RESEARCH PAPER

The Role of Red Ginger (Zingiber officinale var Roscoe) Extract

in Autoimmune Disease and Systemic Inflammation

through IL-4, IL 10, TNF- α

By :

Muhammad Mufti Al Anshori (201510330311141)

Nabila Fitri Kurnia (201510330311127)

Mentari Nur Fadilah (201510330311187)

Naafi Sabbah (201510330311179)

AIRLANGGA MEDICAL SCIENTIFIC WEEK

MEDICAL FACULTY

UNIVERSITY OF AIRLANGGA

2018
The Role of Red Ginger (Zingiber officinale var Roscoe) Extract

in Autoimmune Disease and Systemic Inflammation

through IL-4, IL 10, TNF- α

RESEARCH PAPER

FOR AIRLANGGA MEDICAL SCIENTIFIC WEEK COMPETITION

Muhammad Mufti Al Anshori (201510330311141)

Nabila Fitri Kurnia (201510330311127)

Mentari Nur Fadilah (201510330311187)

Naafi Sabbah (201510330311179)

AIRLANGGA MEDICAL SCIENTIFIC WEEK

MEDICAL FACULTY

UNIVERSITY OF AIRLANGGA
 LETTER OF ORIGINALITY
AMSW 2018

To Comittee of AMSW 2018,

Hereby, I/We, undersigned,

first author : Muhammad Mufti Al Anshori

second author : Nabila Fitri Kurnia

third author : Mentari Nur Fadilah

fourth author : Naafi Sabbah

state that Research Paper , entitled:

The Role of Red Ginger (Zingiber officinale var Roscoe)

Extract in Autoimmune Disease and Systemic Inflammation
through IL-4, IL 10, TNF- α
is an original work of the author/s above and qualified according to the competition
Guideline. This paper has not been published in any competition, congress, or any
other events out of AMSW 2018.

Malang, 29-07-2018

(Muhammad Mufti Al Anshori)

©AMSW2018

ABSTRACT
Muhammad Mufti Al Anshori1, Nabila Fitri Kurnia2, Mentari Nur Fadilah3,
Naafi Sabbah4
Cytokines play key roles in autoimune disease, the deficiency and elevation of some
cytokines trigger immunity problems and some of them are IL-4, IL-10, TNF-α.
For example: IL-4 and IL-10 level decrease in rheumatoid arthritis and psoriasis
however TNF-α level is elevated. Red ginger has been known as a medical herbal
therapy. Red ginger extract that contains gingerol, zingeron and shogaol influence
the role of cytokines and potentially used as anti inflammation. To understand the
role of red ginger extract in cytokines, this study uses 30 white mouses divided into
5 groups : negative control, positif control (induced by Pyrazinamide 9 mg,
Levofloxacin 4,5 mg and Ethambutol 9 mg daily), group 1,2 and 3 (being induced
and treated with red ginger extract 25 mg, 50 mg and 100 mg daily) and it takes 56
days. After termination, plasma IL-4, IL-10, and TNF-α levels were detected using
enzyme-linked immunosorbent assay (ELISA). The result shows : negative control
(IL-4 = 107.600 pg/mL, IL-10 = 202.333 pg/ml, TNF-α = 537.500 pg/ml), positive
control (IL-4 = 126.833 pg/mL, IL-10 = 174.333 pg/ml, TNF-α = 437.000 pg/ml),
group 1 (IL-4 = 122.333 pg/mL, IL-10 = 223.000 pg/ml, TNF-α = 479.167 pg/ml),
group 2 (IL-4 = 130.533 pg/mL, IL-10 = 239.500 pg/ml, TNF-α = 452.500 pg/ml),
group 3 (IL-4 = 136.367 pg/mL, IL-10 = 220.667 pg/ml, TNF-α = 457.500 pg/ml).
This study shows that IL-4 can act as pro or anti inflamatory cytokines and it may
correlate with IL-10. Elevating level of IL-4 accompanied with IL-10 may indicate
reduction of systemic inflammation while elevating level of TNF-α and IL-4 with
reduction of IL-10 may indicate progression of systemic inflammation. In right
doses, red ginger extract can act as anti inflamatory agent and can be used as
autoimmune disease treatment but over theraupetic window, it induces worse
inflammation. From study above, group 2 (red ginger extract 50 mg) has the best
result. To strenghten the potential of red ginger, we need to see Malondialdehyde
(MDA) levels. MDA appears to be a sensitive marker of sytemic inflammation. We
still wait for MDA result to furnish this study.

i
Keywords: autoimmune disease, inflammation, interleukin 4 and interleukin 10,
red ginger, TNF-α

Author information
Affiliations

Medical Faculty of University Muhammadiyah Malang

Muhammad Mufti Al Anshori
Nabila Fitri Kurnia
Mentari Nur Fadilah
Naafi Sabbah

Corresponding author

Correspond to: dr. Thontowi Djauhari, NS, M.Kes

ii
 TABLE OF CONTENTS
ABSTRACT ............................................................................................................. i
TABLE OF CONTENTS ...................................................................................... iiii
LIST OF TABLES .................................................................................................. v
LIST OF FIGURE .................................................................................................. vi
CHAPTER 1 INTRODUCTION ............................................................................ 1
1.1 Background .............................................................................................. 1
1.2 Formulation of problem : ........................................................................ 3
1.3 Purpose ..................................................................................................... 3
1.4 Benefits ..................................................................................................... 3
CHAPTER 2 LITERATURE REVIEW ................................................................. 4
2.1 Autoimmune Disease ............................................................................... 4
2.2 Inflammation ............................................................................................ 5
2.3 Anti-tuberculosis drugs ............................................................................ 7
2.4 Interleukin-10 ........................................................................................... 8
2.5 TNF-𝛼 ..................................................................................................... 11
CHAPTER 3 CONCEPTUAL FRAMEWORK ................................................... 17
CHAPTER 4 RESEARCH METHODS ............................................................... 18
4.1 Types of Research .................................................................................. 18
4.2 Location and Time of Research .............................................................. 18
4.3 Population and Sample Research ........................................................... 18
4.3.1 Population ....................................................................................... 18
4.3.2 Sample ............................................................................................. 18
4.3.3 Large Sample .................................................................................. 18
4.3.4 Sampling Techniques ...................................................................... 20
4.3.5 Characteristics of Research Sample ................................................ 21
4.3.6 Research Variables ......................................................................... 21
4.3.7 Operational Definition .................................................................... 22
4.4 Research Tools and Materials ................................................................ 22
4.4.1 Tools................................................................................................ 22
4.4.2 Materials ............................................................................................... 23
4.5 Research Procedures ............................................................................... 23
4.5.1 Preparation of red ginger extract ..................................................... 23
4.5.2 Determination of Drug Doses ......................................................... 23

iii
 4.5.3 Adaptation process .......................................................................... 25
4.5.4 The process of classifying experimental animals............................ 26
4.5.5 Procedures for measurement of IL-4, IL-10 and TNF-Alpha levels26
4.7 Data Collection Techniques ................................................................... 28
4.8 Data Analysis ........................................................................................ 28
CHAPTER 5 RESULT AND DISCUSSION ....................................................... 30
5.1 Results .................................................................................................... 30
5.1.1 Interleukin-4 .................................................................................... 30
5.1.2 Interleukin-10 .................................................................................. 32
5.1.3 TNF-Alpha ...................................................................................... 34
5.2 Discussion .............................................................................................. 41
CHAPTER 6 CONCLUSION AND SUGGESTION ........................................... 45
6.1 Conclusion .............................................................................................. 45
6.2 Suggestion .............................................................................................. 46
REFFERENCE

iv
 LIST OF TABLES

Table No. Page

2.1 Chemical composition rhizome of red and elephant ginger (mg/g)...............15

4.1 Dosing of Second line drug anti-tuberculosis based on Patient’sWeight

group....................................................................................................................24

4.2 Conversion dose table...................................................................................24

5.1 Result of Interleukin 4 with ELISA..............................................................31

5.1 Result of Interleukin 10 with ELISA............................................................33

5.1 Result of TNF-Alpha with ELISA................................................................35

v
 LIST OF FIGURE

Figure No. Page

2.1 Red Ginger Plant...........................................................................................14

2.2 Chemical Structure of 6 –Gingerol...............................................................16

4.1 Research plot................................................................................................28

5.1 Result of Interleukin 4 graph........................................................................33

5.1 Result of Interleukin 10 graph......................................................................35

5.1 Result of TNF-Alpha graph..........................................................................37

vi
 1

CHAPTER 1

INTRODUCTION

1.1 Background

Autoimmune disorders result from a breakdown of immunologic tolerance

leading to an immune response against self-molecules. In most instances the events

that initiate the immune response to self-molecules are unknown, but a number of

studies suggest associations with environmental and genetic factors and certain

types of infections. Approximately 3% of the populations in Europe and North

America currently suffer from autoimmune diseases, many with symptoms of

multiple disorders (Janeway et al, 2001). This may be an underestimate, as

epidemiologic studies are not available for some of the less common diseases. In

addition, there are suggestions that a number of common health problems such as

atherosclerosis and inflammatory bowel disease may have an autoimmune

component (Eaton et al,2007).

Cytokines and chemokines as key elements of communication network

orchestrate the recruitment, survival, expansion, effector function and contraction

of autoreactive lymphocytes in autoimmunity. Cytokines play key roles in

autoimune disease, the deficiency and elevation of some cytokines trigger immunity

problems and some of them are IL-4, IL-10, TNF-α. For example: IL-4 and IL-10

level decrease in rheumatoid arthritis and psoriasis however TNF-α level is elevated

Cameron, 2011).

Some drugs can also induce inflammation and alergic reaction. Some of
them are pyrazinamide, ethambutol and levofloxacin that usually used as secondline
drug therapy for tuberculosis and it is often have considerable side effects, if it is
 2

used in long time treatment. This is due to the mechanism of inflammatory reactions
induced by drugs that are considered as free radicals and increase ROS (Reactive
Oxygen Species) that can cause oxidative stress and induce elevation of pro
inflamatory cytokines like TNF-α and the result is systemic inflammation (Torun
et al, 2005).

The cost burden to solve inflammation problem and autoimmune disease is

not cheap. A report recently released by the American Autoimmune Related
Diseases Association (AARDA) autoimmune diseases cost US more than $100
billion annually. The used of immunosuppressants and anti inflammatory drugs
therapy has considerable side effects if it is used for long time. Steroid cause
cushing syndrome, elevated blood pressure, decreased of mycardial function,
gynecomastia, increased of liver tumor risk and liver damage while NSAID cause
renal syndrome, hepatocellular damage and serious gastrointestinal damage.
Alternate of drugs that safe and low cost burden is needed. The herbal therapy
known can be used as inflammation therapy, one of them is red ginger (AARDA,
2011), (Gnjidic, 2015), (Sullivan et al, 2017) .

Red ginger has been known as a medical herbal therapy. Red ginger

(Zingiber officinale Roscoe) is one of Zingiberacea medicinal plants that have an

important role in various aspects in Indonesian society. Red ginger rhizome has the

highest volatile content (volatile oil) and non volatile (oleoresin) compared with

other types of ginger, the essential oil content of 2.58 -3.90% and 3% oleoresin, it

is good as a herbal drug. Oleoresin is component of red ginger that consist of

gingerol, zingeron and shogaol. Red ginger rhizomes are commonly used as a cure

for common cold, lowering levels of cholesterol, impotence, preventing depression,

anti-inflammatory, antipyretic, analgesic and others (Hapsoh, 2010),

(Rahayu,2010).
 3

Based on this background, the researches wish to prove and examine the

role of red ginger (Zingiber officinale var Roscoe) extract in autoimmune disease

and systemic inflammation through IL-4, IL 10, TNF-α levels.

1.2 Formulation of problem :

How does red ginger (Zingiber officinale var Roscoe) extract influence in

autoimmune disease and systemic inflammation through IL-4, IL 10, TNF- α levels

1.3 Purpose

To determine the effect of red ginger extract (Zingiber officinale) on

autoimmune disease and systemic inflammation through IL-4, IL-10, and TNFα

levels.

1.4 Benefits

Provides information about the role of red ginger in IL-4, IL-10, and

TNFα that can be utilized as an accessible and inexpensive herbal therapy for

systemic inflammation and autoimmune disease.

 4

CHAPTER 2

LITERATURE REVIEW

2.1 Autoimmune Disease

The immune system is a complex set of cellular, chemical, and soluble

protein components designed to protect the body against foreign substances,

including infectious agents and tumor cells, while not responding to self-molecules.

Foreign or self-molecules (usually proteins or carbohydrates) that evoke specific

immune responses are referred to as antigens. Immune cells are located throughout

the body, either in discretely encapsulated organs such as the spleen and thymus or

as diffuse accumulations of lymphoid and myeloid cells as found in association with

the skin and gut where they are strategically placed to monitor the entry of foreign

substances. Optimal function of the immune system requires that immune cells and

cell products interact with each other in a sequential, regulated manner. The

distinction between self and nonself occurs through complex mechanisms that

depend upon specific recognition molecules present on the surface of

immunocompetent cells, in particular, T and B lymphocytes (Abbas, 2016).

Autoimmune diseases occurs from complex interactions among different

immune cell types, including both T and B lymphocytes and professional antigen-

presenting cells, such as macrophages and dendritic cells. These cellular

interactions result in auto-aggressive responses that target a number of different cell

types in different tissues and organs in a relatively large number of autoimmune

disorders. Although the etiology of most autoimmune diseases is unknown, recent

years have witnesed important advances in our understanding of how the different

immune cell types involved in autoimmunity communicate with one another, how
 5

they trigger autoimmune inflammation and how they cause tissue damage. As key

elements of this communication network, cytokines and chemokines orchestrate the

recruitment, survival, expansion, effector function and contraction of autoreactive

lymphocytes in autoimmunity (Abbas, 2016).

2.2 Inflammation

Inflammation is a complex reaction of the body's immune system in the

vascular tissue that causes the accumulation and activation of leukocytes and

plasma proteins that occur during infection, poisoning or cell damage.

Inflammation is essentially a defense mechanism against infection and tissue repair

but persistent (chronic) inflammation can also cause tissue damage and is

responsible for the mechanisms of some diseases (Abbas et al., 2012). The

occurrence of inflammatory processes is initiated by changes in the blood vessels

that increase leukocyte recruitment and fluid transfer as well as plasma proteins in

the tissues. The process is the first step to destroy foreign objects and

microorganisms and clean the damaged tissue. The body exerts the elements of the

immune system to the foreign body and microorganisms entering the damaged body

or tissue (Judarwanto, 2012).

Inflammation is divided into 2 phases, namely acute inflammation (initial

response to tissue injury) and chronic inflammation. Rapid response to infection

and tissue damage is called acute inflammation. Usually occurs within minutes or

hours and is short duration, lasting for several hours or days; Its main characteristics

are vascular changes, exudation of fluid and plasma proteins (edema) and leukocyte

emigration, especially neutrophils (also called polymorphonuclear leukocytes). If

at the time of acute inflammation has managed to eliminate the cause, it will stop
 6

there. But if the response fails, then the reaction may develop into a prolonged phase

called chronic inflammation (Robbins, 2015).

Chronic inflammation can be thought of as an inflammatory length

(weeks to months, even years), and active inflammation, tissue lesion, and

simultaneous healing. In contrast to acute inflammation, which is distinguished by

vascular changes, edema, and massive neutrophilic infiltrate, chronic inflammation

is characterized by mononuclear cell infiltration that includes macrophages,

lymphocytes, tissue destruction and the proliferation of new blood vessels

(angiogenesis and fibrosis) (Robbins, 2015).

Acute inflammation is one of the reactions of a non-specific immune

system known as innate immunity, and chronic inflammation is more dominant in

adaptive immune reactions or specific immune systems. The presence of irritant

stimuli or tissue injury will trigger the release of inflammatory mediators. This

compound may result in short vasoconstriction in arterioles followed by dilation of

blood vessels, venules and lymph vessels and may increase vascular permeability

in cell membranes. Increased local vascular permeability is affected by complement

through the classical pathway (antigen-antibody complex), lectin (mannose binding

lectin) path or alternative path (Robbins, 2015).

Activated macrophages release various mediators that contribute to the

inflammatory reaction. A few hours after the vascular changes, neutrophils adhere

to the endothelial cells and migrate out of the blood vessels into the tissue cavities,

feeding on pathogens and releasing mediators that play a role in the inflammatory

response. Activated tissue macrophages release cytokines such as TNF-α (tumor

necrosis factor-α) that induce local and systemic changes (Abbas et al., 2012).
 7

2.3 Anti-tuberculosis drugs

2.3.1 Ethambutol

Ethambutol inhibits arabinosil mycobacterial transferase, which is

encoded by the embCAB operon. Arabinosil transferase plays a role in the

arbinoglycan polymerization reaction, an essential component of the microbacterial

cell wall. Resistance to ethambutol is caused by mutations that cause excessive

expression of genes or emb inside the structural genes of embB. Ethambutol is well

absorbed from the gut. After ingestion 25, g / kg, peak blood level of 2-5mcg / ml,

achieved within 2-4 hours. ethambutol is accumulated in renal failure, and the dose

needs to be reduced by half if creatinine clearance is less than 10mL / min.

Ethambutol penetrates the blood brain barrier only if the meninges are inflamed.

Like all other antituberculose drugs, resistance to ethambutol occurs quickly if the

drug is used alone. Therefore, ethambutol is always given in combination with other

antituberculose drugs (Katzung, 2013).

2.3.2 Levofloxacin

Levofloxacin is an optical isomer S (-) which has a broad anti-bacterial

spectrum. Levofloxacin is effective for gram-positive and gram-negative bacteria

(including anaerobes). The bactericidal effect of levofloxacin is at a concentration

proportional to or greater than its inhibitory concentration. The mechanism of

action is by inhibiting DNA-gyrase, a type II II topoisomerase that inhibits bacterial

replication and transcription. Side effects are diarrhea, nausea, bloating, rash,

abdominal pain, dizziness, insomnia, anxiety, constipation, etc. (Ananda, 2015).

 8

2.3.3 Pyrazinamide

Pyrazinamide is a synthetic nicotinamide analogue. This drug is not soluble

in water. Pyrazinamide in the body is hydrolyzed by the enzyme pyrazinamidase

into pyrazinoic acid which is active as tuberculostatic only on acidic medium. In

vitro, the growth of tuberculosis germs in monocytes is perfectly inhibited at 12.5

μg / mL pyrazinamide levels. The mechanism of action of this drug is unknown.

Pyrazinamide is easily absorbed in the intestine and is widespread throughout the

body. Its excretion is primarily through glomerular filtration. The elimination half-

life of this drug is 10-16 hours. The most common and serious side effects are liver

disorders (Istiantoro and Rianto, 2011).

2.4 Interleukin-10

IL-10 is one cytokine that has a four-helical structure of the globular domain

and is bound to the type II cytokine receptor. It is produced mainly by activated

macrophages and further inhibits macrophage function. This is an example of a

negative feedback mechanism (Abbas, 2016).

Interleukin 10 or IL-10 has anti-inflammatory and immunosuppressive

effects by inhibiting the synthesis of immunostimulatory cytokines produced by Th

1 cells such as IL-1, IL-6, IL-8, IL-12 and TNF-Alpha with macrophage activation

as well as from IFN-y. Human IL-10 is produced by B cells and T cells, activated

mast cells, macrophages, monocytes and keratinocytes. (Rofii, 2006:

Baratawidjaja, 2014).

Th 1 and Th 2 are differentiation of Th 0 which is a subclass of T helper

cells. Th 1 and Th 2 can be distinguished from the different types of cytokines they

produce. After activation, Th1 cells will secrete IL-2 and IFN-γ, while Th 2
 9

produces IL-4 and IL-5. IFN-γ proved to inhibit Th 2 response and lately it has been

found a product of Th 2 that has the ability to inhibit cytokine production by Th 1

cells. The substance is then isolated and named IL-10. This substance is found only

in Th 2 cells and it is almost absent in Th 1 cells (Rofii, 2006)

IL-10 has a large inhibitory effect on monocytes including downregulation

of MHC antigen expression class II, IL-1α, IL-1β, IL-6, IL-8, GM-CSF, G-CSF and

TNF α production. IL-10 showed a synergistic effect with IL-2 and IL-4 to increase

thimocyte proliferation, IL-10 also showed action in association with IL-3 and IL-

4 to prolong the survival of mast cells.

The suppressive effect of IL-10 on monocytes and cytokine synthesis by

helper 1 T cells is suspected because IL-10 has a general suppression effect of

immune function. IL-10 is currently used in preclinical research to evaluate it’s

potency as immunosuppressive in various diseases (Lari et al, 2007).

IL-10 deficient mice develop a form of inflammatory bowel disease that is

histologically similar to human IBD. Anti-IL-10 treatment exacerbates mucosal

inflammation in the dextran sodium sulphate (DSS) model of colitis, and IL-10 has

shown promising results in human clinical trials. IL-10 is increased in the mucosa

of IBD patients. Of special note is the efficacy of local delivery of IL-10 using

recombinant Lactobacillus lactis in IL-10-deficient mice and in the DSS-induced

model of colitis, a strategy pioneered by Steidler and co-workers. Although the

levels of IL-10 are increased in the synovial fluid of RA patients it does not appear

to have a pathogenic role. IL-10, however, can prevent collagen-induced arthritis

(Kühn, 1993).
 10

The biological action of IL-10 according to Abul K Abbas:

1. Inhibits the production of IL-12 and TNF by way of ratification of macrophages.

The mechanism of this obstacle is not clear.

2. IL-10 inhibits costimulator and MHC class II on macrophages. Because of this

action there is inhibition of T lymphocyte activation and will end the cellular

immunity reaction.

3. In culture, IL-10 stimulates the proliferation of B lymphocytes. The mechanism

of this stimulus is also unknown. Research on mice proves that IL-10 can work

together with other cytokines to stimulate proliferation.

The two main functions of IL-10 are inhibiting the production of several

types of cytokines (TNF, IL-1, chemokine, and IL-12), and inhibiting macrophage

function in assisting T cell activation. Macrophage function resistance occurs

because IL-10 suppresses expression of MHC molecules class II on macrophages

and decrease the expression of co-stimulators (eg B7-1 and B7-2) B cells and

mastocytes in the mucosa. IL-10 together with TGF-β increase IgA production by

B cells (Abbas, 2016).

.IL-10 can also inhibit infiltration of neutrophils and macrophages into damaged

tissue. In vivo, IL-10 inhibits chemokine expression (monocyte chomoattractant

protein-1, macrophage inflammatory protein-1 alpha) and proinflammatory

cytokines (IL-1, IL-1β, TNF-α). Thus, IL-10 plays an important role in the

infiltration phase of neutrophils and macrophages. The final impact of IL-10

activation is the barrier of non-specific and specific inflammatory reactions

 11

mediated by T cells, therefore IL-10 is also called cytokine synthesis inhibitory

factor and anti-inflammatory cytokine (Abbas, 2016).

2.5 TNF-𝜶

TNF-α is a major cytokine in acute inflammatory responses to gram-

negative and other microbial bacteria. Severe infections can trigger the production

of TNF-α in large quantities that lead to systemic reactions. TNF-α is called TNF-

α on a historical basis and to differentiate it from TNF-β or lymphocytokines. The

main sources of TNF-α are mononuclear phagocytes and activated T cells, antigen,

NK cells and mast cells. Lipopolysaccharide (LPS) is a potent stimulus against

macrophages to secrete TNF-α. IFN-γ produced T cells and NK cells also stimulate

macrophages and increasing TNF-α synthesis (Baratawidjaja and Iris, 2014).

TNF-α is usually undetectable in healthy individuals, but it is often found

in inflammatory and serum infections. TNF-α works against leukocytes and

endothelium, inducing acute inflammation at low levels because TNF-α is a strong

pyrogen. TNF-α also plays a role in systemic inflammation at moderate levels.

TNF-α causes pathological abnormalities of septic shock at high levels, because

TNF-α is cytotoxic (Abbas, 2012). The biological effects of TNF-α according to

(Baratawidjaja, 2014) are :

a. Deploy neutrophils and monocytes to the site of infection and activate the cells

to remove microbes.

b. Encourage the expression of adhesion molecules of vascular endothelial cells for

leukocytes.

c. Stimulates macrophages to secrete chemokine and induce chemotaxis and

leukocyte deposition.
 12

d. Stimulate mononuclear phagocytes to secrete IL-1 with effects such as TNF-α.

e. Induces the same apoptosis of inflammatory cells.

f. Stimulates the hypothalamus that induces heat, so called endogenous pyrogens.

g. The production of TNF-α in large quantities can prevent myocardial contractility

and vascular smooth muscle tone which lowers blood pressure or shock.

h. Septic syndrome syndrome complications caused by gram-negative or gram-

positive bacteria characterized by vascular collapse.

Some of the biological functions of TNF-α consist of cellular proliferation

and differentiation, apoptosis, immunoregulator, lipid metabolism, coagulation and

endothelial function (Abbas, 2012).

2.6 Interleukin 4

Interleukin 4 (IL-4) is a cytokine produced by Th2 cells. The cytokines

are a major stimulus of IgE production and the development of naïve CD4 + cells.

IL-4 stimulates B cells to differentiate into plasma cells that secrete IgE. In addition,

IL-4 also stimulates the differentiation of naive T cells into the Th2 subset. IL-4

may act as anti-inflammatory by suppressing macrophage activation, resulting in

decreased inflammatory mediators (Baratawidjaja, 2014; Sivangala & Sumanlatha,

2015).

IL-4 also serves to prevent the activation of induced macrophages by IFN-

ɤ and is a GF for mast cells especially in combination with IL-3. IL-4 serves as a

non-specific anti-inflammatory cytokine. The role of such cytokines in anti-

inflammatory mediators is to influence the suppression of macrophage activation

(Karnen and Iris, 2014).

 13

In some studies, it was found that interleukin 4 is not only an anti-

inflammatory mediator but rather as a proinflammatory mediator as well. IL-4 is

known as a proinflammatory agent because Th2-type expression and response are

associated with allergies and asthma. IL-4 is also found to have other

proinflammatory functions that is by recruitment of inflammatory cells and increase

VCAM-1 expression on the endothelial surface (Abbas, 2012).

IL-4 proinflammatory activity includes its ability to delay neutrophil

apoptosis and in fact IL-4 also increases production of neutrophils. And contributes

to the formation of other leukocyte cells to inflammation sites. In another study it

was reported that IL-4 had proinflammatory activity as judged for its ability to

attract in vivo leukocytes and local production from some pro-inflammatory

molecules (Abbas, 2012).

2.7 Red Ginger (Zingiber officinale Roscoe)

Taxonomy of red ginger plant is as follows:

Domain : Eukaryota

Kingdom : Plantae

Phylum : Spermatophyta

Subphylum : Angiospermae

Class : Monocotyledonae

Order : Zingiberales

Family : Zingiberaceae

Genus : Zingiberis

Species : Zingiber officinale

Varieties : Zingiber officinale Roscoe var. amarum

(Bermawie, 2010)
 14

Figure 2.1
Red Ginger plant

Red Ginger is an annual plant with a height of 50-100 cm. This plant has a

thick reddish-brown rhizome. The leaves are narrow-shaped lanceolate with a

length of 8-12 inches. The tip of the leaf is pointed, the base is blunt and well-edged

and grows away from the stem. Flowering compound with oval shape, emerged

from the rhizomes, with a long stalk 10-25 cm and there are small leaves on the

base of flowers. Crown flower funnel shape, length 2-2.5 cm, dark purple with

yellow cream spots. Small flower petals, tubular and jagged three. (Azam et al.,

2014).

Ginger contains volatile compounds namely terpenoid and non volatile

consisting of gingerol, shogaol, paradol, zingerone and their derivatives and

flavonoid compounds and polyphenols. Gingerol and shogaol are the main

ingredients of flavonoid compounds in Ginger. These compounds have antioxidant

effects that can prevent free radicals in the body (Stailova et al, 2007) and also have
 15

anti-inflammatory effects that may prevent macrophage activation, thereby

decreasing the release of inflammatory mediators (Ali, Blunden, Tanira, &

Nemmar, 2008 ).

Red ginger has 6-gingerol, 8-gingerol, 10-gingerol and 6-shogaol content

higher than elephant ginger which is 18.03, 4.09, 4.61, and 1.36 mg / g so much

consumed by society as ingredients of medicine (Fathona, 2011; Ermayanti, 2009).

The data of phytochemical content of red ginger rhizome that is known according

to Fathona in table 2.1.

Table 2.1. The content of fitokima rhizome of red and elephant ginger (mg / g)

Kandungan Jahe merah Jahe gajah

6-gingerol, 18.03 mg/g 9.56 mg/g

8-gingerol, 4.09 mg/g 1.49 mg/g

10-gingerol 4.61 mg/g 2.96 mg/g

6-shogaol 1.36 mg/g 0.92 mg/g

(Fathona, 2011)

6-gingerol (1- [40-hydroxy-30-methyoxyphenyl] - 5 - hydroxy - 3 - decanone)

is a natural phenol compound in plants and is a major component of fresh ginger or

untreated ginger. 6-gingerol has various pharmacological effects, including

antioxidant, anti-inflammatory, and anti-cancer effects. 6- gingerol may also inhibit

TPA (12-Otetradecanoylphorbol-13-acetate) which is a mediated tumor promotion

and may inhibit TNF-α. 6-gingerol may also suppress macrophage activation so as

to suppress inflammatory effects (Kyoung, Younghwa, Kwang, 2007).

 16

Figure 2.2
Chemical Structure 6-Gingerol

Several studies have also been done. Some of the pharmacological effects

already possessed by red ginger include antioxidant, antimicrobial, anticacing,

antidiabetic, antiinflammatory, antiplatelet aggregation, analgesic, antiangiogenic,

antipyretic, immunomodulatory, hepatoprotective, renoprotective, gastroprotective

and neuroprotective effects (Dhanik, 2017).

Red ginger works as anti-inflammatory by inhibiting the formation of

prostaglandin, interfere with inflammatory cascade and inhibit vanilloid nociceptor

work. Other studies have described the mechanism of ginger as anti-inflammatory

by suppressing cytokines and pro-inflammatory chemokines produced by

leukocytes. Red ginger is also known to inhibit several genes that induce an

inflammatory response and modulate on biochemical pathways in chronic

inflammation (Qin and Xu, 2008)

 17

CHAPTER 3

CONCEPTUAL FRAMEWORK

Pirazinamid, Etambutol and

levofloxacin as offending
agent

Contact the receptor

Adaptive Immunity Red Ginger Innate Immunity

Gingerol
NK cell
Sel T Naif

Proinflamatory
cytokine release
APC
IL1

Dendritic Macrophage TNFα

cell IL6

Th0/CD4+ TNFα
Local Systemic
Response feedback -

Th2
Th1 Increase Hipotalamus
capillary
permeability

TNFα IL10 IL4 IL5 IL10

IL2 IFN
γ Fever

B cell
Description

: Produce
Plasma cell Memory : Influence
cell : Activate
: Differentiate
: Inhibit
Ig E : Investigated
: Not Investigated
: Content
Hipersensitve
type I
 18

CHAPTER 4

RESEARCH METHODS

4.1 Types of Research

The research type is pure experimental (True experimental design) with Post

Test Only Control Group Design which target are observed the levels of cytokine

interleukin 4, interleukin 10 and TNF-alpha after treatment.

4.2 Location and Time of Research

This research was conducted at Biomedical Laboratory of Medical Faculty

Muhammadiyah Malang University and Biomedical Laboratory of Medical Faculty

Brawijaya Malang with 3 month estimated time.

4.3 Population and Sample Research

4.3.1 Population

The population used in this study was male white mouse (Rattus novergicus

strain wistar) in healthy condition.

4.3.2 Sample

The samples were taken from male white mouse (Rattus novergicus strain

wistar) as experimental animals according to the inclusion criteria.

4.3.3 Large Sample

There were 5 treatment groups in the study, two control groups and 3

experimental groups.

Group 1: Positive Control

 19

Group 2: Negative Controls

Group 3: Red ginger extract 25 mg / 250 g of rat + pyrazinamide, ethambutol and

levofloxacin

Group 4: Red ginger extract 50 mg / 250 g rats + pyrazinamide, ethambutol and

levofloxacin

Group 5: Red ginger extract 100 mg / 250 g of rat + pyrazinamide, ethambutol and

levofloxacin

The calculation of the sample size used in this study is calculated by using the

formula To determine the sample size, using Federer's Formula (Arifin & Zahiruddin,

2017):

(r-1) (t-1) ≥ 15

Information:

t = treatment

r = number of replication

Then:

(r-1) (t-1) ≥ 15

(r-1) (5-1) ≥ 15

(r-1) (4) ≥ 15

4r-4 ≥ 15
 20

4r = 19

r=5

Σ sample of mouse = n x Σ treatment group

=5x5

= 25

The sample formula to anticipate the possibility of the selected

sample has dropout as follows

n '= large number of research samples

n = sample size calculated

f = proliferation of dropout proportions, 20% (f = 0.2)

the number of samples in this study group is

(n ') = 5 / (1-0,2) = 5 / 0.8

= 6, 2

=6

4.3.4 Sampling Techniques

Sampling technique using purposive sampling because not all of mouse in the

population have the same opportunity to be a sample.

 21

4.3.5 Characteristics of Research Sample

a. Inclusion Criteria

1. Age of mouse 2-3 months

2. Weight 150-250 grams

3. Male mouse (Rattus norvegicus strains wistar)

b. Exclusion Criteria

1. Mouse that have been used for research

2. Mouse that sick before the study

3. Mouse that died before the study

c. Drop Out Criteria

1. Mouse sick when treated

2. Mouse die when treated

4.3.6 Research Variables

4.3.6.1 Independent Variable

The independent variable is the variable that influences or becomes the cause

of change or the emergence of the dependent variable (Sugiyono, 2009). The

independent variable in this study were various concentrations of red ginger extract

(Zingiber officinale Roscoe Var Rubrum)

 22

4.3.6.2 Dependent Variables

The dependent variable in this study was interleukin 4, interleukin 10 and TNF-

alpha in male white mouse (Rattus norvegicus strain wistar)

4.3.7 Operational Definition

a. Red Ginger Extract

Red ginger extract is divided into 3 different doses: first dose is 25 mg / 250 gr

of mouse / day, dose 2 is 50 mg / 250 gr of mouse / day, dose 3 is 100 mg / 250 gr

mouse / day. The dose measurements were performed using an electric scale with

accuracy of 0.0001 then dissolved with 0.5% CMC and administered for 56 days using

gastric spuit (Elrokh, 2010). It used ordinal data scale.

b. IL-4, IL-10 and TNF-Alpha

Interleukin 4, Interleukin 10 and TNF-Alpha measured using the IL-10 ELISA

Kit and the material used for measurement using a mouse blood serum specimen. The

results are expressed in units of pg / mL. The data scale used is the ratio scale.

4.4 Research Tools and Materials

4.4.1 Tools

a. Mouse treatment tools: mouse cage 25 cm x 25 cm x 25 cm, water bottle, electric

scale, 3 cc syringe, handscoon, scalpel, glove, sonde.

b. Extracting tool: Oven, incubator, rotary evaporator, filter paper whatman number 2,

blender, knife, and mixer

c. Measurements of IL-4, IL-10 and TNF-Alpha: Micro plate Reader, ELISA kit
 23

4.4.2 Materials

a. Mouse treatment materials: Wistar male rats 2-3 months old white rat with weight

150-150 gr, used standard feed, aquades, 50% alcohol, chloroform

b. TB drugs pyrazinamide, ethambutol and levofloxacin

c. The ingredients of extract of red ginger fruit: 95% ethanol, aquades, and red ginger

with a weight of 2 kg Research Procedure

d. Measurements of IL-10: blood serum of experimental animals

4.5 Research Procedures

4.5.1 Preparation of red ginger extract

The process of making red ginger extract in this study used 96% ethanol as

solvent. The extraction starts from the red ginger weighing then chopping ginger into

tiny shape and then dry them and made them into powder using a blender. Ethanol 96%

was added to extract this powder for approximately 2 (two) hours and then continued

with maceration for 24 hours. After entering the filtration stage, the filtrate and residue

will be obtained. The obtained filtrate will be forwarded to evaporation stage with

Rotary Evaporator at 40 ° C so the viscous extract is obtained then dried it by using a

freeze dryer (-40oC).

4.5.2 Determination of Drug Doses

Drugs used in this study using pyrazinamide, ethambutol and also levofloxacin.

The calculation using the second line drug dose of anti-tuberculosis drug line as

follows:
 24

Table 4.1 Dosing of Second line drug anti-tuberculosis based on Patient's Weight
Group
Anti-tuberculosis Weight
drug < 33 kg 33–50 kg 51–70 kg >70 kg
Pyrazinamide (Z) 20–30 750–1.500 1.500–1.750 1.750–2.000
mg/kg/day mg mg mg
Ethambutol (E) 20–30 800–1.200 1.200–1.600 1.600–2.000
mg/kg/day mg mg mg
Levofloxacin (Lfx) 7,5–10 750 mg 750 mg 750–1.000
mg/kg/day mg
(Reviono,2014

Table 4.2 Conversion dose table

Mouse Rabbit Cat Monkey Dog 12 Human
250 g 1,5 kg 1,5 kg 4 kg kg 70 kg
Mouse 1,0 3,9 4,2 9,2 17,8 56,0
250 g
Rabbit 0,25 1,0 1,08 2,4 4,5 14,2
1,5 kg
Cat 0,23 0,92 1,0 2,2 4,1 13,0
1,5 kg
Monkey 0,11 0,42 0,43 0,1 1,9 6,1
4 kg
Dog 0,06 0,22 1,24 0,52 1,0 3,1
12 kg
Human 0,018 0,07 0,076 0,16 0.32 1,0
70 kg
(Laurance,1964 in Stevani,2016)
Dosage conversion Second-line drug anti-tuberculosis in humans to mouse:

• Pyrazinamide

Dose in humans with 70 kg weight: 1750 mg

Dose in mouse = Dose in humans x conversion factor

= 1750 mg x 0.018

= 31.5 mg / kgBW
 25

Dose in mouse 250 gr = 31.5 mg / kg x 0.2 kg

= 6.3 mg

• Ethambutol

Dose in humans with weight 70 kg: 1600 mg

Dose in mouse = Dose in humans x conversion factor = 1600 mg x 0.018

= 28.8 mg / kgBW

Dose in mouse 250 gr = 28.8 mg / kg x 0.2 kg

= 5.76 mg

• Levofloxacin

Dose in humans with weight 70 kg: 750 mg

Dose in mouse = Dose in humans x conversion factor = 750 mg x 0.018

= 13.5 mg / kgBW

Dose in mouse 250 gr = 13.5 mg / kg x 0.2 kg

= 2.7 mg

4.5.3 Adaptation process

Mouse were adapted for 7 days, then grouped randomly into 5 groups. Each

group consists 6 Mouse. Adaptation aims to make mouse adjust to the new

environment.
 26

4.5.4 The process of classifying experimental animals

The mouse used were 30 individuals divided into 5 groups: one negative group,

one positive control group, one treatment group with drugs and red ginger first dose,

one treatment group with drug and red ginger dose 2, and one treatment group with

giving drug and red ginger dose 3. Each group there are 6 mouse.

4.5.5 Procedures for measurement of IL-4, IL-10 and TNF-Alpha levels

To measure the levels of IL-4, IL-10 and TNF-Alpha it will be done with the

method of Enzyme Linked Immunosorbent Assay (ELISA), the steps are:

1. Mouse were anesthetized with 0.67 concentrated of chloroform ml with inhalation

administration

2. Abdominal and thoracic animals are dissected

3. Taking blood from left ventricle of animal cor 3 ml using 5 m syringe

4. Centrifuge at 3000 rpm for 15 minutes to separate serum from the precipitate within

30 min after taking

5. The obtained serum is stored in a temperature of -20 degrees C, if not directly

analyzed

6. ELISA test to determine the level of IL-4, IL-10 and TNF-Alpha

4.5.6 Handling the experimental animals

After the animals are dissected, it must be ensured that the animal does not

go to recover. Before burying the animal, it is certain that the pulse has stopped. If the

animal tries to recover then euthanasia procedure must be performed, one of them is

cervical dislocation procedure by separating the skull and vertebrae. This technique is

performed by applying pressure to the posterior portion of the skull base and vertebrae.
 27

When the vertebrae is separated from the brain, blinking reflexes disappear

immediately, the excitement of the pain disappears so that the animal does not feel

pain. Furthermore, experimental animals that have been dead will be collected into one

and then buried (Alexandru, 2011).

4.6 Research Plot

A sample of 30 experimental animals that comply inclusion

criteria will be adapted for 7 days

Negative Positive Treatment Treatment Treatment

control Control group dose group dose group dose
group given standard I II III
standard group
standard standard standard
feeding for feeding +
feeding + feeding + feeding +
28 days drugs for 28
drugs + red drugs + red drugs + red
days ginger ginger ginger
extract dose extract dose extract dose
25 mg / 250 50 mg / 250 100 mg /
gr day for gr day for 250 gr day
28 days 28 days for 28 days

Each mouse was anesthetized with 0.67 ml of concentrated

chloroform inhalation

Intake of animal blood:

a. Abdominal and thoracic animal surgery
b. Taking blood from the left ventricle of animal cor as
much as 3 ml using 5 ml syringe
c. After dissected collect all of mouse and then buried
them.

Measure the level of IL-4, IL-10 and TNF-Alpha using ELISA

Data analysis

figure 4.1
Research Plot
 28

4.7 Data Collection Techniques

The data collected in this study were primary data, the results of the research

were interleukin 4, interleukin 10 and TNF-Alpha levels in blood serum of

experimental animals from the treatment group compared with the control group. The

independent variable is the dose of red ginger extract, while the measurement scale is

ordinal scale. The dependent variable are the interleukin-4, interleukin-10 and TNF-

Alpha using ratio scale.

4.8 Data Analysis

In testing the difference of cytokine interleukin-4, interleukin-10 and TNF-

Alpha serum mouse level in each group was tested using one way annova and if it is

obtain significantly different result, then it is followed by Post Hoc Bonferroni test to

understand the significant difference of five treatments and it is used SPSS program 19

(Statistical Product and Service Solution version 19) so it can be known the right dose

of red ginger. One way Anova test and Post Hoc Bonferroni Test were significant when

the p <0,05 was obtained with 95% confidence level (α = 0,05).

a. Normality test by using Shaphiro Wilk Test to understand the normality of data (data

is normal if sig> 0,05). If it is normal, it used One Way Annova Test. If the data is

not normal, then used Kruskal Wallis test and Post hoc Mann Whitney test.

b. Homogeneity test using Levene Test to know the homogeneity of variant obtain data

(data is homogeneous if sig> 0,05). If the data is homogeneous then continue used

Post Hoc Bonferroni test. If the data is not homogeneous then Tamhane's Post Hoc

test is performed.
 29

c. One Way ANOVA to prove a significant difference between control and the group

with extract administration. ANOVA test results are said to have significant

differences if significant (sig) <0.05.

d. The Bonferroni Post Hoc test is a continuation test of the ANOVA test, used to

determine the significant differences between treatment groups in the study.

e. Kruskal Wallis Test, from the normality test results if it is known that there is 1 group

having sig <0.05 which indicates that there are still abnormal groups so that the

ANOVA test will be replaced using Kruskal Wallis

 30

CHAPTER 5

RESULTS AND DISCUSSION

5.1 Results

5.1.1 Interleukin-4

Table 5.1 Result of Interleukin 4 with ELISA

Sample Antibodies Levels (pg/mL)

Control -1 0,651 91,200
Control -2 0,725 106,000
Control -3 0,615 84,000
Control -4 0,854 131,800
Control -5 0,773 115,600
Control -6 0,78 117,000
MEAN 107,600

Control +1 0,861 133,200

Control +2 0,807 122,400
Control +3 0,836 128,200
Control +4 0,785 118,000
Control +5 0,817 124,400
Control +6 0,869 134,800
MEAN 126,833

Ginger dose 1 I 0,818 124,600

Ginger dose 1 II 0,912 143,400
Ginger dose 1 III 0,933 147,600
Ginger dose 1 IV 0,673 95,600
 31

Ginger dose 1 V 0,766 114,200

Ginger dose 1 VI 0,738 108,600
MEAN 122,333

Ginger dose 2 I 0,767 114,400

Ginger dose 2 II 0,793 119,600
Ginger dose 2 III 0,826 126,200
Ginger dose 2 IV 0,906 142,200
Ginger dose 2 V 0,84 129,000
Ginger dose 2 VI 0,954 151,800
MEAN 130,533

Ginger dose 3 I 0,776 116,200

Ginger dose 3 II 0,841 129,200
Ginger dose 3 III 1,056 172,200
Ginger dose 3 IV 0,881 137,200
Ginger dose 3 V 0,808 122,600
Ginger dose 3 VI 0,899 140,800
MEAN 136,367
 32

Figure 5.1
Result of Interleukin 4 graph
5.1.2 Interleukin-10

Table 5.2 Result of Interleukin 10 with ELISA

Sample Antibodies Levels (pg/mL)

Control -1 0,252 209,000
Control -2 0,249 206,000
Control -3 0,228 185,000
Control -4 0,236 193,000
Control -5 0,272 229,000
Control -6 0,235 192,000
MEAN 202,333

Control +1 0,35 307,000

Control +2 0,188 145,000
Control +3 0,181 138,000
Control +4 0,187 144,000
Control +5 0,294 251,000
 33

Control +6 0,304 261,000

MEAN 207,667

Ginger dose 1 I 0,275 232,000

Ginger dose 1 II 0,238 195,000
Ginger dose 1 III 0,282 239,000
Ginger dose 1 IV 0,238 195,000
Ginger dose 1 V 0,261 218,000
Ginger dose 1 VI 0,302 259,000
MEAN 223,000

Ginger dose 2 I 0,263 220,000

Ginger dose 2 II 0,256 213,000
Ginger dose 2 III 0,264 221,000
Ginger dose 2 IV 0,298 255,000
Ginger dose 2 V 0,314 271,000
Ginger dose 2 VI 0,3 257,000
MEAN 239,500

Ginger dose 3 I 0,305 262,000

Ginger dose 3 II 0,253 210,000
Ginger dose 3 III 0,28 237,000
Ginger dose 3 IV 0,247 204,000
Ginger dose 3 V 0,269 226,000
Ginger dose 3 VI 0,228 185,000
MEAN 220,667
 34

Figure 5.2
Result of Interleukin 10 graph
5.1.3 TNF-Alpha

Table 5.3 Result of TNF-Alpha with ELISA

Sample Antibodies Levels (ng/ml)

Control -1 0,719 503,000
Control -2 0,63 414,000
Control -3 0,747 531,000
Control -4 1,222 1006,000
Control -5 0,631 415,000
Control -6 0,572 356,000
MEAN 537,500

Control +1 0,667 451,000

Control +2 0,658 442,000
Control +3 0,572 356,000
Control +4 0,753 537,000
Control +5 0,632 416,000
 35

Control +6 0,636 420,000

MEAN 437,000

Ginger dose 1 I 0,789 573,000

Ginger dose 1 II 0,667 451,000
Ginger dose 1 III 0,608 392,000
Ginger dose 1 IV 0,682 466,000
Ginger dose 1 V 0,695 479,000
Ginger dose 1 VI 0,73 514,000
MEAN 479,167

Ginger dose 2 I 0,683 467,000

Ginger dose 2 II 0,728 512,000
Ginger dose 2 III 0,626 410,000
Ginger dose 2 IV 0,638 422,000
Ginger dose 2 V 0,675 459,000
Ginger dose 2 VI 0,661 445,000
MEAN 452,500

Ginger dose 3 I 0,651 435,000

Ginger dose 3 II 0,648 432,000
Ginger dose 3 III 0,683 467,000
Ginger dose 3 IV 0,635 419,000
Ginger dose 3 V 0,688 472,000
Ginger dose 3 VI 0,736 520,000
MEAN 457,500
 36

600,000

500,000

400,000

300,000

200,000

100,000

0,000
Control- Control+ Ginger 1 Ginger 2 Ginger 3

Figure 5.3
Result of TNF-Alpha graph

5.2 Statistic Results

5.2.1 IL-4

Descriptives

IL_4 N Mean Std. Deviation Std. Error Minimum Maximum

Control- 6 107.6000 17.70017 7.22606 84.00 131.80

Control+ 6 126.8333 6.47261 2.64243 118.00 134.80
Ginger I 6 122.3333 20.28188 8.28004 95.60 147.60
Ginger II 6 130.5333 14.06651 5.74263 114.40 151.80
Ginger III 6 136.3667 19.75831 8.06629 116.20 172.20
Total 122.3083 16.94306 2.44552 84.00 172.20

Based on the descriptive table it is known that the highest value of IL-4 come
from Ginger III group with an average of 136.3667 while the smallest come from
Control - that is 107.6000.
 37

To understand the comparison between groups, it needs normality test to check the
normality of data :

Tests of Normality
Treat Kolmogorov-Smirnova Shapiro-Wilk
Statistic df Sig. Statistic df Sig.
IL-4 Control - .174 6 .200* .962 6 .837
Control + .171 6 .200* .958 6 .807
Ginger I .184 6 .200* .946 6 .712
Ginger II .210 6 .200* .949 6 .731
Ginger III .245 6 .200* .896 6 .351

*. This is a lower bound of the true significance.

a. Lilliefors Significance Correction

From normality test results it is known that all groups have sig> 0.05 which
indicates that normality is fulfilled so ANOVA test can be used.

Test of Homogeneity of Variances

IL_4
Levene Statistic df1 df2 Sig.
.809 7 40 .585

Variety of IL-4 data is homogeneous (sig > 0.05).

ANOVA
IL_4
Sum of Squares df Mean Square F Sig.
Between Groups 3384.277 7 483.468 1.913 .093
Within Groups 10107.880 40 252.697
Total 13492.157 47

The results of the ANOVA test show that there is no IL-4 difference (sig> 0.05)

Multiple Comparisons
Dependent Variable: IL_4
Bonferroni
(I) Treat (J) Treat Mean Difference Std. Error Sig. 95% Confidence Interval
(I-J) Lower Bound Upper Bound
Control + -19.23333 9.17782 1.000 -49.9543 11.4876
Ginger I -14.73333 9.17782 1.000 -45.4543 15.9876
Control -
Ginger II -22.93333 9.17782 .467 -53.6543 7.7876
Ginger III -28.76667 9.17782 .090 -59.4876 1.9543
 38

Control - 19.23333 9.17782 1.000 -11.4876 49.9543

Ginger I 4.50000 9.17782 1.000 -26.2209 35.2209
Control +
Ginger II -3.70000 9.17782 1.000 -34.4209 27.0209
Ginger III -9.53333 9.17782 1.000 -40.2543 21.1876
Control - 14.73333 9.17782 1.000 -15.9876 45.4543
Control + -4.50000 9.17782 1.000 -35.2209 26.2209
Ginger I
Ginger II -8.20000 9.17782 1.000 -38.9209 22.5209
Ginger III -14.03333 9.17782 1.000 -44.7543 16.6876
Control - 22.93333 9.17782 .467 -7.7876 53.6543
Control + 3.70000 9.17782 1.000 -27.0209 34.4209
Ginger II
Ginger I 8.20000 9.17782 1.000 -22.5209 38.9209
Ginger III -5.83333 9.17782 1.000 -36.5543 24.8876
Control - 28.76667 9.17782 .090 -1.9543 59.4876
Control + 9.53333 9.17782 1.000 -21.1876 40.2543
Ginger III
Ginger I 14.03333 9.17782 1.000 -16.6876 44.7543
Ginger II 5.83333 9.17782 1.000 -24.8876 36.5543

Post Hoc results show that there is no single group that has a difference (sig> 0.05)

5.2.2 IL-10

Descriptives
IL_10

N Mean Std. Deviation Std. Error Minimum Maximum

Control- 6 202.3333 15.89549 6.48931 185.00 229.00

Control+ 6 207.6667 74.05854 30.23427 138.00 307.00
Ginger I 6 223.0000 25.40079 10.36983 195.00 259.00
Ginger II 6 239.5000 24.34543 9.93898 213.00 271.00
Ginger III 6 220.6667 27.08259 11.05642 185.00 262.00
Total 48 206.4375 43.48974 6.27720 129.00 307.00

Based on the descriptive table it is known that the highest value of IL-10
come from Ginger II group with an average of 239.5000 while the smallest come
from Control - that is 202.3333.
To understand the comparison between groups, it needs normality test to
check the normality of data :

Tests of Normality
Treat Kolmogorov-Smirnova Shapiro-Wilk
Statistic df Sig. Statistic df Sig.
IL_10 Control- .221 6 .200* .926 6 .552
 39

Control+ .301 6 .095 .830 6 .107

Ginger I .198 6 .200* .929 6 .570
Ginger II .276 6 .169 .867 6 .213
Ginger III .153 6 .200* .988 6 .983
*. This is a lower bound of the true significance.
a. Lilliefors Significance Correction

From normality test results it is known that all groups have sig> 0.05 which indicates
that normality is fulfilled so ANOVA test can be used.

Test of Homogeneity of Variances

IL_10
Levene Statistic df1 df2 Sig.
5.408 7 40 .000

Variety of IL-10 data is not homogeneous (sig < 0.05).

ANOVA
IL_10
Sum of Squares df Mean Square F Sig.
Between Groups 25031.313 7 3575.902 2.240 .051
Within Groups 63862.500 40 1596.563
Total 88893.813 47

The results of the ANOVA test show that there is no IL-10 difference (sig> 0.05)

Multiple Comparisons
Dependent Variable: IL_10
Tamhane
(I) Treat (J) Treat Mean Difference Std. Error Sig. 95% Confidence Interval
(I-J) Lower Bound Upper Bound
Control+ -5.33333 30.92284 1.000 -179.6272 168.9605
Ginger I -20.66667 12.23292 .978 -75.4334 34.1000
Control-
Ginger II -37.16667 11.86990 .302 -89.7810 15.4477
Ginger III -18.33333 12.82012 .997 -76.6508 39.9841
Control - 5.33333 30.92284 1.000 -168.9605 179.6272
Ginger I -15.33333 31.96317 1.000 -182.0524 151.3857
Control+
Ginger II -31.83333 31.82600 1.000 -199.3450 135.6783
Ginger III -13.00000 32.19248 1.000 -178.5134 152.5134
Control - 20.66667 12.23292 .978 -34.1000 75.4334
Control+ 15.33333 31.96317 1.000 -151.3857 182.0524
Ginger I
Ginger II -16.50000 14.36373 1.000 -76.8558 43.8558
Ginger III 2.33333 15.15842 1.000 -61.4100 66.0767
 40

Control- 37.16667 11.86990 .302 -15.4477 89.7810

Control+ 31.83333 31.82600 1.000 -135.6783 199.3450
Ginger II
Ginger I 16.50000 14.36373 1.000 -43.8558 76.8558
Ginger III 18.83333 14.86700 .999 -43.8323 81.4990
Control - 18.33333 12.82012 .997 -39.9841 76.6508
Control + 13.00000 32.19248 1.000 -152.5134 178.5134
Ginger III
Ginger I -2.33333 15.15842 1.000 -66.0767 61.4100
Ginger II -18.83333 14.86700 .999 -81.4990 43.8323

Post Hoc results show that there is no single group that has a difference (sig> 0.05)

5.2.3 TNF-Alpha

Descriptives
TNF_Alpha

N Mean Std. Deviation Std. Error Minimum Maximum

Control- 6 537.5000 238.29624 97.28403 356.00 1006.00

Control+ 6 437.0000 59.18108 24.16057 356.00 537.00
Ginger I 6 479.1667 60.96365 24.88831 392.00 573.00
Ginger II 6 452.5000 36.29187 14.81609 410.00 512.00
Ginger III 6 457.5000 37.00135 15.10574 419.00 520.00
Total 48 455.0625 99.52427 14.36509 333.00 1006.00

Based on the descriptive table it is known that the highest value of TNF-
Alpha come from control- group with an average of 537.5000 while the smallest
come from Control+ that is 437.0000.

To understand the comparison between groups, it needs normality test to

check the normality of data :

Tests of Normality
Perlakuan Kolmogorov-Smirnova Shapiro-Wilk
Statistic df Sig. Statistic df Sig.
Control- .344 6 .025 .741 6 .016
TNF-Alpha Control+ .240 6 .200* .938 6 .641
Ginger I .168 6 .200* .984 6 .969
 41

Ginger II .178 6 .200* .959 6 .808

Ginger III .228 6 .200* .912 6 .447
*. This is a lower bound of the true significance.
a. Lilliefors Significance Correction

From the normality test results it is known that there is 1 group having sig
<0.05 which indicates that there are still abnormal groups so that the ANOVA test
will be replaced using Kruskal Wallis

Kruskal-Wallis Test
Ranks
Perlakuan N Mean Rank
K- 6 27.33
K+ 6 22.33
JI 6 32.17
TNF_Alpha
JII 6 26.58
JIII 6 28.08
Total 48

Test Statisticsa,b
TNF_Alfa
Chi-Square 5.943
df 7
Asymp. Sig. .546
a. Kruskal Wallis Test
b. Grouping Variable:
Perlakuan

Sig values obtained> 0.05 indicate that there is no difference in overall TNF-Alpha
levels. Therefore it is not necessary to continue with Mann Whitney's post hoc.

5.3 Discussion

One of the many health claims attributed to ginger is purported ability to

decrease inflammation, swelling, and pain. Gingerol, shogaol, and other structurally-

related substances in ginger inhibit prostaglandin and leukotriene biosynthesis through

suppression of 5-lipoxygenase or prostaglandin synthetase. Additionally, they can also

 42

inhibit synthesis of pro-inflammatory cytokines such as TNF-Aplha and increase anti

inflammatory cytokines such as IL-10 (Young et al. 2005), (Minghetti et al. 2007).

Red Ginger actually has a potential as antiinflamatory agent even in this study

there is not significant change in used of red ginger against IL-4, IL-10 and TNF-Alpha.

It might because of the inducer pyrazinamide, ethambutol and levofloxacin that

consumed at the same time with high doses of normal theraphy did not show significant

inflammation side effect only in 56 days. So, each category of cytokines need to be

discussed based on the result.

Start from IL-4 that can act as antiinflammatory and also inflammatory

cytokines. IL-4 can downregulate the production of a number of pro-inflammatory

mediators involved in IBD, but they can also lead to Th2-mediated forms of the disease.

Their production is reduced in the inflamed mucosa of Crohn's disease or ulcerative

colitis, yet IL-4-producing Th2 cells appear to have a pathogenic role in colitis. IL-4

also have a pathogenic role in some models of SLE, but it is role in the human disease

process is unclear. IL-4 is absent in the synovial fluid of RA patients. Although

overexpression exacerbates inflammation, it appears to protect against cartilage and

bone destruction (Lubberts, 2001).

The results show that IL-4 levels rise in each elevation of red ginger doses but

it can not be stated that red ginger act as antiinflammatory or inflammatory agents

because IL-4 indicates both of them. So, it needs to observe the levels of IL-10 and

TNF-Alpha to understand the effect of red ginger in inflammation.

 43

Interleukin 10 or IL-10 has anti-inflammatory and immunosuppressive effects

by inhibiting the synthesis of immunostimulatory cytokines produced by Th 1 cells

such as IL-1, IL-6, IL-8, IL-12 and TNF-Alpha with macrophage activation as well as

from IFN-y. IL-10 deficient mice develop a form of inflammatory bowel disease that

is histologically similar to human IBD. Although the levels of IL-10 are increased in

the synovial fluid of RA patients it does not appear to have a pathogenic role. IL-10,

however, can prevent collagen-induced arthritis (Nashan, 2001).

It shows that IL-10 levels increase in red ginger dose 1 and dose 2, it indicates

that the red ginger act as antiinflamation. The red ginger doses 3 , IL-10 levels decrease

if it is compared with dose 1 and dose 2, it indicates that red ginger in this doses induces

inflamation. So it can be stated that in high doses red ginger can induces worse

inflammation.

Tumor Necrosis Factor alpha (TNF alpha), is an inflammatory cytokine

produced by macrophages/monocytes during inflammation and it is responsible for a

diverse range of signalling events within cells, leading to necrosis or apoptosis.TNF-

Alpha has direct cytotoxic effects on the intestinal mucosa in Crohn's disease and

ulcerative colitis but also contributes to the systemic manifestations seen in these

diseases (OõNeil, 2001).

This study shows that TNF-Alpha levels in red ginger dose 1 higher than dose

2, it shows that red ginger dose 2 has more effective dose than dose 1. Compared with

dose 3, red ginger dose 2 has lower TNF-Alpha level so it can be stated that red ginger

dose 3 start giving the inflammation effect.

 44

The correlation between IL-4, IL-10 and TNF-Alpha here can be seen. If IL-4

level increases associated with elevation of IL-10 and reduction of TNF-Alpha level, it

downregulate inflammation progress. However, elevation of IL-4 associated with

elevation of TNF-Alpha and reduction of IL-10 level, it shows that inflammation

progress is getting worse.

In contrast, larger doses of ginger components inhibited oxygen consumption,

which was attributed to disruption of mitochondrial function. In high doses, red ginger

inger could be associated with worsen inflammation symptoms, it might be cause of

thermogenic or heat effect. The thermogenesis can cause dehydration and allergic

reactions. High dose of red ginger might precipitate cardiac arrhythmias, stimulate

irritable bowel syndrome, duodenal ulcer, secretion of bile, and heartburn (Ueki et al,

2008).

The management of red ginger extract dose need to be noticed. Red ginger with

25 mg dose has weak effect againts inflammation, it might be stated that it is in sub

therapy dose condition while red ginger with 100 mg resulting in elevation of IL-4

associated with TNF-Alpha elevation and reduction of IL-10 indicates inflammation

proggress is start increasing again after 50mg dose. Red ginger using 50mg dose has

the best result, this dose shows elevation of IL-4 associated with IL-10 and reduction

of TNF-Alpha level indicate the downregulate of inflammation progress.

 45

CHAPTER 6

CONCLUSION AND SUGGESTION

6.1 Conclusion

Cytokines play key roles in inflammation and autoimune disease. Deficiency

and elevation of some cytokines cause inflammation and trigger immunity problems,

some of them are IL-4, IL-10 and TNF-Alpha. IL-4 can act as antiinflammatory and

also inflammatory cytokines, IL-10 act as antiinflammatory cytokines and TNF-Alpha

act as inflammatory cytokines. There are correlation between IL-4, IL-10 and TNF-

Alpha, if IL-4 level increases associated with elevation of IL-10 and reduction of TNF-

Alpha level, it downregulate inflammation progress. However, elevation of IL-4

associated with elevation of TNF-Alpha and reduction of IL-10 level, it shows that

inflammation progress is getting worse.

Red Ginger actually has a potential as antiinflamatory agent even in this study

there is not significant change in used of red ginger against IL-4, IL-10 and TNF-Alpha.

One of the many health claims attributed to ginger is purported ability to decrease

inflammation, swelling, and pain. Gingerol, shogaol, and other structurally-related

substances in ginger inhibit prostaglandin and leukotriene biosynthesis through

suppression of 5-lipoxygenase or prostaglandin synthetase. It might because of the

inducer pyrazinamide, ethambutol and levofloxacin. So it can be concluded that

pyrazinamide, ethambutol and levofloxacin safe for being consumed in high dose of

normal therapy less than 56 days cause it did not show systemic inflammation

significantly.
 46

Management of red ginger extract dose need to be noticed. Red ginger with 25

mg dose has weak effect againts inflammation, it might be stated that it is in sub therapy

dose condition while red ginger with 100 mg resulting in elevation of IL-4 associated

with TNF-Alpha elevation and reduction of IL-10 indicates inflammation proggress is

start increasing again after 50mg dose. Red ginger using 50mg dose has the best result,

this dose shows elevation of IL-4 associated with IL-10 and reduction of TNF-Alpha

level indicate the downregulate of inflammation progress.

6.2 Suggestion

Further research if the study focused on role of red ginger extract and it is

potential as antiinflammatory, the dose of drugs (pyrazinamide, levofloxacin and

ethambutol) need to be changed into toxic dose to give more significant inflammation

effect. To strenghten the potential of red ginger, we need to see Malondialdehyde

(MDA) levels. MDA appears to be a sensitive marker of sytemic inflammation.

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