Effects of Moringa oleifera aqueous
leaf extract in alloxan induced
diabetic mice
MUOBARAK J. TUORKEY*
Zoology Department, Division of Physiology, Faculty of Science, Damanhour University, Damanhour, Egypt
*Corresponding address: Muobarak J. Tuorkey, PhD; Zoology Department, Faculty of Science, Damanhour University, 14 El-Gomhoria Street,
Damanhour, Al-Behira 22111, Egypt; Phone: +20 198 624 037; Fax: +20 453 368 757; E-mail: physio_mj_tuorkey@yahoo.com
(Received: June 9, 2016; Revised manuscript received: August 29, 2016; Accepted: September 7, 2016)
Abstract: Objective: There is a lack of kn owledge r egarding the underlying mechanisms of the antidiabetic activity o f Moringa oleifera. This study
investigates t he antidiabetic effect of M. oleifera and its impact o n the immune tolerance. Methods: Alloxan-induced diabetes model for mice was
used. A dose of 100 mg/kg of Moringa extract was orally administered to d iabetic treated mice. Glucose and insulin levels were evaluated to
calculate insulin resistance. Tota l antioxidant capacity (TAC), creatinine, and blood urea nitrogen (BUN) levels were measured. The relative
percentage of CD44, CD69, and IFN-γ was investigated by flow cyt ometry. Results: In diabetic mice, insulin resistance by homeos tasis model
assessment of insulin resistance (HOMA- IR) was increas ed 4.5 -fold th an in the control group, and HOMA-IR was decr eased 1.3-f old in the
Moringa treatment group. The level of TAC was declined 1.94-fold i n diabetic mice, and increased 1.67-fold in diab etic treated group. In
diabetic mice, creatinine and BUN levels were significantly reduced 1.42- and 1.2-fold, respectively, in Moringa treatment mice. The relative
percentage of CD44 was not changed i n diabetic mice, b ut the relative per centage of CD69 was found to be increased. INF-γ was decreased
2.4-fold in diabet ic mice and elevated in treated groups . Conclusion: Moringa may ameliorate insulin r esistance, increase TAC, and improve
immune tolerance.
Keywords: blood urea nitrogen, creatinine, insulin resistance, total antioxidant capacity, immune tolerance
Introduction
Diabetes mellitus is one of the most common worldw ide
diseases. As diabetes is a multifactorial disease, its treat-
ment is complicated and requires multiple therapeutic
strategies. Diabetes is associated with the increased
level of blood glucose, which causes hyperglycemia.
The latter triggers oxidative stress that halts the bio-
logical activities, and cause diabetic complications
[1, 2]. Hyperglycemia-mediated oxidative stress plays
a key role in the pathogenesis of diabetic complications
such as nephropat hy
[3]. So, the optimal antidiabetic
drug should combine both hypoglycemic and antioxi-
dant properties. The main drawback of the current drugs
available for diabetes is their potential toxicity in the long
run and lacking efficiency
[4]. Moringa leaves are rich in
proteins, calcium, iron, potassium, vitamins (particularly
C and E), β-carotene
[5], and in antioxidant and
bioactive compounds, such as flavonoids, phenolic acid s,
glucosinolates and isothiocyanates, tannins, and saponins
[6]. Therefore, it seems that Moringa leaves are the first
source of the numerous pharmacological properties at-
tributed to Moringa oleifera leaves. In this regard,
flavonoids and polyphenols are described as natural
antioxidants. Since polyphenols and flavonoids can di-
rectly react with superoxide anions and lipid peroxyl
radical and consequently inhibit or break the chain of
lipid peroxidation
[7]. This radical scavenging activity
of extracts could be related to the antioxidant nature of
polyphenols or flavonoids, thus contributing to their
electron/hydrogen donating ability. Interestingly, the
β-sitosterol isolated from the leaves of M. oleifera is a
plant sterol with close chemical resemblance to choles-
terol, which enables it to block the absorption of
This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium for non-commercial purposes, provided the original author and source are credited.
Interventional Medicine & Applied Science, Vol. 8 (3), pp. 109–117 (2016)
ORIGINAL PAPER
DOI: 10.1556/1646.8.2016.3.7 109 ISSN 2061-1617 © 2016 The Author(s)
Unauthenticated | Downloaded 08/26/22 10:55 AM UTC
cholesterol by competitive inhibition [8]. Since Moringa
has an impact on the immune system, it could stimulate
both cellular and humoral immune responses
[9, 10].
Oxidative stress has emerged in the pathogenesis of
many diseases including diabetes
[11, 12]. Individual
oxidative stress markers including the measurement of
antioxidant enzymes-superoxide dismutase, catalase, glu-
tathione reductase, glutathione peroxidase, ceruloplas-
min, and proteins such as metallothioneins have been
used for decad es for mentoring the potency of the
antioxidant defense system. Recently, a new test to
measure the total antioxidant status was introduced,
which has been designated as total antioxidant capacity
(TAC)
[13]. The major advantage of this test is to
measure the TAC of all antioxidants in a biological
sample and not just the TAC of a single compound.
Since the measure of TAC considers the cumulative
action of all the antioxidants present in plasma and body
fluids, thus providing an integrated parameter rather
than the simple sum of measurable antioxidants
[14].
Furthermore, assessment of plasma TAC could provide a
clear picture about the physiological status, and may help
to identify the state and potential of oxidative stress in
the organism. The aim of this study was to investigate
the ameliorative effects of low doses of aqueous Moringa
extract on diabetes and its impact on the TAC and
immune tolerance, which have not yet been studied. In
another study on the same model with alloxan, I found
that Moringa promotes the activity of both CD4
+
and
CD8
+
T cells in diabetic treated mice that may occur
through the Sca-1
+
CD117
+
stem cell factors, which play
an important role as hematopoietic regulators (data not
shown). Also, administration of Moringa leaf extract
enhanced the percentage of the endothelial pro-
genitors (CD34
+
CD117
+
) and mature endothelial
cells (CD34
+
CD117
−
). Moringa also increased the per-
centage of blood-derived circulating angiogenic cells
(Sca-1
+
/CD34
+
). This study investigated the levels of
CD44 as a marker for the T-cell activation, the trans-
membrane CD69 protein, which is supposed to be
highly up-regulated in all immune cells, and INF-γ,
which is a potent activator of macrophages.
Materials and Methods
Moringa oleifera aqueous extract preparation
Moringa aqueou s extract was p repared by mixing 1 0 g
of dried and powdered M. oleifera leaves with 100 mL
of distilled water for 24 h and then stored at 4 °C.
Afterward, the mixture was filtered twice through a
2-μmporefilter paper. The aqueous extract stock
solution (100 mg/mL) was stored at 4 °C for up
to 5 days, or freshly prepared for each set o f
experiment.
Animals care and treatments
Forty albino mice (20 ± 5 g) were adapted in the labora-
tory for 2 weeks under the same natural environmental
condition of temperature and photoperiod and with free
access of food and water. All the procedures were in
accordance with the protocol of National Animal Care
and Use Committee and Guidelines for the Care and Use
of Experimental Animals. Mice were randomly divided
into four groups (10 mice each) as follows: control group,
diabetic untreated group, mice received oral administra-
tion of M. oleifera aqueous extract (100 mg/kg), and
diabetic treated groups with 100 mg/kg of M. oleifera
aqueous extract given by an oral gavage for 14 days after
diabetes induction (mice lived for 21 days). Diabetes was
induced with two intraperitoneal injections of alloxan
(Sigma-Aldrich), previously dissolved in ice-cold phos-
phate-buffered saline, pH 6.8 (Merck Millipore). The first
dose was 150 mg/kg as recommended by Bromme et al.
[15], and the second dose was 100 mg/kg given 2 days
after the first dose to ensure the induction of diabetes
throughout the experimental duration. Diabetes thresh-
old was plasma glucose level >250 mg/dL (Diagnostics,
Indianapolis, IN, USA).
Plasma Measurements
Insulin resistance by homeostasis model assessment of insulin
resistance (HOMA-IR)
Blood was taken from the tail vein of the fasting mice
(12 h); the glucose level was estimated by LifeScan
OneTouch® UltraEasy meter. Insulin level was deter-
mined according to the instructions of kit’s manufacturer
(Mercodia-10-1251-01). HOMA-IR was determined
using the following formula: HOMA-IR = fasting glu-
cose value (mg/dL) × fasting insulin value (μU/mL)/
405
[16].
Biochemical analysis
The TAC was measured in the plasma according to a
previously described method
[17], and its activity was
expressed in mM/mg protein. The total protein was
determined using Bio-diagnostic kit according to a pre-
viously described method
[18]. The levels of creatinine
and blood urea nitrogen were determined by the com-
mercially available kits (Bio-Diagnostic Co., Egypt).
Flow cytometry analysis
Peripheral blood mononuclear cells and splenocytes from
mice were incubated with primary antibody, including
Tuorkey
ISSN 2061-1617 © 2016 The Author(s) 110 Interventional Medicine & Applied Science
Unauthenticated | Downloaded 08/26/22 10:55 AM UTC
CD44 (156-3C11) mouse mAb, CD69 (Clone H1.2F3),
and IFN-γ (bs-0480R). Stained cells were resuspended in
Flow Cytometry Staining Buffer and analyzed by flow
cytometry. FACSCanto II (BD Biosciences, SanJose,
CA, USA) was used for acquisition. CellQuest (BD
Biosciences, SanJose, CA, USA) and FlowJo software were
used for data analysis. The absolute numbers of cells were
calculated using the following formula: the percent of
cells × the total number of white blood cells/100.
Statistical and data analysis
The data were analyzed with Sigma Plot 10 software
(Systat Software Inc., San Jose, CA, USA), and Prism 3.0
package (GraphPad Software Inc., San Diego, CA, USA).
One-way analysis of variance (ANOVA) Newman–Keuls
multiple test was used as a post-hoc comparison test. The
significant difference was set at P < 0.05.
Results
As shown in Fig.
1A, a significant increase was noted in the
level of glucose in diabetic mice (321.2 ± 33.93 mg/dL)
compared with the control group (140.8 ± 13.61 mg/dL).
The level of glucose was significantly higher in diabetic
mice treated with Moringa by 1.7-fold when compared
with the control group. However, due to treatment with
Moringa, the level of glucose was decreased by 1.28-fold
compared with the diabetic group. Therefore, treating
diabetic mice with Moringa significantly reduced hyper-
glycemia, maintaining mean glucose levels at 249.2 ±
11.77 mg/dL. The level of insulin in plasma reflects the
function of the pancreatic beta cells and the sensitivity of
tissues to insulin through glucose uptake. As shown in
Fig.
1B, the insulin level was significantly declined from
14.35 ± 1.35 mg/dL in the control group to 5.35 ±
0.84 mg/dL in the diabetic group. The level of insulin
was significantly increased to 9.800 ± 1.530 mg/dL
in the diabetic group because of treatment with
Moringa. Surprisingly, an increase occurred in mice
treated with Moringa alone (20.00 ± 1.673), and that
was statistically significant compared with the control
group.
HOMA-IR analysis
HOMA-IR was calculated from the values of serum
glucose (mg/dL) and serum insulin (μU/mL) in mice
fasted over night (Fig.
2). The level of insulin resistance
was significantly increased from 7.172 ± 0.815 (mg/dL ×
mU/mL) in the control group to 13.09 ± 0.965 (mg/dL ×
mU/mL) in the diabetic untreated group. As a result of
Moringa treatment, insulin resistance was significantly
decreased to 10.31 ± 0.466 (mg/dL × mU/mL) com-
pared with the diabetic untreated group, although it was
still significantly higher compared with the control group.
Total antioxidant capacity
The TAC was prominently declined to 0.27 ±
0.057 mM/mg protein in the diabetic untreated mice
when compared with the control group, which recorded
0.526 ± 0.026 mM/mg protein (Fig.
3). There was no
significant difference between the mice received Moringa,
which found to be 0.463 ± 0.02 mM/mg protein, com-
pared with the control group. Treatment of diabetic mice
with Moringa significantly enhanced and restored the
TAC to 0.453 ± 0.029 mM/mg protein.
Fig. 1. Fasting glucose (A) and insulin (B) levels in different mice involved in this study. Data were expressed as mean ± SE of 10 mice in each
group. *P < 0.05, **P < 0.01, and ***P < 0.001, NS: statistically non-significant
The antidiabetic effect of Moringa oleifera
Interventional Medicine & Applied Science 111 ISSN 2061-1617 © 2016 The Author(s)
Unauthenticated | Downloaded 08/26/22 10:55 AM UTC
Creatinine and urea levels
As shown in Fig.
4, the level of plasma creatinine was
significantly increased in diabetic untreated mice (0.49 ±
0.066 mg/dL) compared with the control group (0.11 ±
0.0089 mg/dL). There was a non-significant difference
in the mice received Moringa (0.49 ± 0.066 mg/dL)
when compared with the control group. Due to treat-
ment of diabetic mice with Moringa, the level of creati-
nine was significantly reduced to 0.344 ± 0.078 mg/dL.
On the other hand, the level of urea was significantly
enhanced from 6.050 ± 0.27 mg/dL in the control group
to 11.12 ± 1.24 mg/dL in the diabetic untreated
group. The level of urea in the mice received Moringa
was recorded 7.02 ± 0.511 mg/dL, reflecting a non-
significant difference when compared with the control
group. The level of urea was significantly declined
to 9.16 ± 0.96 mg/dL when compared with the diabetic
untreated group.
Flow cytometry analysis
The Fluorescence Minus One gating boundaries for
CD44, IFN-γ, and CD69 molecules are shown in
Fig.
5. As shown in Fig. 6, there was no significant
difference in the percent of CD44 molecules when the
diabetic untreated group was compared with the control
group (73.74 ± 0.98 and 70.30 ± 1.70, respectively).
Compared with all groups, the maximum percent of
CD44 molecules was recorded in the diabetic treated
group with Moringa (87.20 ± 2.048).
Although there wa s an increase in the percent of the
expression of CD69 PE.Cy7 in the diabetic untreated
group, which recorded 4.138 ± 0.512, when compared
with the control group 1.43 ± 0.092, but that increase
was not significant (Fig.
7). When compared with the
control group, Moringa promotes the activity of CD69
PE.Cy7 expression as indicated in the mice received
Moringa and in the diabetic mice treated with Moringa,
which were recorded 5.49 ± 0.87% and 8.33 ± 1.30%,
respectively.
INF-γ production was significantly decreased in dia-
betic mice, which recorded 2.059 ± 0.417%, compared
with the control group that recorded 5.03 ± 0.80%. Mor-
inga significantly enhanced the productio n of INF-γ in
mice to 9.70 ± 0.57% in normal mice and 12.24 ± 1.34%
in diabetic treated mice (Fig.
8).
Discussion
Insulin is a critical hormone for the process of cellular
glucose uptake, and thus mainstreaming the normal levels
of blood glucose. Diabetes mellitus is a multifactorial
disease marked by hyperglycemia due to the impairment
in insulin secretion and/or periphera l insulin resistance
[19]. Insulin resistance is defined as a reduced respon-
siveness of insulin on a targe t cell or a whole organ, which
results in reducing insulin-mediated glucose utilization in
peripheral tissues, accompanying glucose intolerance and
insulin intolerance
[20]. Although HOMA-IR is a marker
usually used in human type 2 diabetes, not routinely
Fig. 3. Total antioxidant capacity in different mice involved in this
study. Data were expressed as mean ± SE of 10 mice in
each group. *P < 0.05, **P < 0.01, and ***P < 0.001,
NS: statistically non-significant
Fig. 2. HOMA-IR analysis in different mice involved in this study.
HOMA-IR was calculated from glucose (mg/dL) and
insulin (μU/mL) levels using the following formula:
HOMA = fasting glucose value (mg/dL) × fasting insulin
value (μU/mL)/405. Data were expressed as mean ± SE
of 10 mice in each group. *P < 0.05, **P < 0.01, and
***P < 0.001, NS: statistically non-significant
Tuorkey
ISSN 2061-1617 © 2016 The Author(s) 112 Interventional Medicine & Applied Science
Unauthenticated | Downloaded 08/26/22 10:55 AM UTC
evaluated in mice, this study determined it in order to
investigate whether treatment with Moringa could aid us
to overcome the insulin resistance. HOMA-IR in diabetic
mice was increased about 4.5-fold than that in the control
group. As a result of treatment of diabetic mice with
Moringa, HOMA-IR was decreased 1.3-fold compared
with the diabetic untreated mice. The antihyperglycemic
and antioxidant abilities of Moringa leaves extract may be
associated with its ability to improve insulin sensitivity in
diabetic mice.
On the other hand, oxidative stress has recently
emerged and involved in the etiology of many diseases
including diabetes. Oxidative stress causes membrane
damage leading finally to membrane rupture in different
cellular types. Cells possess an efficient antioxidant defense
system against destructive damage induced by oxidative
Fig. 4. The level of creatinine (A) and blood urea nitrogen (B) levels in different mice involved in this study. Data were expressed as mean ± SE of
10 mice in each group. *P < 0.05, **P < 0.01, and ***P < 0.001, NS: statistically non-significant
Fig. 5. The Fluorescence Minus One gating boundaries for CD44, IFN-γ, and CD69 molecules
The antidiabetic effect of Moringa oleifera
Interventional Medicine & Applied Science 113 ISSN 2061-1617 © 2016 The Author(s)
Unauthenticated | Downloaded 08/26/22 10:55 AM UTC