Effect of Therapy on Mangosteen (Garcinia Mangostana L.) Bark

Extract on Serum Blood Blood Protease Activity and Expression of

Malondialdehyde (MDA) on Rattus Norvegicus Traumatic Brain

Injury Model

N. Indah Ratnasari

, A. Aulanni’am

, and C. Mahdi

Institute Biosains, Brawijaya University

Faculty of Veterinary Medicine, Brawijaya University

Chemistry Department, Faculty of Mathematic and Natural Science, Brawijaya University

Keywords: Garcinia mangostana L., malondialdehyde (MDA), protease activity, inflammation, oxidative stress,

traumatic brain injury

Abstract: Traumatic brain injury is a cause of impaired cognitive and physical function that is permanent or temporary

and is accompanied by loss or change of level of consciousness and cause damage or death of cells in the

brain. Cell death or apoptosis can cause inflammation and oxidative stress andproduce reactive oxygen

species (ROS). The use of mangosteen peel extract serves to prevent inflammation and inhibit the

production of oxidative stress. This study aims to determine the role of mangosteen peel extract therapy in

brain organ expression and protease activity in rat blood serum. Besides that, minocycline is used as a test

standard and comparison of mangosteen peel extract. The results of this study formed 4 treatment groups

namely the negative group, the positivegroup TBI model, the TBI group with mangosteen skin extract

therapy dose 0.5 mL/day for 5 daysand the TBI group with minocycline therapy 0.5 mL/day dose for 5 days.

The results showed that the treatment of mangosteen peel extract after brain injury could reduce the

expression ofmalondialdehyde (MDA) by 32.29% and protease activity in rat blood serum by 47.62%.

Theresults of statistical analysis of MDA exposure and protease activity showed that there were

verysignificant differences between treatment groups (p <0.01). This study can be concluded

thatmangosteen peel extract therapy can be used when a traumatic brain injury occurs.

1 INTRODUCTION

Brain injury is one of the highest causes of death in

traffic accidents. Indonesia itself has a 50% increase

in mortality rate every year. Brain injury is a major

factor causing death in traffic accidents (Warpani,

2002).

Rats (Rattus novergicus) are animals in the form

of mammals. This white rat belongs to the Muridae, the

genus Rattus with the order of Rodentia. Also, it is an

experimental animal that is widely used in research.

This is because rats have a high similarity of 95% of

the same genes as humans. This white rat also has a

short generation rate of 1-year rats equal to 30 years of

humans with high reproduction and can manipulate

genomes directly (Armitage, 2004). This then becomes

the basis of research to observe brain injuries that

occur in humans by making mice as a model of

Traumatic Brain Injury. Mangosteen extract is

known to contain active xanthones, flavonoids, etc.

Where xanthone is a substance that has important

benefits such as anti-inflammatory, antioxidant, anti-

cancer, and also anti-cardioprotective (Miryanti et

al., 2011). The anti-inflammation contained in the

mangosteen extract is very important to be used in

traumatic brain injury because it can prevent the

onset of increased inflammation. Increasing

inflammation will indicate the body's physiological

response to an interruption by external factors

(Murfu’ati et al., 2014). Besides that, the flavonoid

content in mangosteen peel extract also has an

important effect which can inhibit the work of

enzymes involved in the formation of ROS (Redha,

2010). Based on the results of research that has been

done, it is stated that flavonoids are a group of

phenolic compounds that have antioxidative

Ratnasari, N., Aulanni’am, A. and Mahdi, C.

Effect of Therapy on Mangosteen (Garcinia mangostana L.) Bark Extract on Serum Blood Blood Protease Activity and Expression of Malondialdehyde (MDA) on Rattus norvegicus Traumatic

Brain Injury Model.

DOI: 10.5220/0009587401330140

In Proceedings of the 6th International Conference on Advanced Molecular Bioscience and Biomedical Engineering (ICAMBBE 2019) - Bio-Prospecting Natural Biological Compounds for

Seeds Vaccine and Drug Discovery, pages 133-140

ISBN: 978-989-758-483-1

133

properties and play a role in preventing cell damage

and cellular components caused by free radicals

(Simamora, 2009).

Some things to be able to prove that mangosteen

peel extract is a therapy that can provide positive

changes for traumatic brain injury that is proven by

using levels of malondialdehyde (MDA). This

substance is widely known and is also often used as

a biological marker of lipid peroxidation and

oxidative indicators. Also, it is easily obtained in the

blood circulation and is the main product of the

reaction of free radicals with phospholipids. The

results of these reactions are produced in constant

accordance with the proportion of lipid peroxidation

that occurs, so it is a good indicator to see the speed

of lipid peroxidation in vivo. The higher levels of

MDA are expected to increasingly show the level of

damage that occurs in traumatic brain injury (Yigit

et al., 1999).

Also, observations were made on protease

activity. Protease activity itself is the ability of

protease to break down proteins. Protease is secreted

into tissues involved in the mechanism of tissue

damage. Excessive protease activity can damage

tissue cells found in the brain (Hantoko and Drajat,

2003). Increased uncontrolled protease activity

causes an inflammatory process so it is expected to

be inhibited by mangosteen peel extract therapy.

2 MATERIAL AND METHOD

2.1 Preparation of Animals Trying

Mice (Rattusnovergicus)

Rats were divided into 4 treatment groups, each group

consisted of 5 mice. Before being treated, mice were

adapted to the laboratory environment for 7 days by

standard feeding to all mice.

Group 1 was a negative control group of rats,

namely the group of

rats without TBI induction and

the administration of minocycline therapy and

mangosteen extract. Group 2 was a positive control

rat group, namely the TBI induction rat group.

Group 3 was a group of TBI induction mice and

treated with minocycline at a dose of 0.5 mL/day.

Group 4 is a group of TBI induction mice and

mangosteen extract treated with a dose of 0.5

mL/day. The research scheme can be seen in

appendix 1.

Table 3.1: Design of the Rat Treatment Group(Rattus

norvegicus)

The research sample used was a rat animal

(Rattus norvegicus) male Wistar stain with a

bodyweight of 300-350 grams. Calculation of the

number of samples can use the Federer formula as

follows [45]:

t (n - 1) ≥ 15

4 (n – 1) ≥ 15

4n – 4 ≥ 15

4n ≥ 19

n ≥ 4, 75 (be rounded 5)

Note:

t : number of treatment groups

n : number of repetitions needed

Based on the estimation calculation from the

sample above, then for the four treatment groups

required at least 5 replications in each group so that

the total number of rat animals needed was 20.

Rats were kept by

the

treatment group and kept

in a room temperature of

22-24

C and humidity 50-

60% with

adequate ventilation, where each cage

consisted of 5 mice. The mouse cage is made of a

plastic tub with a size of 17.5

x 23.75 x 17.5 cm

which is equipped with a cover of

wire.

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134

The variables observed in this study are as

follows:

1. Independent variable

: treatment of falling brain

load, therapeutic

dose o

mangosteen extract

2. Dependent variable : brain

organ,

serum

protease

activity,

MDA

immunohistochemistry

3. Control variables : sex, age, body weight

of Rattus norvegicus

strain Wistar rats.

2.2 Providing Mangosteen and

Minocycline Skin Extract Therapy

Minocycline therapy was given to the KB TB post-

treatment group at a dose of 0,5 mL/day for 5 days.

While mangosteen therapy was given to the KC

treatment group at a dose of 0,5 mL/day for 5 days.

2.3 Preparation of Animal Rats (Rattus

novergicus) TBI Models

Ketamine at a dose of 100 mg / KgBB and xyla at a

dose of 10mg / kg BW are anaesthetized through

intramuscular injection of the thigh muscle. Then the

rats were placed face down on the surgical board and

fixed the four extremities using a paper clip. The

rat's head was disinfected using 70% alcohol and the

rat's head was shaved. The scalp of the rat is opened

with scissors from the middle to between the two

ears toward the frontal until the skull is visible. Then

the mouse head is positioned just below the cylinder

sleeve with a distance of 1 cm. The iron cylinder

weighing 40 grams and 4 mm diameter was dropped

perpendicularly from a height of 180 cm 1 time.

Then the scalp is cleaned, stitched back and given a

topical 10% gentamicin ointment and intramuscular

analgesic.

2.4 Intake of Rat Brain

Rats were treated with euthanasia using ketamine at a

dose of 0,2 mL and placed on a surgical board. Next,

cut the back of the rat's neck or cut in the direction

of the back to the abdomen entirely so that you can

see the boundary between the skull and skin.

The scalp of mice in the TBI lesion area is

completely removed. Then the mouse skull is cut as

needed from the direction of the neck intersection.

The skull was opened with the power of a finger to

open and obtained a cross-section of the brain and its

limits. Nerves that are still connected to the brain are

cut. Then the brain is carefully removed and placed

in an organ bottle containing 10% formalin Solution.

2.5 Retrieval of Rat Blood Serum

Rats were placed in a dorsal lying position on the

surgical board. Then dissected in the abdominal

cavity and taken as much as 5 cc of blood plasma in

the superior vena cava of the heart. Then put in a red

vacutainer.

2.6 Making Slides of Brain Tissue

Histopathology Preparations

The rats head was cut into 2 cm x 1 cm x 3 cm size

and brain tissue was fixed using 10% formalin, then

soaked for 18-24 hours. Then washed with running

water for 15 minutes and dehydrated using acetone

Solution for 1 hour 4 times. Then the stage of

clearing (clearing) using xylol for 30 minutes as

much as 4 times. Furthermore, the stage of

immersion (impregnation) using liquid paraffin with

a temperature of 55

C for 1 hour 4 times. Casting

(blocking) is done on the paraffin block and sliced

on a network that has been embedded in the paraffin

block using a rotary microtome with a thickness of

3-5 microns and placed on a glass object.

2.7 MDA Measurement by

Immunohistochemistry

Preparation slides before being deprived are heated at

C for 60 minutes. The preparations were depolished

with xylol for 2 times 10 minutes, put into absolute

ethanol for 2 times 10 minutes. Then put into

multilevel ethanol (95%, 90%, 80%, and 70% and

distilled water) for 5 minutes each. The slide

preparation was immersed in a Chamber containing

citrate buffer pH 6.0. Then the Chamber is heated in a

water temperature of 95

C for 20 minutes. Slides are

removed from the water bath, wait until the room

temperature ± 20 minutes. Slides were washed with

PBS for 6 minutes.

On the first day the slides were ready for IHC,

3% H

2O2 in methanol was incubated for 15 minutes

and washed with PBS pH 7.4 3 times 2 minutes. In

Unspecific Blocking Protein Background Sniper

drops, incubated 15 minutes at room temperature

then washed with PBS pH 7.4 3 times 2 minutes.

Primary antibody (MDA) drops were Solutiond in

PBS + 2% BSA buffer, incubated overnight at 4oC.

Then on the second day, the two slide preparations

were removed, waited for room temperature, then

Effect of Therapy on Mangosteen (Garcinia mangostana L.) Bark Extract on Serum Blood Blood Protease Activity and Expression of

Malondialdehyde (MDA) on Rattus norvegicus Traumatic Brain Injury Model

135

washed with PBS pH 7.4 3 times 2 minutes.

Secondary antibody is dropped, incubated 30

minutes at room temperature and washed PBS pH

7.4 3 times 2 minutes. Furthermore, SA-HRP

(StrepAvidin-Horse Radish Peroxidase) was added

and incubated for 20 minutes at room temperature.

Washed with PBS pH 7.4 3 times 2 minutes and

rinsed with distilled water. Added DAB (Chromagen

DAB: DAB buffer = 1:50) and incubated for 3-10

minutes at room temperature.

Washed with PBS pH 7.4 3 times 2 minutes and

washed with aqua dest 3 times 2 minutes.

Counterstrain (Mayer's Hematoxylin) was given

with a tap water in a ratio of 1:10 and incubated for

5-10 minutes at room temperature, then rinsed with

tap water. Mounting glass cover. Furthermore, dried

until dried. Furthermore, observations were made

using a microscope. MDA levels in rat brain tissue

can be calculated with a portrait microscope at the

Anatomy Pathology Laboratory, Faculty of

Medicine, Brawijaya University. The preparations

are placed on a microscope with 10x ocular

magnification and 10x objective magnification.

After brain tissue is seen, the objective

magnification is increased to 40x. Observations were

made on 10 different fields of view so that the

results obtained are objective.

2.8 Measurement of Protease Enzyme

Activity (serum) Protein Isolation

Blood serum is prepared first and added a little

quartz sand. After homogenate added with PBS-

Tween: PSMF (9: 1) Solution as much as 1 mL and

transferred into a sterile effendorf tube. Followed by

vortexing for 15 minutes (6000 rpm), and 10

minutes sonicated with a sonicator. Then the

supernatant is taken and added to absolute cold

ethanol in a ratio of

1 and left overnight to form a precipitate. After

that centrifuge for 15 minutes (10.000 rpm), the

sediment is taken and dried until the ethanol odour

disappears. Then the precipitate was added with a

0.02 M Tris-HCl pH 6.5 cold Solution with a

volume ratio of 1: 1.

2.9 Making the Tyrosine Raw Curve

The first step in making the tyrosine standard curve

is to prepare 10 volumetric flasks and each filled

with 20 ppm tyrosine standard Solution 1, 2, 3, 4, 5,

6, 7, 8, 9, 10 mL for concentrations of 2, 4, 6, 8, 10,

12, 14, 16, 18, 20 ppm. Next, add distilled water to

the boundary mark, then the tube is closed with

aluminium foil and shaken. Then the absorbance is

measured in each standard Solution at the maximum

wavelength. The blank used is aquades.

2.9 Measurement of Protease Activity

in Protein Isolation

The first step that must be done is to mix 500 ppm

casein as much as 200 µL, phosphate buffer Solution

pH 7 as much as 300 µL and protease enzyme as

much as 100 µL then let stand 60 minutes at 37

C in

the incubator. Then a 400% TCA Solution of 400%

was added and allowed to stand for 30 minutes at

C (room temperature). Then it is rotated with a

centrifuge at 4000 rpm for 10 minutes. The

supernatant was taken as much as 100 µL and

diluted 5 times the sample volume with phosphate

buffer then measured its absorbance value at a

maximum tyrosine of 280 nm. The blank used was

made by the same procedure as the determination of

activity, but for the addition of TCA treatment was

carried out as soon as possible after the addition of

the enzyme Solution. One unit of activity is as much

as tyros avoided the breakdown of 1 mL of the

protease enzyme.

Measurement of protease enzyme activity was

carried out based on the Walter (1984) method using

the formula :

Enzyme Activity



Tirosin

fp

MrTirosin

xq

q = incubation time

(mL) fp = dilution factor

p = amount of enzymes (mL)

2.10 Data Analysis

Analysis of quantitative immunohistochemical data

using MDA levels in rat brain and calculation of

protease activity as an anti-inflammatory marker on

brain tissue was performed statistically using a one-

way analysis of variance (ANOVA) variance test.

Then the Honestly Significant Difference (BNJ)

or Tukey test is performed to determine whether

there is a significant difference with a significance

level of 5% using Microsoft Office Excel and

statistical package for the social science (SPSS)

version 16.0 for Windows 7.

ICAMBBE 2019 - 6th ICAMBBE (International Conference on Advance Molecular Bioscience Biomedical Engineering) 2019

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3 RESULT AND DISCUSSION

In this research, therapy on traumatic brain injury

rats using mangosteen extract and minocycline as

gold standard. Observations made were the

expression of malondialdehyde (MDA) in rat brain

organs and serum protease activity in the blood.

3.1 MDA Expression of White Rat

(Rattus novergicus) Brain TBI

Model in Mangosteen Skin Extract

Therapy

Figure 1. Negative Control (Healthy)

Figure 2. TBI Positive Control

Figure 3. TBI Control and Mangosteen Skin Extract

Therapy

Figure 4. TBI Control and Minocycline Therapy

In rats, traumatic brain injury occurs aninflammatory

process and activates inflammatory mediators such

as T cells. T cells express CD4 + to recognize

antigens. T cellsactivate macrophages to produce

proinflammatory cytokines [47]. Reactive Oxygen

Species (ROS) are also produced during the

inflammatory process due to free radicals. Oxidative

stress on the central nervous system is very deadly

because the human brain mainly uses oxidative

metabolism. Although the brain weighs only 2% of

body weight, the brain uses about 50% of all body

oxygen. Another very dangerous factor is oxidative

stress in the brainwith a high PUFA

(polyunsaturated fatty acid) content, affecting almost

50% of all brain tissue structures. Poly Unsaturated

Fatty Acid is degraded by free radicals which are

ROS products such as hydroxyl radicals (-OH),

superoxide radicals (O2-), hydrogen peroxide

(H2O2) then form malondialdehyde (MDA) [48].

High MDA levels indicate that cells experience

oxidative stress [49] and are indicated by changes in

the appearance of mouse brain cells in brown colour.

Mangosteen peel extract has antioxidant and anti-

inflammatory properties. Antioxidants will inhibit

the process of activation of inflammatory cells so

that the activation of macrophages in producing

cytokines is also reduced and inhibited the

production of free radicals. Inhibition of free

radicals by antioxidants by the bioactive content of

mangosteen through the process of inhibiting the

oxidation reaction to reduce the expression of MDA.

The content of bioactive compounds possessed by

mangosteen peel extract can reduce MDA

expression better than minocycline because

minocycline is only a derivative of tetracycline so

that minocycline has only one benefit to reduce

MDA expression in TBI model mice. Minocycline is

also a drug that has long been used for the treatment

of brain injuries. This can be proven in Table 4.1.

Table 4.1 Average number of cells expressing

malondialdehyde (MDA) in TBI mouse brain organs.

Treatment group

MDA Expression

Average (%)

Negative

Control

7,87 ± 0,09

(Healthy) (A)

TBI Positive Control 12,48 ± 0,29

(B)

TBI Control and 2,16 ± 0,37

Mangosteen

Skin

Extract Therapy (C)

TBI Control and 8,45 ± 0,53

Minocycline

Therapy (D)

Effect of Therapy on Mangosteen (Garcinia mangostana L.) Bark Extract on Serum Blood Blood Protease Activity and Expression of

Malondialdehyde (MDA) on Rattus norvegicus Traumatic Brain Injury Model

137

Figure 5. Graph of the number of MDA expressions in rat

brain

The average MDA level in group A was 7.87 ±

0.09. These values indicate the standard value of

MDA levels in mice under normal circumstances.

The mean value of MDA levels in group B is the

highest value of 12.48 ± 0.29 and testing by BNJ test

shows that there are significant differences

compared to treatment groups A, C, and D.

The results of statistical analysis (Appendix 11)

indicate that group B provide a real influence on the

increase in MDA expression in rat brain. The results

showed that a drop of iron cylinder weighing 40

grams and a diameter of 4 mm can cause

inflammation in the rat brain. Therapeutic groups

namely group C and group D showed a decrease in

MDA expression after administration of mangosteen

and minocycline skin extract therapy. But in the

therapy done, mangosteen skin therapy is better than

minocycline therapy. This can be caused because

there is a high antioxidant content in mangosteen

peel extract which can counteract free radicals which

is indicated by a decrease in MDA levels in the

brains of mice that have been injured. Antioxidants

in the content of mangosteen peel extract functions

as a revenger (catcher) of free radicals so that it can

help reduce high levels of free radicals in the rat

brain due to traumatic brain injury. The mechanism

of inhibition of free radicals by antioxidants

mangosteen rind extract is inhibiting the oxidation

process by inhibiting the initiation and propagation

of oxidation reactions from free radicals and ROS.

Antioxidants in mangosteen peel extract namely

xanton can contributehydrogen atoms to capture

hydroxyl radicals (OH) so as not to become reactive

so that it inhibits free radicals. The xanton

component works through the capture of O-peroxide

nitrite (ONOO-) formed from nitric oxide (NO) with

superoxide (O2-) which is a free radical. The

antioxidant content of mangosteen peel extract

inhibits the initiation process so as to prevent the

formation of lipid radicals that are unstable due to

the loss of one hydrogen atom (H) from the lipid

molecules due to hydroxyl radicals (OH-), prevent

the propagation process so that free radicals will not

react with oxygen and automatically does not

directly reduce MDA levels in rat brain.

This is also strengthened by the presence of other

bioactive compounds including flavonoids where

these compounds can ward off free radicals by

reducing free radical compounds so that they

become stable compounds. Binding of free radicals

by flavonoids will prevent chain radical reactions

that damage protein function and normal tissue

structure. Flavonoids are compounds that can be

easily modified to stop radicals so that they can

prevent oxidative stress in cells and increase the

enzyme protease in tissues

3.2 Effect

of Mangosteen Skin Extract

Therapy on TBI Model Blood Serum

Protease Activity

Protease activity is the ability of proteases to

hydrolyze peptide bonds in proteins. The method

used in the analysis of protease activity is

spectrophotometry. The method is based on

enzymatic hydrolysis by proteases tested from a

casein substrate Solution at pH 7, and followed by

deposition of non-hydrolyzed substrates using

trichloroacetic acid (TCA) 4% to stop the reaction.

The result of hydrolysis from the casein substrate

Solution is L-tyrosine. Therefore, in the

measurement of protease activity, the tyrosine

standard curve is used with λ = 275 nm because the

final product (product) is measured by UV

spectrophotometer at a maximum wavelength of 275

nm. The protease activity unit of rat blood serum

(Rattus norvegicus) is defined as the number of

tyrosine units produced by hydrolysis of peptide

bonds in proteins by protease isolated from rat

duodenum (Rattus norvegicus) under optimum

conditions in pH 6.5, temperature 370 C, and time

incubation of 60 minutes.On the tyrosine standard

curve obtained by the equation of the line y=0.015x

- 0.013. The line equation is used to calculate the

measured tyrosine concentration from the study so

that the protease activity can be

calculated for each

treatment. A complete calculation of protease activity

is presented in Attachment 9.

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4 CONCLUSIONS

Based on the results of research that has been done

can be concluded that:

1. Mangosteen skin extract therapy can repair

damage to the cortex of the rat brain.

2. Mangosteen peel extract therapy can reduce

malondialdehyde (MDA) expression in rat brain

by 2.16 ± 0.37.

3. Mangosteen peel extract therapy can reduce the

activity of protease enzymes by 47.62%.

ACKNOWLEDGEMENT

We would like to thanks to Prof. Dr. drh.

Aulanni’am., DESS and Prof Dr. Ir Chanif Mahdi.,

MS who gave us a research, , thus we can write a

paper based on several study cases.

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