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# Pharm D undergraduate;
† Assistant Professor;
*Corresponding author: E-mail: yzhindi@uqu.edu.sa;
Journal of Pharmaceutical Research International
33(58A): 336-343, 2021; Article no.JPRI.79896
ISSN: 2456-9119
(Past name: British Journal of Pharmaceutical Research, Past ISSN: 2231-2919,
NLM ID: 101631759)
Oxidative Effects in Streptozotocin-induced Male
and Female Mice: The Effect of Garlic Oil and
Melatonin
Abeer Al-Rashedi a#, Banan Al-Johari a#, Rahaf Al-Abbasi a#, Raghdh Sindi a#,
Rawan Bin Hameed a#, Sulafah Al-Saadi a#, Sara Al-Awam a#,
Mawada Bukhari a#, Rahaf Al-Talhi a# and Yosra Al-Hindi b*†
a Faculty of Pharmacy, Umm Al-Qura University, KSA, Saudi Arabia.
b Clinical Pharmacy, Faculty of Pharmacy, Umm Al-Qura, Saudi Arabia.
Authors’ contributions
This work was carried out in collaboration among all authors. All authors read and approved the final
manuscript.
Article Information
DOI: 10.9734/JPRI/2021/v33i58A34124
Open Peer Review History:
This journal follows the Advanced Open Peer Review policy. Identity of the Reviewers, Editor(s) and additional Reviewers,
peer review comments, different versions of the manuscript, comments of the editors, etc are available here:
https://www.sdiarticle5.com/review-history/79896
Received 10 October 2021
Accepted 14 December 2021
Published 15 December 2021
ABSTRACT
Background: Recent studies have revealed that a hyperglycemia-induced overproduction of
superoxide can be the first event in the activation of all pathways involved in the pathogenesis of
complications of diabetes. Supplementation of garlic was found to decrease diabetes-induced
oxidative stress complications. Studies shown also that melatonin attenuates diabetes‐induced
oxidative stress in diabetic induced rabbits and rats.
Objective: In this present study, oxidative stress in diabetic model and the effect of garlic oil or
melatonin treatment were examined in both genders' male and females' mice.
Methods: 96 mice were randomly divided into 5 groups including control (C), diabetic (D),
melatonin 10 mg/kg (D+M), garlic extract 100 mg/kg (D+G) and combined melatonin and garlic
(D+M+G). All treatments were given orally daily for 16 weeks after induction of hyperglycemia by
streptozocin (STZ). Fasting blood glucose and antioxidant levels were estimated.
Results: Streptozotocin induced diabetic mice, showed a significant increase of plasma glucose,
lipid peroxide and uric acid. Accordingly, significant decreases in the levels of antioxidants
Original Research Article
Al-Rashedi et al.; JPRI, 33(58A): 336-343, 2021; Article no.JPRI.79896
337
ceruloplasmin were found in the plasma of diabetic mice. Treatment of diabetic mice with garlic oil
or melatonin for 16 weeks significantly increased plasma levels of ceruloplasmin activities. Lipid
peroxides, uric acid, blood glucose was decreased significantly after treatment with garlic oil or
melatonin.
Conclusion: The results suggest that garlic oil or melatonin may effectively normalize the impaired
antioxidants status in streptozotocin induced diabetes in both males and females mice.
Keywords: Diabetes; Garlic; Melatonin; STZ; mice; antioxidants.
1. INTRODUCTION
Nowadays, researches are focused on the
relation of antioxidants activity and diabetes [1].
The oxygen species like hydrogen peroxide,
superoxide anions, singlet oxygen and hydroxyl
radicals could be developed through ionizing
radiation and aerobic metabolism of either
endogenous or exogenous materials [2].
Evidence have shown that diabetes has been
related to an increased free radical production
[3]. The mechanisms of this state could be
contributed to the free radical development in
diabetes that can increase the non-enzymatic
and auto-oxidative glycosylation, and moreover
metabolic stress due to differences in the
metabolism of energy, inflammatory processes,
and antioxidant activation [4].
A lot of focus has been developed in using herbal
medicine and other natural products in treating
diseases that have a state of oxidative stress like
diabetes and metabolic syndrome [5]. Garlic is
one of the elements that were used as herbal
therapy for long time and have been seen to help
in eliminating cardiovascular risks and diabetes
[6]. Moreover, melatonin was found to have a
great antioxidant ability towards diseases [7].
To our knowledge, no previous researches if any
used the combination of garlic and melatonin in
diabetic subjects to measure antioxidants
activity. Therefore, due to the frequent use of
antidiabetic drugs with their associated side
effects, we hypothesized that combination of low
doses of garlic and melatonin might have
beneficial effects on glycemic and antioxidants in
diabetes mellitus. Also, this combination can be
used with traditional antidiabetic drugs to
reduce their doses and potential side effects. The
aim of this study was to investigate the effects
of low doses of melatonin and garlic extract
both individually and combined on blood
glucose levels as well as antioxidants in
streptozotocin induced diabetic male and female
mice.
2. MATERIALS AND METHODS
2.1 Reagents
Reagents and chemicals including were
purchased from Sigma-Aldrich (MO, USA),
unless otherwise stated.
2.2 Preparation of Garlic Extract
Black garlic (BG) was supplied from Ueisung-
Nongsan (Korea), and extracted by heating with
water twice under reflux at 80°C (yield, 12.8%).
The resulting solution was freeze-dried after
evaporation and kept at 4°C until use. The BG
was fermented with edible Saccharomyces
cerevisiae (KCTC 7910) by 2-stage cultivation. At
the first stage cultivation, the microorganism was
cultivated in a medium containing 3% (wt/vol)
malt extract for 36 hours at 28°C to enhance cell
growth. The cell mass was obtained by
centrifugation and recultivated in a medium
containing 5% (wt/vol) of the BG extracts under
the same conditions to increase the
concentration of physiologically active
substances, such as polyphenol and allycystein,
which play a key role in antioxidation. After
cultivation, the culture solutions were extracted
by heating after filtration to remove the cells. The
solution was then freeze-dried after evaporation
and kept at 4°C until needed. Then it was
dissolved directly in distilled water and
administered orally at a dose of 100 mg/kg, once
per day for 16 weeks after induction of diabetes
[8]. The S-allyl cysteine sulfoxide is responsible
for anti-peroxide effect of garlic.
2.3 Melatonin
Melatonin from (Sigma-Aldrich Co., St. Louis,
MO) 10 mg/kg daily dissolved in 0.04% ethanol
added to the drinking water at night [9].
2.4 Animals
All procedures concerning animal care and
treatment were approved by Umm Al-Qura
Al-Rashedi et al.; JPRI, 33(58A): 336-343, 2021; Article no.JPRI.79896
338
university’s Biomedical and research Ethics
Committee (HAPO-02-K-012-2021-10-788). For
this study total number of 96 males and females
of C57BL/6J (B6) strain mice were purchased
from Harlan (Charles River Laboratories,
Wilmington, Massachusetts, USA). Mice were
maintained in a temperature-controlled room (23
± 1°C) under a 12-h to 12-h light to dark cycle.
Mice were individually housed in standard cages
with ad libitum water and standard chow (CRM
pellets, SDS diets, U.K.).
Measurements then started at the age of 10
weeks with ranging body weight of 25.7g to
30.33g and were taken over a period of 16
weeks. We choose this period of time because
diabetes is a chronic disease and we wanted to
explore this at acute and long-term use of these
herbal medicines. After 16 weeks mice were
fasted overnight and euthanized by CO2 and
blood samples were taken by cardiac puncture.
2.5 Induction and Assessment of
Diabetes
A method of inducing type 2 DM with
hyperglycemia and relatively low insulin levels
can be produced STZ. Mice were
intraperitoneally (i.p.) injected with STZ (50
mg/kg of BW) in 0.1 M citrate buffer (pH 4.2) on
two consecutive days. NA (120 mg/kg of BW) in
saline was i.p. injected 30 min before the STZ
injection on the first day after overnight fasting.
Seven days after the second i.p. injection, mice
that exhibited an 8-h fasting blood glucose (FBG)
level of 200 mg/dL were recognized as being
hyperglycemic. The others that exhibited an FBG
level of <200 mg/dL were injected with STZ and
monitored until the FBG level reached 200
mg/dL. FBG was monitored with a glucometer
(Dragon Pharmaceutical Co, New Taipei,
Taiwan).
2.6 Experimental Design
Mice were randomly classified into 4 groups
(n=16 each, 8 Males and 8 Females); one group
served as a control non-diabetic group and 3
groups were injected with STZ to induce
diabetes, then they received their respective drug
treatments daily for 16 consecutive weeks
according to the following design (Fig. 1):
• Group 1(C); control non-diabetic mice
treated with saline orally and citrate buffer
intraperitoneally.
• Group 2 (D); nontreated induced diabetic
mice.
• Group 3 (D+M); induced diabetic mice
treated with melatonin [9] (10 mg/kg/day in
drinking water)
• Group 4 (D+G); induced diabetic mice
treated with prepared garlic extract [8] (100
mg/kg/day in drinking water)
The doses of melatonin and garlic were chosen
based on previous experimental studies that
examined wide range of doses in diabetes. The
lowest doses were selected for both melatonin
and garlic due to the potential side effects of
large doses.
At 2nd and 16th weeks, the levels of FBG were
examined. After 16 weeks antioxidants were
examined.
Fig. 1. Experimental design of the study
Al-Rashedi et al.; JPRI, 33(58A): 336-343, 2021; Article no.JPRI.79896
339
2.7 Assessment
2.7.1 Fasting blood glucose measurements
At week 2 and 16 all mice were fasted overnight
and blood were taken from their tails in order to
measure their FBG and insulin levels per the
manufacturer’s instructions. Fasting blood
glucose was measured with a One Touch II
glucose meter (Lifescan, Inc., Johnson &
Johnson, Milpitas, CA).
2.7.2 Antioxidant's measurements
Lipid peroxide levels were measured in plasma,
hemolysate and tissue homogenates as
thiobarbituric acid reactivity (TBARS) [10]. The
product of the reaction between malondialdehyde
and thiobarbituric acid was measured as
described by Thayer. Ceruloplasmin activity was
determined using a para-phenylenediamine
dihydrochloride method [10]. Uric acid was
determined by enzymatic colorimetric method
using commercial kit (Biocon,
BurbachyGermany) [10].
2.7.3 Statistical analysis
All results are expressed as group means ±
SEM. Results were analyzed by one-way
analysis of variance, followed by Tukey’s post-
hoc test to assess significance, using a criterion
of P value of less than 0.05. The statistical
analysis was carried out using GraphPad Prism
version 5 (GraphPad Software Inc., California,
USA).
3. RESULTS
3.1 Effects of Melatonin and Garlic on
Fasting Blood Glucose in Diabetic
Mice
Fig. 2 describes the effect of garlic, melatonin
and their combination on fasting blood glucose
on both male and female mice. It is significant
that after 16 week there were a vast
improvement in all treatments in decreasing
blood glucose levels in diabetic mice (P<0.005).
3.2 Effects of Melatonin and Garlic on
Antioxidant levels in Diabetic Mice
Fig. 3 describes the effect of garlic, melatonin
and their combination on antioxidants levels on
both male and female mice. It is significant that
after 16 weeks of treatments there was an
increased plasma level of ceruloplasmin
activities. Lipid peroxides and uric acid were
decreased significantly after treatment with garlic
oil or melatonin or their combinations (P<0.005).
Al-Rashedi et al.; JPRI, 33(58A): 336-343, 2021; Article no.JPRI.79896
340
Fig. 2. Effects of Melatonin and Garlic on Fasting Blood Glucose in Diabetic Mice; A) Plasma
Glucose measurements in week 2 of the study. B) Plasma Glucose measurements in week 16
of the study C; control, D; diabetic induced mice, D+M; diabetic mice treated with melatonin,
D+G; diabetic mice treated with garlic, D+M+G; diabetic mice treated with combination of
melatonin and garlic
*P<0.05 compared to control mice, **P<0.05 compared to diabetic mice
Al-Rashedi et al.; JPRI, 33(58A): 336-343, 2021; Article no.JPRI.79896
341
Fig. 3. Effects of Melatonin and Garlic on antioxidant levels in Diabetic Mice; A) Levels of lipids
peroxidase; B) Ceruloplasmin and C) Uric acids after 16 weeks of treatments
*P<0.05 compared to control mice, **P<0.05 compared to diabetic mice, +P<0.001 compared to D+M+G
4. DISCUSSION
Diabetes can cause multiple changes
systemically and also upon the cellular levels
[10,11]. In our present study, diabetic mice both
genders induced by streptozotocin showed the
expected raise in their plasma glucose levels and
the decrease in antioxidant levels. However,
treatment of diabetic mice with garlic oil or
melatonin for 16 weeks significantly increased
plasma levels of ceruloplasmin activities. Lipid
peroxides, uric acid, blood glucose was
decreased significantly after treatment with garlic
oil or melatonin. Our results were in agreement
of other researchers whom used garlic or
melatonin alone, they showed a lower levels of
blood glucoses and other parameters as
cholesterol and lipid profile [11,12,13,14].
Researches tried to explain the possible
mechanism of this blood glucose lowering, for
garlic they approved multiple pathways of anti-
inflammatory and antihyperlipidemic and
antioxidant effects. On the other hand, for the
melatonin they believed it works on brain site and
can achieves this action through direct
detoxification of reactive oxygen and reactive
nitrogen species and indirectly by stimulating
antioxidant enzymes at the same time inhibiting
the activity of enzymes as prooxidant's.
Moreover, melatonin shown to chelates transition
metals, that have an involvement in the
Fenton/Haber–Weiss reactions; therefore,
melatonin can decrease toxic hydroxyl radical
that results in the inhibition of oxidative stress.
However, more investigation for a clearer
explanation is in need [15]. Evidence showed
also that when given melatonin to rats taking
cytotoxic drugs that there state of antioxidants
were improved [16,17,18,19].
There is evidence from a study done on
antiperoxide effect of garlic oil on various tissues
and blood cell counts of arsenic exposed albino
mice found that there was a significant reduction
in the arsenic levels. That indicates garlic pearls
can be administered to persons who are working
in places of coal burning, mining and metal ore
smelting, as the diallyl disulphide (DADS) that is
the active principle of garlic has a possible
protective role on LPO status of many tissues
and blood cell counts of arsenic exposed mice
[20].
From the benefits shown in our study of using the
combination of garlic and melatonin together in
diabetic subjects, we recommend starting a trail
with diabetic patients using the combination of
melatonin and garlic to investigate in depth their
beneficial effect.
5. CONCLUSION
Our results from this present study suggest that
garlic oil or melatonin may effectively normalize
the impaired antioxidants status in streptozotocin
induced diabetes in both males and females
mice. The effects of these antioxidants of both
agents may be useful in delaying the
complications of diabetes such as retinopathy,
nephropathy and neuropathy due to imbalance
between free radicals and antioxidant systems.
Al-Rashedi et al.; JPRI, 33(58A): 336-343, 2021; Article no.JPRI.79896
342
DISCLAIMER
The products used for this research are
commonly and predominantly use products in our
area of research and country. There is absolutely
no conflict of interest between the authors and
producers of the products because we do not
intend to use these products as an avenue for
any litigation but for the advancement of
knowledge. Also, the research was not funded by
the producing company rather it was funded by
personal efforts of the authors.
CONSENT
It is not applicable.
ETHICAL APPROVAL
All procedures concerning animal care and
treatment were approved by Umm Al-Qura
university’s Biomedical and research Ethics
Committee (HAPO-02-K-012-2021-10-788).
The study highlights the efficacy of " Herbal "
which is an ancient tradition, used in some parts
of India. This ancient concept should be carefully
evaluated in the light of modern medical science
and can be utilized partially if found suitable.
DATA AVAILABILITY
The datasets generated and/or analysed during
the current study are available from the
corresponding author on reasonable request.
COMPETING INTERESTS
Authors have declared that no competing
interests exist.
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© 2021 Al-Rashedi et al.; This is an Open Access article distributed under the terms of the Creative Commons Attribution
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