The Effect of Alpha-mangostin in Glucose Level, Cholesterol Level

and Diameter of the Islets of Langerhans of STZ-induced Diabetic

Mice

Saikhu Akhmad Husen

1,2

, Salamun

, Arif Nur Muhammad Ansori

, Raden Joko

Kuncoroningrat Susilo

, Suhailah Hayaza

and Dwi Winarni

1,2

Department of Biology, Faculty of Science and Technology, Universitas Airlangga, Surabaya, Indonesia

Animal Histology Laboratory, Faculty of Science and Technology, Universitas Airlangga, Surabaya, Indonesia

Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, Indonesia

Keywords: Alpha-mangostin, antidiabetic, antilipidemic, diabetic mice, streptozotocin.

Abstract: The increasing prevalence of Diabetes Mellitus (DM) worldwide is an issue of major socio-economic

concern. DM is a complex and a multifarious group of disorders that disturbs the metabolism of

carbohydrates, fat, and protein. Medicinal plants play an important role in the management of DM,

especially in developing countries. The aim of this study is to investigate the antidiabetic and antilipidemic

effects of alpha-mangostin in STZ-induced diabetic mice. We conducted the study using the male BALB/C

mice. The mice were divided into two groups: the normal control (KN) and the STZ-induced diabetic mice.

Streptozotocin (STZ) induction was performed using multiple low-doses of 30 mg/kg bw injected for five

consecutive days. The diabetic mice were grouped again into three subgroups: diabetic control (KD),

metformin HCL treated diabetic mice (KM), and diabetic mice which were treated using alpha-mangostin at

2 mg/kg bw (P1), 4 mg/kg bw (P2), and 8 mg/kg bw (P3). Before and after STZ injection, the blood glucose

and the cholesterol levels were observed. The blood glucose and the cholesterol levels were also measured

on the 1

, 7

, and 14

day of alpha-mangostin treatments. Treatment was given for 14 days. At the 15

day,

the pancreases were collected and then processed into histological slides. The results of this experimental

study indicated that alpha-mangostin has hypoglycemic and hypolipidemic activities which can ameliorate

the damaged islets of Langerhans in STZ-induced diabetic mice. Therefore, we concluded that alpha-

mangostin is a promising antidiabetic and antilipidemic agent due to its antioxidant activity.

1 INTRODUCTION

DM is a metabolic disorder that affects about 6% of

the world population. DM is characterized by the

prolonged hyperglycemic conditions due to the

reduction of both insulin secretion and insulin

sensitivity (Kang et al., 2014). DM is divided into

type-1 DM (insulin dependent DM) and type-2 DM

(non-insulin dependent DM). Type-1 DM is an

autoimmune disease which causes the immune

system to attack pancreatic cells, thus damaging a

person's ability to produce insulin. Type-2 DM is a

metabolic disorder that is characterized by an insulin

resistance, a decrease of cell sensitivity to insulin,

and a relative lack of insulin due to the damage

suffered by pancreatic islet β-cells (American

Diabetes Association, 2013). The decrease of cell

sensitivity to insulin is a typical condition, as well as

the cause of type-2 DM. Progressive decrease in

insulin secretion is generally a result of decreased

tissue sensitivity to insulin (McClung et al., 2004;

Husen et al., 2017a; Husen et al. 2017b).

Beside the prolonged hyperglycemic conditions,

one of the factors which causes DM is obesity due to

the increased levels of fat in the body caused by

hyperlipidemic and escalating levels of cholesterol

in the blood (Husen et al., 2016; Husen et al.,

2017a). The increased blood cholesterol levels can

be followed by the dilated levels of free fatty acids,

which generate the enlarged superoxide production

by mitochondria and the higher risk of cell exposure

by the reactive oxygen species (ROS). A growth in

superoxide production will lead to an increase in

nitric oxide (NO) caused by the induction of NO

synthase enzymes. This condition leads to the

Husen, S., Salamun, ., Ansor i, A., Susilo, R., Hayaza, S. and Winarni, D.

The Effect of Alpha-mangostin in Glucose Level, Cholesterol Level, and Diameter of the Islets of Langerhans of STZ-induced Diabetic Mice.

DOI: 10.5220/0007547005610566

In Proceedings of the 2nd International Conference Postgraduate School (ICPS 2018), pages 561-566

ISBN: 978-989-758-348-3

561

production of reactive nitrogen species (RNS),

which will oxidize the sulfhydryl groups of proteins,

especially amino nitrate acids such as tyrosine,

which can increase lipid peroxidation and cause a

harmful DNA damage to cells (Novelli et al., 2010;

Husen et al., 2017a).

The condition of hyperlipidemia in the people

with obesity can boost the oxidative stress in the

body, which can lead to some complications. People

with obesity also experience heightened levels of

cholesterol in the body (hypercholesterolemia)

caused by excessive accumulation of fat in the body.

One of the negative effects of obesity is the insulin

resistance, which is the inability of insulin to

produce the normal biological functions, which

causes decreased tissue sensitivity to insulin. The

cell resistance to cellular action of insulin is

developed in people with obesity, which is

characterized by the reduced ability of insulin to

support the glucose intake in fat and muscle

resulting in the hyperglycemic conditions (Husen et

al., 2017a; Husen et al., 2017b). The condition of

hyperglycemia leads directly to the increased levels

of ROS and RNS. ROS and RNS can directly

oxidize and destroy DNA, proteins, and lipids. High

levels of ROS and RNS also can damage the

macromolecules indirectly, which causes oxidative

stress. Oxidative stress occurs when there is an

imbalance between the number of highly reactive

molecules (ROS and RNS) with the existing

antioxidants (Novelli et al., 2010; Husen et al., 2018;

Ansori et al., 2018).

Antioxidants are substances that can prevent the

negative effects of free radicals by providing

electrons to enable to suppress damages of lipids,

cell membranes, blood vessels, DNA, and other

damages caused by the reactive compounds, such as

ROS and RNS. To reduce the occurrence of the free

radical’s effect, extra antioxidants from the outside

(exogenous), such as vitamin E, vitamin C and other

antioxidants obtained from consuming various types

of fruits and vegetables that contain high

antioxidants, are needed. One type of antioxidants

which still provides a chance to overcome free

radicals up until today is alpha-mangostin. The

alpha-mangostin compound is a pigment of Garcinia

mangostana, which is able to contribute hydrogen

atoms and stabilize the free radicals by resonance,

which is hard to participate in other radical reactions

(Husen et al., 2018). In addition to neutralizing free

radicals, antioxidants are expected to reduce

oxidative stress, mainly in various cells affected by

the prolonged hyperglycemic conditions, such as

hepatocyte cells in the liver and renal tubular cells

(Vallon, 2011; Ansori et al., 2018).

Indonesia has a high number of biodiversity,

which contains various natural potentials that can be

utilized for the treatment of various diseases

(Wahyuni et al., 2017; Ansori et al., 2018). One of

Indonesia's original flora which currently has great

potential to be developed as a medicinal raw

material is mangosteen (Ansori et al., 2018). The

pericarp of the mangosteen fruit contains an active

compound known as xanthone. Beside having anti-

hypertensive and anti-inflammatory potential,

xanthone compounds also play an important role as

a powerful antioxidant compared to vitamin C and

vitamin E in preventing free radicals and cell

damage, as well as inhibiting cell degeneration

processes (Chin et al., 2008). The xanthone

compounds contained in the pericarp of the

mangosteen, especially alpha-mangostin

compounds, have been proven ameliorate the

damaged pancreatic islet β-cells so that insulin can

be produced optimally (Husen et al., 2017b; Husen

et al., 2018). Based on the above background

information, it has now been widely reported that the

raw mangosteen pericarp extract is able to lower

glucose and blood cholesterol levels. However,

presently there has been no scientific explanation

about the potential of alpha-mangostin to reduce

blood glucose and cholesterol levels, as well as to

ameliorate the pancreatic islet β-cells damages

caused by the prolonged hyperglycemic conditions.

Thus, this study was aimed to investigate the effects

of alpha-mangostin in glucose level, cholesterol

level, and diameter of the islets of Langerhans of

STZ-induced diabetic mice.

2 MATERIALS AND METHODS

This study was conducted at the Animal Laboratory,

Animal Histology Laboratory, and Molecular

Genetics Laboratory, Faculty of Science and

Technology, Universitas Airlangga. The ethical

clearance for this study was obtained from the

Committee of Animal Care and Use, Faculty of

Veterinary Medicine, Universitas Airlangga (701-

KE). The used samples were adult male mice of

BALB/C strain aged 3-4 months with weights

ranging from 30 to 40 g. The study materials consist

of alpha-mangostin and STZ (purchased from

Sigma), buffer citrate solution pH 4.5, phosphate-

buffered saline (PBS), solvent extract of

carboxymethylcellulose (CMC), standard

antidiabetic drugs (Metformin HCl 100 mg/kg bw),

ICPS 2018 - 2nd International Conference Postgraduate School

562

lard, xylazine and ketamine, and glucose (10% D-

glucose in aquadest). The main tools used were mice

cage made in plastic with lid of gauze wire, drinking

bottle, feeding plate, husk, microscope, Petri dish,

analytical scale, injection needles which have lead

tackle at the end, 1 mL insulin injection needle for

diabetic induction, Accu-Check® Active Test,

EasyTouch® GCU Multi-Function Monitoring

System, glass tools, and rotary vacuum evaporator

(Buchi).

The study samples consisted of 24 male mice,

distributed to the normal control group (KN) and

diabetic group (induced by STZ). The fasting blood

glucose and fasting blood cholesterol were measured

before and after the STZ induction. The

measurement of fasting blood glucose was

performed on the 7

and 14

day after induction of

STZ. Meanwhile, the measurement of blood glucose

levels was performed using a glucometer to

determine the diabetic condition of mice. Only the

mice which had the fasting blood sugar level of

more than 140 mg/dL were used as a diabetic mice

group. The grouping of experimental animals was

performed as follows: non-diabetic mice were used

as the normal control group (KN), the diabetic mice

induced by STZ were divided into 2 control groups,

namely the diabetic control group (KD), the diabetic

control group which was given Metformin HCl of

dose 100 mg/kg bw (KM), and, the last one, was the

alpha-mangostin treatment group. Furthermore, the

alpha-mangostin treatment group was divided into 3

subgroups, which were P1 which was given 2 mg/kg

bw, P2 which was given 4 mg/kg bw, and P3 which

was given 8 mg/kg bw. All treatments were

administered for 14 days.

The measurements of fasting blood glucose and

fasting blood cholesterol levels were performed in

all groups of mice before and after STZ induction,

the measurement of which then continued on the 1

, and the 14

day of the alpha-mangostin

treatment. The measurement of fasting blood

glucose was done by using Accu-Check® Active

Test, while the measurement of cholesterol levels

was performed using EasyTouch® GCU Multi-

Function Monitoring System. The measurements of

fasting blood glucose and cholesterol levels were

performed after the mice were fasted for 6-8 hours.

3 RESULTS AND DISCUSSION

The mean of mice’s fasting blood glucose level and

fasting blood cholesterol level data, before and after

STZ induction, are presented in Figure 1. The mean

of mice’s fasting blood glucose and fasting blood

cholesterol level data after alpha-mangoostin

treatment is presented in Figure 2, while the mean

data of diameter of the islets of Langerhans after the

administration of alpha-mangostin is shown in

Figure 3.

Figure 1: Blood glucose and cholesterol level (mg/dL)

before and after STZ induction. The different letter

indicated a significant difference.

The Effect of Alpha-mangostin in Glucose Level, Cholesterol Level, and Diameter of the Islets of Langerhans of STZ-induced Diabetic Mice

563

Figure 2: Blood glucose level and cholesterol level

changes of each mice groups after treatments. The same

letters above diagrams indicated an insignificant

difference, while different letter indicated a significant

difference. KN: normal control group; KD: diabetic group

without metformin HCl; KM: diabetic group with

metformin HCl 100 mg/kg bw; P1: diabetic treatment

group with alpha-mangostin 2 mg/kg bw; P2: diabetic

treatment group with alpha-mangostin 4 mg/kg bw; P3:

diabetic treatment group with alpha-mangostin 8 mg/kg

bw.

Figure 3: Diameter of the islets of Langerhans of each

mice group after treatments. Same letters above the

diagrams indicate insignificant difference, while different

letters indicate significant difference. KN: normal control

group; KD: diabetic group without metformin HCl; KM:

diabetic group with metformin HCl 100 mg/kg bw; P1:

diabetic treatment group with alpha-mangostin 2 mg/kg

bw; P2: diabetic treatment group with alpha-mangostin 4

mg/kg bw; P3: diabetic treatment group with alpha-

mangostin 8 mg/kg bw.

Figure 4: Histological structure of pancreatic gland and

diameter of the islets of Langerhans of each mice group

after treatments. KN: normal control group; KD: diabetic

group without metformin HCl; KM: diabetic group with

metformin HCl 100 mg/kg bw; P1: diabetic treatment

group with alpha-mangostin 2 mg/kg bw; P2: diabetic

treatment group with alpha-mangostin 4 mg/kg bw; P3:

diabetic treatment group with alpha-mangostin 8 mg/kg

bw; LI: diameter of the islet of Langerhans; Bar: 100 µm.

Lard was administered for 3 weeks to obtain

hyperlipidemic condition within the mice.

Hyperlipidemic state has a great chance to cause

insulin resistance, so it is very easy to occur after

administration of STZ. This study began with the

administration of lard followed by STZ. The

measurements of fasting blood glucose level and

fasting blood cholesterol level (mg/dL) were done

before and after the STZ injection. The data is

presented in Figure 1, it shows that STZ injection

with a dose of 30 mg/kg body weight for five

constitutive days was able to significantly increase

the mice’s blood glucose levels significantly, from a

mean blood glucose level of 120.625±19.354 to

183.500±39.419. STZ is a nitric oxide (NO) donor

and NO was found to bring about the destruction of

pancreatic islet cells and contribute to DNA damage

in the cells (Kröncke et al., 1995). This condition

indicates that STZ as an oxidant agent is capable of

destroying pancreatic islet β-cells, which leads to the

decrease of insulin production. Therefore, it

generates an increased level of fasting blood glucose

(Husen et al., 2016). Meanwhile, on the blood

cholesterol levels, the STZ injection was also able to

increase the mice’s blood cholesterol level

significantly from a mean of 152.40±24.294 before

injection to 167.000±27.325 after injection. This

ICPS 2018 - 2nd International Conference Postgraduate School

564

means that STZ is a highly reactive free radical

which is able to increase ROS and RNS levels in

cells, especially for insulin-sensitive cells and tissues

such as the pancreatic gland. In the current work,

diabetes was induced in laboratory mice via

intraperitoneal injection of STZ. Streptozotocin

(STZ) is considered to be toxic to insulin producing

beta cells within pancreas, and thus it is widely used

to induce experimental diabetes in laboratory

animals (Aldahmash et al., 2015). Ansori et al.

(2018) showed pathological changes in kidney of

STZ-induced diabetic mice.

The increased levels of blood cholesterol after

STZ injection caused by the prolonged

hyperglycemic conditions were the result of the

cellular damage of pancreatic islet β-cells and the

decrease in insulin levels in blood. These conditions

led to the increase of gluconeogenesis and lipolysis

in striated muscle and fat tissues, as well as fat

mobilization of adipose tissue, which caused the

increased level of cholesterol in blood. The

breakdown of fatty tissue both within the striated

muscle cells and within the tissues of the body can

lead to the increased levels of cholesterol in the

blood (Husen et al., 2016; Husen et al., 2017a). In

the prolonged hyperglycemic conditions, the

administration of exogenous antioxidant compounds

such as alpha-mangostin was expected to provide

hope for ameliorating of the pancreatic islet β-cells

damaged by free radicals, such as ROS and RNS.

Based on this study, it was found that the

administration of alpha-mangostin antioxidants

affects average fasting blood glucose levels in the

diabetic mice. The dose of 2 mg/kg body weight had

the highest response compared to the other treatment

groups with doses of 4 and 8 mg/kg body weight.

Those results showed a condition in which the

lowest dose of the alpha-mangostin antioxidant

active substance was able to provide a more positive

response, compared to the larger dose groups. It has

been proven that the antioxidant compounds of

alpha-mangostin is a powerful antioxidant and has

the ability to restore the homeostatic condition of

glucose levels in the blood, called hormesis.

Hormesis is a term in toxicology that demonstrates

the phenomenon of response to the low doses

stimulation and inhibition at the high doses and

results in a curve formed of inverted J or U (Husen

et al., 2017b).

The glucose cannot be processed into energy

because of the hyperglycemic condition. Therefore,

the energy must be made from other sources, such as

fat and protein. Energy is obtained through the

increased catabolism of protein and fat. Along with

these conditions, there is a stimulation of lipolysis

and the increase levels of free fatty acids and blood

glycerol. This leads to the increasing production of

acetyl-CoA by the liver, which in turn is converted

to acetoacetic acid and ultimately reduced into β-

hydroxybutyric acid or decarboxylated into acetone

(Husen et al., 2016; Husen et al., 2017a). Due to the

formation of energy from proteins and fats, the

cholesterol levels formed in the chain of fat and

protein metabolism increase. In patients with DM,

hyperglycemic conditions lead to the increased

production of ROS and RNS due to the increase of

NADPH oxidation in endothelial tissue. ROS and

RNS are highly reactive molecules that can directly

oxidize and destroy DNA, proteins, and lipids and

can cause an oxidative stress. An oxidative stress

occurs when there is an imbalance between the

number of highly reactive molecules (ROS and

RNS) with the existing antioxidants (Husen et al.,

2018; Ansori et al., 2018).

Interestingly, this study has proved that most

diabetic mice have high cholesterol levels, as shown

in the KD group, due to the interference of fat

metabolism which causes high levels of acetate as

one of the cholesterols formed in one reaction

catabolism. Excessive energy sources lead to an

excessive acetate formation, and fat in the body will

increase as well. The increased fat metabolism

causes the occurrence of abnormal fat metabolism

with cholesterol deposits in the blood vessel wall.

This condition can lead to an atherosclerosis and a

decrease protein in the body. Various diseases are

often associated with the increased cardiovascular

risk parameters such as hypertriglyceridemia,

hypercholesterolemia, and high-density lipoprotein

(HDL) (Höhn et al., 2014).

4 CONCLUSIONS

We found that the administration of STZ can

increase the fasting blood glucose levels and the

fasting blood cholesterol levels in STZ-induced

diabetic mice significantly. In addition, the

administration of alpha-mangostin can reduce the

average of fasting blood glucose level and fasting

blood cholesterol level, as well as ameliorate the

pancreatic islet β-cells damaged by STZ

administration. Therefore, we concluded that alpha-

mangostin is a promising antidiabetic and

antilipidemic agent due to its antioxidant activity.

The Effect of Alpha-mangostin in Glucose Level, Cholesterol Level, and Diameter of the Islets of Langerhans of STZ-induced Diabetic Mice

565

ACKNOWLEDGEMENTS

The authors would like to thank the Dean of Faculty

of Science and Technology and Head of Institute of

Innovation and Research Universitas Airlangga for

the opportunity given to conduct this research,

which is funded by a grant from Directorate General

of Higher Education, Ministry of Research,

Technology, and Higher Education of the Republic

of Indonesia awarded to Saikhu Akhmad Husen

(Associate Professor in Faculty of Science and

Technology, Universitas Airlangga). Moreover, the

authors would liketo especially thank the PMDSU

Scholarship - Batch III (Ministry of Research,

Technology, and Higher Education of the Republic

of Indonesia) awarded to Arif Nur Muhammad

Ansori, Raden Joko Kuncoroningrat Susilo, and

Suhailah Hayaza.

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