Effectiveness of Malang Apple Cider (Malus sylvestris) against the

Decreased Number of Mast Cells in Allergy-induced Rat (Rattus

novergicus) Lung

Dian Yuliartha Lestari

and Faiq Shabri Maulana

Department of Pathology Anatomy, Faculty of Medicine, University of Muhammadiyah Malang, Jalan Bendungan Sutami

No. 188A, Malang, East Java, Indonesia

Keywords: Malang apple cider, Mast Cell, Ovalbumin, Allergic Reaction

Abstract: Allergy is a state of hypersensitivity induced by exposure to certain antigens that cause reactions immunology.

Mast cells have a role in triggering allergies. There is a new innovation as an anti-allergy, one of them is apple

because it contains quercetin. Quercetin are believed to have an influential effect on human health, that of

anti-allergy and anti-inflammatory activity. This study to determine the effectiveness of Malang Apple Cider

(Malus sylvestris) on decreasing the number of mast cells in the lung of an allergic induced rat (Rattus

novergicus). This study was experimental research using Post Test Control Group Design. Samples were

white male Wistar Strain Rats which were divided into 5 groups, each group contains 5 rats. The positive

control group was induced with ovalbumin through intraperitoneal and inhalation. Besides ovalbumin

induction, treatment groups were also given apple cider with doses of 15%, 20%, and 25%. Lung tissue was

taken and then paraffin blocks were created and stained using Methylene blue staining and evaluated with the

microscope in 40 x magnification. Data analyzed using One Way ANOVA. Statistical results showed there

was no significant (p = 0,079 > or p 0.05). This decline is due to the levels of Quercetin in apple cider can

prevent mast cell degranulation. However, the decrease was not significant occurs due to several factors,

including the strength of apple cider, the absorption in the intestine, stress factors, and environmental

circumstances.

1 INTRODUCTION

Every year, the prevalence of allergies shows a

significant increase in almost all countries. It began to

become a serious health problem. In recent years, the

incidence of allergies has continued to increase

significantly both domestically and abroad. The

World Allergy Organization (WAO) said that 22% of

the world’s population suffer from allergies and

continues to increase every year (Candra et al, 2011).

Allergy is a complex chronic inflammatory

process that is influenced by genetic, environmental

and internal control factors. Activation of CD4 T cells

with predominance of T helper 2 (Th2) will induce an

inflammatory process, with one of the cytokines

involved is interleukin-4 (IL-4). Interleukin 4 plays a

role in the differentiation of CD4 T cells into Th2

cells, switching B-lymphocytes to produce

immunoglobulin (Ig) E and increasing the production

of eosinophils. IgE produced will be attached to the

surface of mast cells and will bind to allergens. The

bonds between mast cell, IgE and allergen can cause

mast cell rupture, and secrete chemical mediators.

The effect of this chemical mediators cause

vasodilatation, edema, and spasm of smooth muscle

(Abbas et al, 2016).

Mast cells is one part of connective tissue that has

pseudopodia for mobilization. Basically, mast cells

are found in all organs, especially in the lung mucosal

tissue. Based on Merijanti research (2011), quercetin

can inhibit histamine production and release from

mast cells and basophil cells. This is because

quercetin has a strong affinity for mast cells and

basophils. Quercentin is one of the flavonoids that has

an antioxidant effect. That is why these antioxidants

can reduce the chance of someone infected with

various allergens and also help cure allergies

(Merijanti, 2011).

Quercetin can be found in apples. The quercetin

content in 100-gram apples is 4.4 mg. Malang and

124

Lestari, D. and Maulana, F.

Effectiveness of Malang Apple Cider (Malus sylvestris) against the Decreased Number of Mast Cells in Allergy-induced Rat (Rattus novergicus) Lung.

DOI: 10.5220/0009124401240128

In Proceedings of the 2nd Health Science International Conference (HSIC 2019), pages 124-128

ISBN: 978-989-758-462-6

Batu are the main apple producing regions in

Indonesia (Yuwono, 2015). About 80% of apple

production in East Java is concentrated in Malang and

Batu. Therefore, author want to prove the efficacy of

Malang apple juice (Malus sylvestris) ad an anti-

allergic, especially to mast cell responses.

2 METHODS

This research is an experimental study using posttest

control group design approach. The samples used

were male Rattus novergicus strain wistar aged 2-3-

month, body weight 150-250 grams, in good

condition, characterized by active movements, thick

fur, and clear eyes. The sampling technique used

simple random sampling. In this study, there were 25

samples of rats divided into 5 groups namely positive

control group, negative control group, and 3 treatment

group with different dosage.

2.1 Preparation Ovalbumin Solution

Preparation of ovalbumin solution as an allergen can

be done by using 50 ml of purebred chicken egg

whites then stirred until there are no lumps (Barlianto,

2009).

2.2 Cider Apple Preparation

Apple cider has been made through a juicing process.

The apple juice than dissolved in distilled water. The

apple cider dose used in this study was: 15% (15 ml

apple juice and 85 ml distilled water), 20% (20 ml

apple juice and 80 ml distilled water), and 25% (25

ml apple juice and 75 ml distilled water)

(Widyaningtyas, 2014).

2.3 Administration of Ovalbumin and

Apple Cider

Rats used as many as 25, were divided into 5 groups

and each group consisted of 5 rats. The negative

control group was given standard BR-1 feed of 40

gr/day/rat and drinking distilled water for 30 days.

The positive control group and treatment group

besides being given standard food, it given the

administration of 70 mg ovalbumin and 14 mg

Al(OH) 3 in 1.4 cc of normal saline intraperitoneally

on the first day, 7

day and 14

day. Then it given 7%

ovalbumin in 10 ml of normal saline by inhalation

using nebulizer for 30 minutes in the 19

day and 22

day (Laesa, 2010). The treatment group was also

given apple cider orally based on the dose determined

(15%, 20% and 25%) on days 15

– 22

. At the end

of the treatment, all rats were killed to observe its

mast cell in the lungs.

2.4 Mast Cell Observation

Rats that have been killed, pulmonary organs were

taken and made paraffin block preparation. To

observe mast cell, methylene blue has been stained.

Observation were made by counting mast cells using

a microscope with 400x magnification in 5 visual

fields.

2.5 Data Analysis

The data obtained in this study were analyzed using

the normality test, homogeneity test, and One-Way

ANOVA test that processing using SPSS 23 for

windows with 95% confidence level.

3 RESULTS AND DISCUSSION

Table 1 shows the number of mast cells in male white

rats in the negative control (C-), positive control (C+)

and treatment groups (T1, T2, T3) using methylene

blue staining. During the study, 3 rats were dropped

out for various reasons, so the data used only 3

samples per group.

Histopathology examination in each study group

can be seen in Figure 1 - 5. Mast cells were identified

as dark blue using methylene blue staining.

Homogeneity test was performed with p > 0.05

and ANOVA test with p = 0.079. Based on the One-

Way ANOVA result, there was no significant

difference between each group.

Effectiveness of Malang Apple Cider (Malus sylvestris) against the Decreased Number of Mast Cells in Allergy-induced Rat (Rattus

novergicus) Lung

125

Table 1: The Number of Mast Cells in Each Group.

Group The Number of Mast Cell in Rats Average Saphiro-

Wilk*

1 2 3 4 5

C- 23 22 21 17 Drop out 20.91 -

C+ 31 33 45 35 29 34.80 0.346

T1 30 22 30 27 Drop out 27.58 0.147

T2 22 36 27 24 Drop out 27.66 0.283

T3 26 30 22 25 29 26.80 0.833

Note :

C- : negative control group

C+ : Positive control group

T1 : 1

Treatment group, with 15% apple squeeze

T2 : 2

Treatment group, with 20% apple squeeze

T3 : 3

Treatment group, with 25% apple squeeze

*Significant if >0.005

Figure 1: Mast cell in negative control group.

Figure 2: Mast cell in positive control group.

This study uses ovabulmin as an allergen. The

results showed that exposure to ovalbumin for 29

days could increase the average mast cell count in

rats. This increase occurs because ovalbumin which

acts as an allergen will spread to the digestive tract,

skin and respiratory tract through the blood

circulation. When allergens enter, they will be

processed by Antigen Presenting Cells (APCs) in

endosomes. Then APC will present Class II Major

Histocompability Complex (MHC) to T helper (Th0)

lymphocyte cells. Th0 cells will release Interleukin

(IL-4) which converts Th into Th2. So Th2 cells

induce B lymphocytes to produce immunoglobulin E

(IgE). This causes an increase in production of IgE.

IgE in the circulation will be attached to receptors

with high affinity on the surface of mast cells. The

bond between IgE and allergens will reduce the

stability of mast cell membranes, resulting in mast

cell degranulation. Degranulation of these mast cells

results in the release of chemical mediators into the

circulation, and gives rise to clastic manifestation of

allergies (Busse and Lemanske, 2001).

Figure 3: Mast cell in treatment group 1 (15%).

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Figure 4: Mast cell in treatment group 2 (20%).

Figure 5: Mast cell in treatment group 3 (25%).

Ovalbumin will also activate mast cells and

CD4+Th2 cells in reaction to allergies. Activated

CD+Th2 cells will produce inflammatory mediators

such as histamine, leukotriene, and cytokines such as

IL-4, IL-5, IL-10 and IL-13 (Abbas et al, 2016). IL-4

and IL-10 can stimulate mast cell growth. Cytokines

produced by CD4+Th2 cells play a rle in mast cell

hyperplation and are very influential in allergic

responses (Li Weber and Krammer, 2003).

The result of the study between the positive

control group and treatment group showed a decrease

in the number of mast cells in the treatment group

(Table 1). This is caused by the treatment group that

is given apple cider which contain flavonoids,

especially quercetin, has an anti-inflammatory effect

by affecting Th2 production and by maintain stability

of the mast cell wall membrane thereby inhibiting

mast cell degranulation and inhibiting the release of

inflammatory of mediators like histamine

(Chirumbolo, 2010).

Statistical test results using One Way Anova

obtained p=0.079, which means there is no significant

difference between the control group and the

treatment group. Many factors can influence this,

including the duration of poor apple cider which is

only 1 week, so that the flavonoid effect is optimal.

Another thing that can be a factor also is the extract

content and the cider content, will have more effect

when using extracts because the extract compound

content is strengthened so that the effects that arise

will be more optimal (Rachmadian, 2011).

Genetic factors can affect the genetic

predisposition to hypersensitivity reactions (atopic).

Atopic individuals have an innate tendency to

produce IgE antibodies to specific allergens and

excessive allergen responses (Ningrum et al, 2016).

In this study, suggest that there are several factors

that influence insignificant results where quercetin

which is given orally is not absorbed optimally in the

intestine because quercetin contained in apple cider

will bind cholesterol carriers when it passes through

the brush border membrane. It will reduce the effect

of quercetin as an anti-inflammation (Witosari,

2014).

Other studies said that quercetin is an aglycone

where the bind is very weak by sugar. However, this

study did not examine how much sugar levels in the

sample (Kelly, 2011).

Another factor that can influence in this study but

not examined in this study is the rat’s level stress

during the research process. Stress experienced by

rats can cause increases suppressive effects on

eosinophils and other pro-inflammatory cytikines

(Chang, 2010).

4 CONCLUSIONS

There was a mast cell levels decrease in the treatment

group given apple cider (Malus sylvestris) in

ovalbumin-induced wistar male rats. However,

statistically the result of the reduction was not

significant.

ACKNOWLEDGEMENTS

The research authors would like to thank the medical

students as well as research assistant. The researcher

also wishes to thank the Public Health Faculty,

University of Muhammadiyah Malang and the HSIC

committee for giving authors the opportunity to write

this article.

Effectiveness of Malang Apple Cider (Malus sylvestris) against the Decreased Number of Mast Cells in Allergy-induced Rat (Rattus

novergicus) Lung

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