/gm dismutase (nmol/min/mg
Groups protein) protein) (IU/mg protein) protein)

Normal control (% tween  in . ± . . ± . . ± . . ± .
.% NaCl,  mL/kg)
DM control (% tween  in . ± .∗∗ . ± .∗∗ . ± .∗∗ . ± .∗∗
.% NaCl,  mL/kg)
DM + AHELE  mg/kg . ± .b . ± .b . ± .b . ± .b
DM + AHELE  mg/kg . ± .b . ± .b . ± .b . ± .b
DM + GLIB . mg/kg . ± .b . ± .b . ± .b . ± .b

DM control: Diabetic mellitus rats; DM + AHELE : Diabetic mellitus rats treated with -mg/kg Amaranthus hybridus
ethanol leaf extract; DM + AHELE : Diabetic rats treated with -mg/kg Amaranthus hybridus ethanol leaf extract; DM +
GLIB .: Diabetic rats treated with .-mg/kg glibenclamide; MDA: Malondialdehyde.
Values are given as mean ± SEM,  rats in each group;
∗∗ p < . as compared with the normal control group.
b p < . as compared with the diabetic control group.

in size, atrophy of β-cells, vacuolar degenerative changes, and mild polymorpho nuclear leu-
cocytes infiltration. Treatment with low- and high-dose AHELE depicted maximum cellu-
lar regeneration of pancreatic β-cells with well-granulated and an increased number of islets
(Figures 1(c) and (d)). The islets were well preserved and recovering from infiltration of lym-
phocytes.

Figure . (a–d) Photomicrographs of pancreatic sections of control and diabetic rats treated with Ama-
ranthus hybridus ethanol leaf extract. (a) Normal control, (b) diabetic control, (c) diabetic rats treated with
-mg/kg Amaranthus hybridus ethanol leaf extract, (d) diabetic rats treated with -mg/kg Amaranthus
hybridus ethanol leaf extract.
 JOURNAL OF DIETARY SUPPLEMENTS 11

Figure . (a–d) Photomicrographs of liver sections of control and diabetic rats treated with Amaranthus
hybridus ethanol leaf extract. (a) Normal control, (b) diabetic control, (c) diabetic rats treated with -mg/kg
Amaranthus hybridus ethanol leaf extract, (d) diabetic rats treated with -mg/kg Amaranthus hybridus
ethanol leaf extract.

Histopathological studies of liver

Figure 2(a) presents the section of a normal control rat liver showing normal cellular archi-
tecture with distinct hepatic cells in the form of radial laminae, hepatic sinusoids in between
radial hepatocytes, and well brought out central vein. STZ-induction led to disarrangement
of normal hepatocytes with several large areas of atrophy, severe inflammatory cell infiltra-
tion, cytoplasmic vacuolization, fatty degeneration, and sinusoidal dilation in diabetic control
rat livers (Figure 2(b)). Figure 2(c) presents the section of the liver tissue of AHELE-treated
(200 mg/kg) animals, showing moderate accumulation of fatty lobules and cellular necrosis.
Figure 2(d) presents the section of the liver tissue of AHELE-treated (400 mg/kg) animals
showing normal lobular pattern with a mild fatty change, feathery necrosis almost compara-
ble to the normal.

Discussion
Diabetes mellitus is probably the fastest growing metabolic disease in the world, and as knowl-
edge of the heterogeneous nature of the disease increases, so does the need for more chal-
lenging and appropriate therapies (Ramdas et al., 2012b). The present research provides an
evidence of the beneficial effects of Amaranthus hybridus leaf extract in glucose, lipid profiles,
and oxidative defense system in STZ -induced diabetic rats.
Acute oral toxicity study revealed that oral administration of AHELE did not show
any mortality up to 2,000 mg/kg. In glucose-fed hyperglycemic rats, blood glucose was
12 T. BALASUBRAMANIAN ET AL.

significantly lower in the AHELE-treated group than in the control group 30, 60, and 120 min
after glucose loading, suggesting that the extract (200 and 400 mg/kg) significantly increased
the tolerance for glucose and may be able to reduce the intestinal absorption of glucose (James,
Lionel, & Timothy, 1995; Sharma, Khare, & Feroz, 1983).
In this study, we induced an experimental diabetes mellitus in Wistar rats by STZ injec-
tion. STZ is transported into β-cells of pancreas via glucose transporter (GLUT2), and causes
DNA damage, leading to increased activity of poly(ADP-ribose) polymerase (PARP-1) to
repair DNA. However, exaggerated activity of this enzyme results in depletion of intracellular
NAD(+) and ATP, and the insulin-secreting cells undergo necrosis (Szkudelski, 2001). The
sub-acute anti-hyperglycemic studies clearly demonstrated that AHELE (200 and 400 mg/kg)
significantly produced a dose-dependent anti-hyperglycemic effect in diabetic rats through-
out the course of study. The fundamental mechanism underlying hyperglycemia in diabetes
mellitus due to insulin deficiency involves the over production of glucose by excessive hepatic
glycogenolysis and gluconeogenesis, decreased hepatic glycogenesis, and/or decreased utiliza-
tion of glucose by the tissue (Erah et al., 1996; Latner, 1958). Significant decrease in the levels
of fasting blood glucose in diabetic rats treated with AHELE (200 and 400 mg/kg) may be by
the stimulation of residual pancreatic mechanism, which is probably similar to glibenclamide
(Tripathi, 2003) or by increasing peripheral utilization of glucose by tissues, or by increas-
ing glycogenesis and decreasing glycogenolysis in the liver. Histopathological studies further
revealed that the reduced islet β-cells were restored to near-normal conditions on treatment
with AHELE in STZ-induced diabetic rats. This finding suggests that the anti-hyperglycemic
activity of AHELE may be due to potentiation of insulin secretion by regeneration of β-cells.
Diabetes mellitus is often associated with hyperlipidemia with increased risk of coronary
heart disease (Betteridge, 1997). In the present study, increase in serum total cholesterol,
triglycerides, and LDL levels was observed in STZ-induced diabetic control rats. Insulin
deficiency in diabetes mellitus is often linked with hypercholesterolemia and hypertriglyc-
eridemia due to metabolic abnormalities (Shirwaikar, Rajendran, & Kumar, 2004). The
increased concentration of cholesterol could result in a relative molecular ordering of resid-
ual phospholipids, resulting in a decrease in membrane fluidity (Bopanna et al., 1997). It is
well known that under normal circumstances, insulin activates enzyme lipoprotein lipase,
which hydrolyzes the triglycerides and insulin deficiency in diabetic condition resulting in
hypertriglyceridemia. The coronary risk and especially premature atherosclerosis in diabetic
patients is well established by the elevated levels of LDL cholesterol (Temme, Van Hoydonck, &
Schouten, 2002). However, treatment with AHELE (200 and 400 mg/kg) significantly reduced
the serum total cholesterol, triglycerides, and LDL in STZ-induced diabetic rats. This result
implies that AHELE may prevent or be helpful in reducing the complications of lipid profile
as well as in improving lipid metabolism in diabetes. HDL protects or reverses atherosclero-
sis by their ability to serve as acceptor particles for macrophage cholesterol efflux, prevention
of endothelial dysfunction, and maintenance of endothelial integrity (Linsel & Tall, 2005).
Treatment with AHELE showed marked elevation in the HDL level as compared with that
in the diabetic controls, which will prevent the chances of atherosclerosis and coronary heart
disease.
Diabetes mellitus is known to impair the normal capacity of liver to synthesize glyco-
gen (Hornbook, 1975). In the present study, treatment with AHELE (200 and 400 mg/kg)
for 14 days significantly increased the hepatic glycogen content in diabetic rats, suggesting
the activation of glycogen synthase for which the substrate glucose-6-phosphate could have
been readily provided by increased hexokinase activity or due to inhibition of glycogenolysis
 JOURNAL OF DIETARY SUPPLEMENTS 13

and gluconeogenesis in liver (Bouche et al., 2004; Pradeep et al., 2010). AHELE reduced the
increased levels of SGOT, SGPT, and SALP observed in diabetic control rats. This might sug-
gest the protective action of AHELE in reversing any organ damage due to induction of exper-
imental diabetes that is manifested by elevation in the levels of SGOT, SGPT, and SALP. More-
over, the administration of AHELE caused a significant reduction in serum creatinine levels
in diabetic rats when compared with diabetic control animals. This indicates that AHELE
reverses damage in the kidney tissues of diabetic rats.
We also examined antioxidant capacity of plant extracts, since antioxidants have been
reported to prevent oxidative stress and diabetic complications. One of the most often used
biomarker to investigate the oxidative damage on kidney and liver is thiobarbituric acid reac-
tive substances (TBARS) or simply MDA, a major lipid peroxidation product. It can react
with the free amino group of proteins, phospholipids, and nucleic acids, leading to structural
modification (Pandey & Rizvi, 2010). In the present study, the hepatic and renal TBARS levels
were significantly lower in the AHELE-treated groups compared with the diabetic control rats.
These findings support that AHELE may exert antioxidant activities and protect the tissues
from lipid peroxidation.
Glutathione, a tripeptide present in all the cells, is an important antioxidant. GSH can par-
ticipate in the elimination of reactive intermediates by reduction of hydrogen peroxide in the
presence of GPx (Meloy, 2002). SOD and CAT are enzymes that destroy peroxides and play
a significant role in providing antioxidant defense to organisms (Bolzanet & Bianchi, 2002;
Gosh et al., 2008). In our study, it was observed that AHELE caused a significant increase in
hepatic and renal SOD, CAT, and GSH activities of diabetic rats. This means that AHELE can
reduce reactive oxygen free radicals and improve the activities of hepatic and renal antiox-
idant enzymes to protect cellular damage during diabetes mellitus. In the present study,
histopathological findings also provided supportive evidence for the antioxidant potential of
AHELE during diabetes. There are studies reporting the antioxidant activities of A. hybridus
(Fernand et al., 2012). The phytochemical constituents, flavonoids, tannins, saponins, and
terpenoids, are known to have bioactive antidiabetic principles (Meliani et al., 2011). More-
over, the significant antioxidant and anti-hyperglycemic activity of AHELE on STZ-induced
diabetic rats may be attributed to the presence of biologically active compounds, such as
flavonoids, saponins, tannins, and terpenoids, in A. hybridus.
According to these results, AHELE could be a supplement, as an antioxidant therapy, and
may be beneficial for correcting the hyperglycemia and preventing diabetic complications
due to lipid peroxidation and free radicals. As the present study is a preclinical one, further
proceedings with human volunteers may pave a way for the usage of this drug in human
beings.

Conclusion
Food and food supplements have increasingly become attractive alternatives to prevent or
treat diabetes and its complications. The observed anti-hyperglycemic effect appears to be due
to the antioxidant properties of A. hybridus, which may pave the way to finding a new drug to
be used for fighting diabetes mellitus. Our research established pharmacological evidence to
support the folklore claim that Amaranthus leaves are antidiabetic agents. However, further
studies are necessary to find out active phytochemicals as well as the exact mechanisms of
action involved in antidiabetic potential of A. hybridus.
14 T. BALASUBRAMANIAN ET AL.

Acknowledgments
The authors are thankful to the Centre for Medicinal Plant Research (CMPR), Department of AYUSH,
Government of India, Kottakal, Malappuram District, Kerala for identification of plant material. The
authors thank Principal, Al Shifa College of Pharmacy, Kerala, for providing facilities to carry out the
experiments of this research work.

Declaration of interest
The authors report no conflicts of interest. The authors alone are responsible for the content and writing
of the article.

About the authors

T. Balasubramanian, MPharm, PhD, professor and head of the department, Department of Pharma-
cology, Al Shifa College of Pharmacy, Kerala, India.

M. Karthikeyan, MPharm, associate professor, Department of Pharmacology, Al Shifa College of Phar-

macy, Kerala, India.

K. P. Muhammed Anees, BPharm, research scholar, Department of Pharmacology, Al Shifa College of

Pharmacy, Kerala, India.

C. P. Kadeeja, BPharm, research scholar, Department of Pharmacology, Al Shifa College of Pharmacy,

Kerala, India.

K. Jaseela, BPharm, research scholar, Department of Pharmacology, Al Shifa College of Pharmacy,

Kerala, India.

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