The Effect of Apple Fruit Extract (Malus Sylvestris Mill) as an Antioxidant and Anti-Inflammatory on Allergic Rhinitis
Phase: NA
Conditions: Rhinitis, Allergic, Perennial
Sponsor: Universitas Sebelas Maret

STUDY PROTOCOL AND STATISTICAL ANALYSIS 
 
The Effect of Apple Fruit Extract (Malus Sylvestris Mill) as an Antioxidant and 
Anti-Inflammatory on Allergic Rhinitis 
 
ClinicalTrials.gov Identifier: 
Not yet assigned 
 
Document Type: 
Study Protocol and Statistical Analysis 
 
Document Date: 
20 January 2026 
 
Sponsor: 
Universitas Sebelas Maret 
 
Principal Investigator: 
Niken Dyah Aryani Kuncorowati, MD, Otolaryngologist (ENT Specialist) 
 
Institution: 
Universitas Sebelas Maret / Sebelas Maret Hospital 
 

 
THE INFLUENCE OF APPLE FRUIT EXTRACT (MALUS SYLVESTRIS 
MILL) AS AN ANTIOXIDANT AND ANTI-INFLAMMATORY IN 
ALLERGIC RHINITIS 
(Study on the levels of Malondialdehyde, Nuclear Factor Kappa Beta, Tumor 
Necrosis Factor Alpha, Interleukin 6, Interleukin 8, clinical symptoms of nasal 
congestion, sneezing, itchy nose, and rhinorrhea) 
 
CHAPTER I 
 
INTRODUCTION 
 
A. Background of the Problem 
 
Allergic Rhinitis (AR) is a chronic disease that can affect the quality of life of its 
sufferers. As many as 57% of AR sufferers still experience symptoms despite 
receiving antihistamine therapy and intranasal corticosteroids (ICS). Allergic 
Rhinitis is an inflammatory process in the nasal mucosa that is initiated by 
hypersensitivity reactions due to exposure to allergens mediated by 
Immunoglobulin E (IgE) with several characteristic symptoms consisting of: nasal 
congestion, runny nose or watery nasal discharge (rhinorrhea), itching in the nose, 
and sneezing. The occurrence of AR can be triggered by contact with allergens. 
Several allergens that can cause AR symptoms are often found in both home and 
outdoor environments. Examples of allergens include: house dust mites, pollen, and 
fur or hair from pets (Akhouri & House, 2022). 
 
Based on a survey by the World Health Organization (WHO), approximately 400 
million people worldwide suffer from allergic rhinitis, and the incidence rate is 
expected to continue to rise. According to the ISA AC phase III study, the 
prevalence of children aged 13 to 14 years with allergic rhinitis ranges from 1.4% 
to 39.7% of the global population. According to the World Allergy Report, the 
incidence of allergic rhinitis in low- and middle-income countries in the Asia- 
Pacific region is 5% – 45% of the total population (Nurhutami et al., 2020). Allergen 
avoidance is often difficult to achieve due to the daily activities or jobs of sufferers 
that do not allow for avoiding allergens, thus necessitating long-term use of 
antihistamines. 
 
Data from the AR patients in the ENT outpatient unit at the Sebelas Maret 
University Hospital, Surakarta, shows that the number of new AR cases in 2023 is 
103 (37%) cases, and old cases are 170 (62%) cases. The number of new cases up 
to April 2024 is 159 (36%) cases, and old cases are 273 (63%) cases. This data 
indicates that the number of old cases each year is greater than the number of new 
cases, which shows. 
 
2 that long-term patients still experience complaints and still come for treatment. 
This may be caused by the standard therapy used not being maximal in addressing 

 
complaints comprehensively, thus complementary therapy is still needed to address 
RA symptoms. Complementary treatment is a group of additional medical care 
using modalities or products that are not included in conventional medicine, which 
are used on patients with allergic diseases including RA. Various complementary 
therapies for RA include acupuncture, herbal medicine, homeopathy, and physical 
therapy. 
 
Pathogenetically, inflammation in RA begins with the sensitization phase, rapid 
allergic reaction phase (RAFC), and slow allergic reaction phase (RAFL). In RAFC, 
mast cell degranulation occurs, resulting in the production of several mediators such 
as histamine, prostaglandin, and leukotriene. In RAFL, infiltration of several 
inflammatory cells such as eosinophils occurs due to ICAM 1 (Intercellular 
adhesion molecule 1) molecules. Subsequently, several inflammatory cytokines 
such as interleukin (IL) IL 4, IL 6, Tumor Necrosis Factor Alpha, and others are 
produced. The pathway for cytokine and inflammatory molecule formation is 
through cytokine gene transcription by NFkB (Nuclear factor kappa B) in the cell 
nucleus. The activators of NFkB include oxidant free radicals, namely ROS 
(Reactive oxygen species) such as air pollution. In research that has been conducted, 
an increase in NFkB expression and mRNA ICAM 1 in the nasal mucosa of RA 
patients was observed (Zhao et al, 2008). 
 
Reactive oxygen species originating from motor vehicle pollution can strengthen 
allergic inflammation by increasing IgE production through B cell stimulation, 
enhancing allergen-induced specific IgE production, stimulating the production of 
cytokines IL 4 and IL 6, stimulating Th2, inducing the influx of inflammatory cells 
by increasing ICAM 1 expression, enhancing chemokine production, and inducing 
NFkB in expressing IL 8, IL 6, and TNF α. Hwang et al (2006) concluded that 
exposure to pollutants NOx, CO, and SO2 would increase the incidence of RA. 
Then Sequeira et al (2012) stated that there is a role of oxidative stress in RA with 
moderate to severe degrees, so strategies to maintain the balance of ROS and total 
antioxidant capacity can be beneficial in RA. 
 
Apple Fruit Extract 
 
Apple fruit extract derived from raw apples has been reported to have anti-allergic 
activity. In human atopic dermatitis, itching, sleep disturbances, and peripheral 
eosinophil counts have been reported to significantly decrease in the group 
receiving oral apple polyphenols at 10 mg/kg for 8 weeks compared to the control 
group treated with placebo (Enomoto et al, 2006). The mechanism by which apple 
fruit extract inhibits allergic reactions is provided by the observation that apple 
polyphenols and procyanidin fractions prevent degranulation and histamine release 
by inhibiting the increase in calcium concentration in antigen-stimulated mouse 
basophils (RBL 2H3 cells). Apple fruit extract is suggested to prevent degranulation 
by enhancing granulocyte stability and inhibiting histamine release. Additionally, 
apple fruit extract and its procyanidin fractions may also suppress inflammation, as 
they  have  been  reported  to  have  a  dose-dependent  inhibitory  effect  on 

 
hyaluronidase activity, an enzyme associated with inflammation and released by 
degranulation. 
 
CHAPTER II LITERATURE REVIEW 
 
A. Theoretical Basis 
Allergic Rhinitis 
a. Definition 
 
Rhinitis comes from the word rino which means nose or nasal and itis which means 
inflammation or inflammatory. Allergic rhinitis is an inflammatory disease of the 
nasal mucosa characterized by symptoms such as frequent sneezing, a blocked nose, 
nasal pruritus or itching in the nose, and clear rhinorrhea or discharge from the nose 
(Bousquet et al., 2020). The inflammatory reaction in allergic rhinitis is caused by 
an allergic reaction in patients with a history of atopy who have previously been 
sensitized to the same allergen (Kasim et al., 2020). In addition to symptomatic 
symptoms in the nose, patients with allergic rhinitis may also experience allergic 
conjunctivitis, nonproductive cough, dysfunction of the eustachian tube, and 
chronic sinusitis (Akhouri & House, 2022). 
 
b. Classification 
 
Initially, allergic rhinitis (AR) was divided into several classifications based on the 
time of exposure, becoming seasonal (occurring in certain seasons), perennial 
(occurring throughout the year), and occupational. This classification then changed 
to the latest clarification according to the guidelines of The Allergic Rhinitis and Its 
Impact on Asthma (ARIA). The ARIA guidelines classify allergic rhinitis based on 
the duration of symptoms and the severity of symptoms. The classification of AR 
based on symptoms according to ARIA is as follows: 
 
1. Intermittent: classified as allergic rhinitis with symptoms ≤4 days per week 
or lasting ≤4 weeks. 
 
2. Persistent: defined as allergic rhinitis with symptoms >4 days per week 
and duration >4 weeks. 
 
Whereas based on the severity of allergic rhinitis symptoms, it can then be classified 
into: 
 
1. Mild: Can sleep normally No disturbances during daily activities, sports, 
and relaxation Works and studies normally No bothersome complaints 
2. Moderate to Severe: Sleep disturbed There are disturbances during daily 
activities, sports, and relaxation Disturbances while working and studying 
There are bothersome complaints 

 
Figure 2.1. Classification of Allergic Rhinitis According to ARIA (Klimek et al., 
2019) 
 
Note: Classification of AR based on symptoms according to ARIA. 
 
Intermittent: classified as allergic rhinitis with symptoms ≤4 days per week or 
duration ≤4 weeks, Persistent: defined as allergic rhinitis with symptoms >4 days 
per week and duration >4 weeks. 
Epidemiology of Allergic Rhinitis 
 
Allergic Rhinitis affects about 25 to 40% of children and adults globally. About 
80% of AR symptoms appear before the age of 20 years and peak at ages 20 to 40 
years before gradually declining. (Nur Husna, 2022) The incidence rate of AR in 
children during the first 5 years of life is reported to be 17.2%, with the peak age 
at diagnosis between 24 and 29 months (2.5%). Meta-analysis studies have shown 
specific gender differences in the prevalence of AR, with a male predominance in 
childhood and a female predominance in adolescence. (Pinart, 2017). The 
incidence of AR is also influenced by the living environment. This is mainly due 
to increased pollutant levels (e.g., traffic-related pollutants) that can exacerbate 
clinical AR. (Nur Husna, 2022) 
 
Pathophysiology of Allergic Rhinitis 
 
Type I hypersensitivity is an allergic reaction mediated by IgE antibodies in 
response to allergens. Type I hypersensitivity reactions occur rapidly (+/- 20 
minutes after exposure to allergens), activating mast cells and inflammation as well 
as their infiltration into tissues. (Gangwar, 2015) The allergic response in AR can 
be divided into two phases: the early phase and the late phase. The early phase 
begins within 20 minutes after exposure to harmful allergens. Antigen-presenting 
cells such as dendritic cells in the mucosal surface uptake, process, and present 
peptides from allergens on major histocompatibility complex (MHC) class II 
molecules. The antigen-MHC class II complex serves as a ligand for the T cell 
receptor on naive CD4+ T cells, resulting in the differentiation of naive CD4+ T 
cells into allergen-specific Th2 cells. Cytokines such as IL-4 and IL-13 released 
from activated Th2 cells interact with B cells to produce allergen-specific IgE. This 
allergen-specific IgE binds to high-affinity Fc receptors for IgE (FcεR) present on 
mast cells, leading to mast cell activation. (Nur Husna,2022). The cross-linking of 
FcεR on mast cells causes the release of allergy mediators consisting of histamine, 
protease, and lipid mediators such as leukotriene (LT) C4, and prostaglandin D2 
(PGD2) which cause blood vessel leakage, bronchoconstriction, inflammation, and 
intestinal hypermotility (Justiz, 2018). These mediators induce mucosal edema and 
characteristic watery rhinorrhea of allergic rhinitis (AR) by causing blood vessels 
to leak. Histamine is the main mediator in AR where it activates H1 receptors on 
sensory nerve endings and causes sneezing, pruritus, and reflex secretion responses, 
and also interacts with H1 and H2 receptors on mucosal blood vessels, causing 

 
swelling of blood vessels (nasal obstruction) and plasma leakage (Nur Husna, 
2022). 
 
After 4 to 6 hours of allergen exposure, the final phase of the allergic response 
begins. In this phase, inflammation of the nasal mucosa occurs with the entry and 
activation of various inflammatory cells such as T cells, eosinophils, basophils, 
neutrophils, and monocytes into the nasal mucosa (Nur Husna, 2022). The 
recruitment of these inflammatory cells is triggered by cytokines such as IL-4 and 
IL-5. These cytokines regulate the expression of adhesion molecules such as 
vascular cell adhesion molecule 1 (VCAM-1) on endothelial cells that facilitate the 
entry of inflammatory cells. Activation of structural cells in the nasal mucosa, such 
as epithelial cells and fibroblasts, can promote the release of additional chemokines 
(e.g., eotaxin, RANTES, and TARC) that facilitate cellular entry from peripheral 
blood (Nur Husna, 2022). 
 
Helper T cells 2 (Th2) activate a type 2 response by stimulating B cells to proliferate 
and differentiate into plasma cells through the production of Th2 cytokines 
including IL-4, IL-5, IL-6, and IL-13. Th2 cells are the main contributors to IgE- 
producing B cells, and Th2 cells play a key role in the pathogenesis of allergic 
rhinitis. Together with eosinophils and basophils, Th2 cells infiltrate the nasal 
mucosal tissue, producing the final phase allergic response. IL-4 is a key cytokine. 
 
17 degrees allergy and the initial sensitization dose can be determined, known to be 
slightly more sensitive compared to serological tests and more cost-effective. 
 
Contraindications of SET include patients with uncontrolled/severe asthma, 
unstable cardiovascular disease, pregnancy, and/or concurrent beta blocker therapy. 
H2 receptor antagonists, tricyclic antidepressants, and anti-IgE monoclonal 
antibodies omalizumab can interfere with allergic reactions in skin tests. Therefore, 
it is recommended to stop use before testing (Schoeman et al., 2021). 
 
Another in vitro test that can be performed is the eosinophil count. Eosinophil levels 
may be normal or increased. Total IgE tests (Pristopaper radioimmunosorbent test) 
often show normal values unless the patient shows signs of allergy to more than one 
type of disease such as bronchial asthma or urticaria (Schoeman et al., 2021). 
 
Mallus sylvestris Mill (Apple Fruit) 
 
The apple fruit has the Latin name Mallus sylvestris mill and is a perennial fruit 
plant originating from the western Asia region with a subtropical climate. Apple 
plants can grow and bear fruit well at altitudes of 700 to 1200 m above sea level, 
with an optimal altitude of 1000 to 1200 m above sea level. Apple plants have a 
taproot, which is a root that is perpendicular to the ground that supports the plant 
and absorbs nutrients from the soil. The bark is usually brown and scaly. The simple 
leaves are oval-shaped and usually have fine teeth along their edges. Apple flowers 
are striking with five white petals, often tinged with pink, and many stamens. The 

 
flowers are pollinated by bees and other insects, and most varieties require cross- 
pollination for fertilization (Botannica, 2023). 
 
Apple plants have very varied fruits, which are green, red, and also reddish with 
oval or round shapes. The skin of the apple fruit is thin and rough and has large 
pores. However, after.perfect ripe will become shiny and also smooth the surface 
of the fruit. 
 
Apple is one type of fruit that is high in fiber and phytochemical content, especially 
phenolic and flavonoid (Lobo, 2019). 
 
Flavonoids are one group of phenolics that have antioxidants obtained from food 
sources derived from plants believed to be protection against oxidative stress. One 
of the most important flavonoid contents is quercetin. The quercetin content in 
apples is quite high, in 100 grams of apple contains about 4.42 mg of quercetin 
aglycone and 13.2 quercetin glycosides (Mahardinar, 2015). 
 
The compounds most commonly found in apple skin consist of procyanidins, 
catechin, epicatechin, chlorogenic acid, phloridzin, and quercetin conjugates. Inside 
the apple flesh, there are several catechins, procyanidins, epicatechins, and 
phloridzin, but these compounds are found in much lower concentrations than in 
the skin (Patricia et al., 2016). 
 
Apples are produced globally. Apples are consumed raw and in processed products, 
such as dried fruit, juice, apple cider, brandy, jam, and vinegar. Apple consumption 
reduces the risk of coronary heart disease, which is related to the presence of high 
concentrations of polyphenols in apples (Wojdyło & Oszmiański, 2020). 
 
Apples contain five different structural subclasses, including phenolic acids and 
flavonoids, which consist of phenolic carboxylic acids (e.g., chlorogenic acid), 
anthocyanins (e.g., cyanidin glycosides), flavonols (e.g., quercetin glycosides), 
dihydrochalcones (e.g., phloridzin), flavan 3-ols (e.g., epicatechin) as well as their 
oligomers and polymers, and procyanidins. Chlorogenic acid is the most abundant 
polyphenol in apples as a single polyphenol. Among these polyphenols, 70% of it 
consists of flavan 3-ols and procyanidins. The concentration of procyanidins in 
apples is significantly correlated with antioxidant activity, and various clinical 
studies in animals and humans show that flavan 3-ols and procyanidins are involved 
in various health benefits, such as the prevention of dementia, cardiovascular 
diseases, type II diabetes, and cancer (Shoji et al., 2021). 
 
The Taxonomic Position of Flavonoids 
 
The taxonomic position of flavonoids is viewed from the level of oxidation of the 
structure and the substitution pattern of the C bonds. In one class, different from 
one another, are flavonoids with substituents on bonds A and B. Flavonoids have 
the following taxonomic position: flavon, flavanon, isoflavon, flavonol, flavanonol, 

 
flavan 3 ol, and anthocyanidin (Simamora, 2009). According to Simamora (2010), 
the basic structure of flavonoids can be seen in Figure 2.5 as follows: 
 
4.  Antioxidant Polyphenols in Apples 
 
Quercetin (3,31,41,5,7 pentahydroxyflavone) is a natural polyphenol flavonoid 
found in several fruits and vegetables, such as: apples, tomatoes, grapes, red onions, 
onions, legumes, and grains. Quercetin is an antioxidant flavonol from the flavonoid 
group and becomes the glycoside Que. This glycoside can conjugate with glucose, 
xylose, or rutinose and then attach to one of the Que hydroxyl groups, which 
ultimately creates various forms of glycoside Que. Nutritional flavonoids like Que 
are a type of antioxidant that is better than vitamins C and E. Que and its derivatives 
have major biological effects in the development of cycles and cellular signal 
transduction pathways. (Jafarinia et al., 2020). 
 
he chemical structure of quercetin is an anti-flavan with carbon atoms, namely 15 
atoms in three rings (C6 C3 C6), which are marked with labels A, B, and C. 
Quercetin is one of the flavonoids needed by the body, around 5 to 40 mg per day. 
Quercetin can be metabolized in the intestines and liver. Quercetin has 3 rings and 
a 5-hydroxyl group. 
 
An important benefit of quercetin is as an anti-inflammatory, antioxidant, and its 
ability to inhibit lipid peroxidation, platelet aggregation, and capillary permeability. 
Quercetin is known to be able to inhibit lipopolysaccharide (LPS) induced 
production of tumor necrosis factor alpha (TNF α) in macrophages. LPS can induce 
the mRNA levels of TNF α and IL 1α, producing inflammatory enzymes such as 
cyclooxygenase (COX) and lipoxygenase (LOX). Based on this, quercetin as an 
antioxidant plays an important role in addressing inflammation (Jafarinia et al., 
2020). 
 
The antioxidant mechanism possessed by quercetin is reflected in its effects on 
glutathione (GSH), signal transduction pathways, ROS, and enzyme activity 
(Deepika and Maurya 2022). Quercetin can enhance the antioxidant capacity in the 
body by regulating GSH levels. This is because free radicals are produced by the 
body during metabolic processes, leading to genetic mutations, membrane cell 
damage, and causing various diseases such as heart disease, liver disease, diabetes 
mellitus, and accelerating body aging. 
 
When the body produces free radicals, Superoxide Dismutase (SOD) will 
immediately convert O2 into H2O2, which will then be catalyzed into non-toxic 
H2O and GSH. Quercetin can convert H2O2 into H2O, and GSH will be oxidized 
and then oxidize glutathione (Michala and Pritsa, 2022). 
 
Research by Granado Serrano et al. shows that Que increases the antioxidant 
capacity of cells by enhancing the intracellular p28 MAPK pathway, increasing 
GSH levels, and providing hydrogen donors to scavenge free radicals present in the 

 
body. It was also found that Que inhibits atherosclerosis by blocking Nf kB 
signaling, thereby increasing NADPH expression. By affecting signal transduction, 
Que can modulate enzymes or antioxidant substances and enhance antioxidant 
properties, thus preventing disease progression. Que can prevent oxidative damage 
by inhibiting oxidative stress. Oxidative stress is caused by an imbalance between 
antioxidants and oxidants in the body and reactions that tend toward oxidation, 
where once oxidized, it will lead to increased secretion of protein enzymes and 
neutrophil infiltration. Que can regulate the balance of antioxidants and oxidants to 
suppress oxidative stress. Two enzymes, namely acetylcholinesterase and 
butyrylcholinesterase, are related to oxidative properties, while the OH group on 
the lateral side of the benzene ring in Que binds to amino acid residues on both 
active sides of the enzyme, thus preventing oxidative effects (Michala and Pritsa 
2022) (Qi et al., 2022). 
 
Anti-Inflammatory Apple Polyphenols 
 
Inflammation is a normal biological process in response to tissue injury, microbial 
pathogen infection, and chemical irritants. This process begins with the migration 
of immune cells and the release of mediators, followed by the recruitment of 
inflammatory cells, the release of pro-inflammatory cytokines, and tissue repair. 
Flavonoids, such as hesperidin, apigenin, luteolin, and quercetin, have anti- 
inflammatory and analgesic effects and can influence the enzyme systems involved 
in the inflammatory process. Flavonoids can inhibit the expression of inducible 
nitric oxide synthase isoforms, cyclooxygenase, and lipoxygenase, which produce 
cytokines, prostanoids, leukotrienes, and other mediators of the inflammatory 
process. They also inhibit phosphodiesterase involved in cell activation. Flavonoids 
can reverse the inflammatory effects induced by carrageenan and inhibit adhesion, 
aggregation, and secretion of platelets (Wang et al., 2018). 
 
Cytokines pro-allergic, IL 4 and IL 13, produced by Th2 and ILC2 at the early stage 
of the sensitization phase, promote the occurrence of isotype class switching of IgE 
in B cells and their transformation into plasma cells that secrete allergen-specific 
IgE in large quantities. This IgE then binds to high-affinity FcεRI receptors on the 
surface of mast cells and basophils, causing an allergic sensitization state. 
 
Polyphenols are suspected to affect the recruitment, maturation, and function of B 
cells; however, these effects have not been thoroughly investigated and explained. 
On the other hand, the capacity to inhibit antigen-specific IgE production in a dose- 
and time-dependent manner has been well documented in in vitro and in vivo 
studies for several polyphenols such as curcumin, rosmarinic acid, quercetin, ferulic 
acid, tea catechins (EGCG, ellagitannins, and gallic acid), and red wine 
polyphenols. 
 
Zhang et al. illustrated the modulatory effects of polyphenols with the example of 
dihydromyricetin, a natural flavonoid, which effectively suppresses the 
sensitization phase by reducing B cell populations and antigen-specific IgE 

 
production as well as blocking the interaction of IgE with FcεRI. Similarly, 
phlorotannins (i.e., eckol, dieckol) and tea catechins can interact with FcεRI by 
directly binding to the α chain, thereby blocking the possibility of specific antigen 
IgE binding to FcεRI and thus suppressing the mast cell sensitization phase. 
 
Furthermore, evidence has been provided that phlorotannins, saponins, catechins, 
as well as quercetin, kaempferol, and resveratrol may contribute to the weakening 
of allergic reactions by reducing the expression of FcεRI receptors, which are 
crucial for persistent sensitization and subsequent degranulation during the effector 
phase (Satitsuksanoa, 2021). 
 
Considering the fundamental role of allergic inflammation in the development and 
progression of respiratory allergic diseases, it can be assumed that polyphenols, due 
to their anti-inflammatory and immunomodulatory properties, may be beneficial in 
the prevention and treatment of asthma and allergic rhinitis. In addition to anti- 
allergic actions, in vitro studies indicate that polyphenols may also function as anti- 
mucus secretion agents and possess antioxidant and antifibrotic activities. 
 
so that it not only targets allergic inflammation, but also oxidative stress caused 
by inflammation and structural changes in the oral mucosa. The respiratory tract 
causes airway hyperreactivity and airway remodeling (Debinska, 2023). 
 
The Role of Apple Polyphenol Antioxidants in Reducing Signs and Symptoms of 
Allergic Rhinitis 
 
Quercetin has multiple biological activities. Quercetin plays a role in slowing the 
progression of diseases, such as: allergic diseases, malignancies, metabolic 
syndrome, and autoimmune diseases. Quercetin is capable of inhibiting 
inflammation by suppressing apoptosis and epithelial cell cytokines. Quercetin 
alleviates the symptoms of allergic rhinitis through the role of inhibiting 
neuropeptide production (Th1/Th2 cytokine imbalance) and eosinophil activation. 
Significant symptoms of allergic rhinitis include sneezing and frequent nose 
rubbing. Quercetin is known to reduce the frequency of sneezing and nose rubbing, 
thus improving RA symptoms (Xia et al., 2023). 
 
Allergic rhinitis is an inflammatory nasal disease due to allergens mediated by IgE, 
driven by Th2 cells. IgG1 antibodies, IL-4, and IL-5 serve as markers for Th2 cells, 
while IgG2 antibodies and Interferon γ serve as markers for Th1 cells. Histamine 
promotes cytokine release in Th2 cells and decreases Th1 cell production. Quercetin 
can inhibit the increase of IgE, IgG1, histamine, IL-4, and IL-5 and the decrease of 
IgG2a and IFN γ, indicating that quercetin prevents Th1/Th2 imbalance. Quercetin 
is also capable of inhibiting the increase of inflammatory cells, such as: eosinophils, 
neutrophils, lymphocytes, and macrophages (Xia et al., 2023). 
 
The imbalance of Treg/Th17 cells has been shown to be closely related to 
inflammation. Foxp3, IL-10, and TGF β are important regulators for Treg cells, 

 
while IL-17 is a regulator for Th17 cells. IL-6 and TNF α are pro-inflammatory 
factors that are also regulators of Treg/Th17 balance. These findings indicate that 
quercetin enhances the balance of Treg/Th17 cells (Xia et al., 2023). 
 
NF κB Pathway 
 
The NF κB pathway is a classic pro-inflammatory signaling pathway that increases 
the expression of pro-inflammatory cytokines and chemokines. The entry and exit 
of nuclear NF κB regulate the inflammatory response. When exposed to drugs, the 
nuclear translocation of p65 undergoes significant changes. Cyclooxygenase 2 is a 
pro-inflammatory chemokine that can be transcriptionally regulated by the NF κB 
pathway. Previous research has reported that the NF κB pathway significantly 
participates in the development of RA, particularly the effects of drugs on the 
Treg/Th17 or Th1/Th2 balance. In this regard, it was found that quercetin reduces 
the regulation of COX 2, p IKBα, nuclear p65, and increases the levels of IKBα and 
cytoplasmic 65, indicating that quercetin inactivates the NF κB pathway (Xia et al., 
2023). 
 
In summary, oral administration of quercetin can alleviate RA symptoms by 
inhibiting inflammation, Th1/Th2 imbalance, Treg/Th17 imbalance, and the NF κB 
pathway. Therefore, quercetin can weaken the inflammatory response of RA by 
enhancing the Th1/Th2 and Treg/Th17 imbalances and inactivating the NF κB 
pathway (Xia et al., 2023). 
 
a. Side Effects 
 
High doses of quercetin consumed together with certain types of drugs can affect 
the pharmacokinetics of those drugs. Quercetin has a weak inhibitory effect on CYP 
enzymes. Quercetin, when consumed in large amounts, will affect the transport of 
drugs mediated by organic anion transporting polypeptide (OATP), for example, 
statin drugs (Mohos et al., 2020). 
 
b. Weaknesses 
 
Quercetin has not been widely developed in the treatment of RA because quercetin 
has poor bioavailability, poor water solubility, and is unstable when passing through 
the gastrointestinal tract and when facing first-pass drug metabolism effects (Rajesh 
& Dhanaraj, 2023). If obtained from natural sources, a specific dose must be 
achieved for quercetin to provide benefits. 
 
Formulation of Quercetin 
 
The formulation of quercetin in apple polyphenols can be found in powder form, 
and functions as an antioxidant, anti-inflammatory, prevents platelet aggregation, 
and enhances relaxation of cardiovascular smooth muscle. 

 
Complementary Therapy 
 
Complementary therapy is an additional therapy using certain modalities that are 
not included in conventional medicine (Ibnu et al., 2019). The goal of this 
complementary therapy is to improve the degree of health. Complementary therapy 
includes promotive, preventive, curative, and rehabilitative programs while 
considering quality, safety, and effectiveness, and is based on biomedical science. 
Complementary therapy serves as an additional therapy outside of the main therapy 
and functions to improve quality of life and enhance the immune system (Ismail et 
al., 2018). 
 
Apple polyphenols are extracted and purified from raw apples and mainly consist 
of procyanidins, and the concentrated procyanidin fraction that has been purified 
has been confirmed to have higher activity in tests using rat cells. Mast cell and 
basophil stabilization, immune tolerance control has been suggested as a possible 
mechanism underlying the action of polyphenols (Sonji et al., 2006). 
 
Apple polyphenols consist of 50% to 60% procyanidins, which are oligomers and 
polymers of catechins, along with low molecular weight polyphenols such as 
chlorogenic acid and catechins. However, macromolecular procyanidins are not 
absorbed and are largely considered to be excreted. The percentage of T cells in 
intestinal epithelial cells of food allergy index has been shown to decrease in rat 
models induced by continuous oral consumption of ovalbumin, but this decrease is 
prevented by the administration of procyanidin fractions derived from apples. In 
contrast, the decrease is not inhibited when catechins alone are administered. These 
results indicate that the procyanidin fraction derived from apples can induce oral 
immunotolerance (Akiyama et al., 2005). 
 
Malondialdehyde 
 
Malondialdehyde is a small and reactive organic molecule widely distributed within 
human cells, formed by three carbon molecules with two aldehyde groups at carbon 
positions 1 and 3. 
Malondialdehyde exists in various forms in aqueous solution due to its tautomeric 
chemical properties that depend on pH. At pH higher than pKa 4.46, the dominant 
form is the enolate anion, which shows low chemical reactivity, whereas at lower 
pH occurring under oxidative stress conditions, MDA appears in equilibrium 
between protonated enol (α β unsaturated carbonyl) aldehyde and dialdehyde form 
(Morales & Munné Boschb, 2019). 
 
Malondialdehyde is one of several products of lipid peroxide decomposition formed 
in fats and oils, food, and tissues. This substance is the most widely investigated 
product due to its reactivity with various biological macromolecules and its 
relationship with the pathophysiology of several disease conditions. 

 
Malondialdehyde is enzymatically formed as a product of the cyclooxygenase 
reaction in the synthesis of prostaglandins and thromboxanes. Undoubtedly, there 
are a number of free MDA in food, as indicated by its accumulation as a volatile 
product during cooking, but the amount present is difficult to determine due to its 
artifact formation (Wang et al., 2022). 
 
MDA derivatives excreted in urine are interesting to know as potential indicators 
of the form of MDA in food and its reaction properties in vivo. The cross-linked 
glycine adenin related to MDA is obtained by reacting siximal 9 ethyladenine with 
methyl ester glycine, which indicates that cross-linking of nucleic acid amino acids 
mediated by MDA can occur in vivo (Draper et al., 2019). 
 
Stimulation by bacterial and viral antigens can cause PMN cells to produce and 
release ROS in large amounts, leading to oxidative stress. Proteins can become 
targets of ROS attack, such as those arising from lipid peroxidation and/or 
degradation products. Malondialdehyde or 4-hydroxynonenal derived from lipid 
peroxidation can form stable cross-linking products with certain amino acids. 
Malondialdehyde can be formed during the peroxidation of polyunsaturated fatty 
acids through the action of thromboxane synthase in human platelets on 
prostaglandin PGH2, PGH3, and PGG2, as well as through the action of polyamine 
oxidase and amine oxidase on spermine (Jové et al., 2020). 
 
Malondialdehyde is metabolized in the liver into semialdehyde malonic acid, 
which is an unstable molecule and spontaneously decomposes into acetaldehyde, 
which is then converted into acetate by aldehyde dehydrogenase and finally into 
carbon dioxide and water. After this, the next steps include conformational changes, 
enzymatic activity, or binding and inactivation of receptors, increased susceptibility 
to proteases, and changes in immunogenicity (Cherian et al., 2019). Reactive 
oxygen species have a very short lifespan, making them difficult to detect; however, 
tissue damage related to ROS can be observed by the end products of lipid 
peroxidation such as MDA. The level of tissue damage can be assessed by 
measuring the concentration of lipid peroxidation products and the levels of 
endogenous antioxidants in the body. Increased lipid peroxidation can burden the 
antioxidant defense system and trigger cell apoptosis and pathological processes 
that lead to increased serum MDA levels, reflecting increased free radical 
production. Increased MDA and other biomarkers of oxidative stress contribute to 
increased morbidity and mortality. 
 
Cytokines Involved in Allergic Rhinitis 
 
Cytokines are peptides produced in response to microbial stimuli and other antigens 
and act as mediators in immune and inflammatory reactions. Cytokine secretion 
occurs quickly and only briefly. Their action is often pleiotropic, meaning one 
cytokine acts on various types of cells and produces various effects. Cytokines often 
influence the synthesis and effects of other cytokines (Subowo, 2009). 

 
Cytokine products related to inflammation include TNF α, IL 1, IL 6, and IL 8. 
These pro-inflammatory cytokines are released by activated tissue macrophages 
(Baratawijaya, 2006). 
 
Cytokine Products 
 
In RAFL, there is infiltration of a number of inflammatory cells such as eosinophils 
due to ICAM 1 (Intercellular adhesion molecule 1) molecules. Subsequently, a 
number of inflammatory cytokines such as interleukin (IL) IL 4, IL 6, TNF α, and 
others are produced. The pathway for cytokine and inflammatory molecule 
formation is through cytokine gene transcription by NFkB (Nuclear factor kappa B) 
in the cell nucleus. Activators of NFkB include oxidant free radicals such as ROS 
(Reactive oxygen species) like air pollution. Zhao et al (2008) found an increase in 
NFkB expression and mRNA ICAM 1 in the nasal mucosa of patients with allergic 
rhinitis. 
 
Tumor Necrosis Factor Alpha 
 
Tumor Necrosis Factor Alpha is the first pro-inflammatory cytokine identified in 
allergic rhinitis. The concentration of TNF α is related to age; older children have 
higher levels and concentrations of TNF α. Children who experience recurrent 
effusion otitis media caused by allergic rhinitis also have higher levels of TNF α 
than children who are undergoing tympanostomy for the first time. 
 
Interleukin 6 
 
Interleukin 6 is one of the most important pro-inflammatory cytokines in the body's 
immune response. The IL 6 cytokine is a four-helix protein that binds to the 
membrane-bound IL 6 receptor or IL 6 receptor (IL 6R), and the IL 6 and IL 6R 
complex is the second receptor protein that joins. The 130 kDa glycoprotein 
(gp130) transduces intracellular signals through Janus kinase (JAK)/signal 
transducer and activator of transcription (STAT). Interleukin 6 shows measurable 
affinity only for IL 6R, but not for gp130, and IL 6R itself does not bind to gp130. 
Only the IL 6 and IL 6R complex binds gp130 and induces its dimerization. All 
cells in the body express gp130, but only a few cells, such as liver cells and some 
white blood cells, express IL 6R. Therefore, cells that only express gp130 and not 
IL 6R are not stimulated by IL 6 (Rose John, 2020). 
 
Interleukin 8 
 
Interleukin 8 (IL 8) is a member of the chemokine family that is related to the CXC 
motif. It is associated with two chemokine receptors, CXC 1 (CXCR1) and 2 
(CXCR2), which are included in the superfamily of G protein-coupled receptors 
with high affinity. CXCR1 is also associated with granulocyte chemotactic protein 
2 (GCP 2), neutrophil-activating peptide 2 (NAP2), and epithelial neutrophil- 
activating peptide 78 (ENA 78) with low affinity. Many types of cells, such as 

 
fibroblasts, keratinocytes, endothelial cells, neutrophils, mast cells, monocytes, 
macrophages, and megakaryocytes, produce IL 8 in vitro, which performs various 
biological activities. 
 
IL 8 plays an important role in acute inflammation because it acts as a potential 
chemotactic agent and neutrophil activator by activating CXCR1 and CXCR2 
(Emadi et al., 2005). The reduced IL 8 response induced by LPS in neutrophils has 
been confirmed in human whole blood translational and non-animal models. This 
was achieved by exposing neutrophils in vitro to a pro-inflammatory 
microenvironment containing N-formyl Met Leu Phe, LPS, or IL 8, resulting in a 
response accompanied by an increase in cell size and pH that leads to a decrease. 
The detailed kinetics of this degradation can be shown almost in real time by 
measuring flow cytometry. Overall, we provide a new mechanistic overview of the 
interaction between IL 8 and neutrophils and explain how IL 8 changes during 
systemic inflammation. 
 
Meanwhile, MKK6 phosphorylates another MAPK, p38, which leads to the 
phosphorylation of MK 2 and stabilizes IL 8 mRNA. The activation of the C/EBP 
signaling pathway is largely still unknown (Matsushima et al., 2022). 
CHAPTER III 
RESEARCH METHOD 
A. Type of ResearchThe type of research used is Clinical Experimental, designed 
with a Randomized Controlled Trial double blind, with pre and post test design on 
samples of patients with persistent allergic rhinitis. 
B. Place and Time of ResearchThe research is conducted at the Outpatient Clinic 
of Ear, Nose, Throat Health Science Surgery at the Sebelas Maret University 
Hospital and the biomedical laboratory of the Faculty of Medicine UNS for the 
examination of MDA, NF kB, TNF α, Interleukin 6, Interleukin 8. The research will 
be carried out after obtaining research approval from the dissertation proposal 
examination board and the Research Ethics Commission. 
 
C. Subjects/Sample of ResearchOutpatient patients who have undergone anamnesis 
and physical examination of ENT. 
 
1. Restriction CriteriaInclusion Criteria: 
 
1) Patients aged 18 – 60 years who have signed informed consent 
 
2) Diagnosed with persistent allergic rhinitis with Skin Prick Test or 
Positive IgE Examination. 

 
Exclusion Criteria: 
 
1) The presence of complications in the nose such as nasal septum deviation, 
polyps, rhinosinusitis 
 
2) Have undergone specific immunotherapy for 2 years 
 
3) Use of medications that affect allergy test results: antihistamines, corticosteroids, 
antihypertensive medications, and decongestants 
 
4) Pregnancy and Breastfeeding. 
 
Sample Size 
 
The sample size uses the Frederer formula. 
Frederer Formula = (n - 1) (t - 1) ≥ 15 
Explanation: n: sample size of each groupt: number of groups 
According to the Frederer formula, the number of samples required: 
(n - 1) (t - 1) ≥ 15(n - 1) (3 - 1) ≥ 15(n - 1) 2 ≥ 152n = 17, n = 8.5 
The number of samples used must be greater than or equal to 9 samples per group. 
In this study, 10 test samples will be used in each group. This is done to facilitate 
the author in data analysis calculations, so the total number of samples used in this 
study is 40 samples. 
 
Sampling Technique 
 
Randomized Control Trial pre and post-test design. Eligible participants/patients 
who meet the inclusion and exclusion criteria will be randomized. Participants will 
be grouped into four groups, namely the control group and the treatment group, and 
then the outcomes will be observed. 
 
F. Implementation/Research Procedure 
 
1. Recruitment of research subjects and randomizationAll patients with 
persistent allergic rhinitis, Randomization with closed envelopes, Therapy 
according to the persistent allergic rhinitis algorithm 
2. Source of apple fruit extractApple fruit extract is made in the Pharmacy 
Laboratory of Muhammadiyah University of Surakarta. 

 
3. Preparation of apple fruit extract Polyphenol extract is derived from the fruit 
of Mallus sylvestris mill obtained from apple farmers in Malang. Dried 
apples are extracted, and re-extracted twice with 50% ethanol. The obtained 
material is concentrated using a rotary evaporator (Rotavapor, Buchi 
Labortechnik AG, Switzerland) in a water bath at 40 °C. Furthermore, the 
extract is adsorbed using Amberlite XAD 16 resin (Brentag, Essen, 
Germany), and after the evaporation of ethanol, the collected fraction is 
dried in a vacuum oven SPT 200 (Zeamil, Krakow, Poland) (Mihailović et 
al., 2018). 
4. Measurement of Malondialdehyde (MDA)Measurement of MDA using the 
Enzyme Linked techniqueItchy noseClinical symptoms in allergic 
rhinitis.TNSS 
Checked 
on 
the 
First 
day 
and 
the 
14th 
dayOrdinalRhinorrheaClinical symptoms in allergic rhinitis.TNSS Checked 
on the First day and the 14th dayOrdinal. 
 
G.  Statistical Analysis 
 
Statistical analysis was conducted using IBM SPSS Statistics for Windows, version 
25.0 (IBM Corp., Armonk, NY, USA). Data normality was evaluated using the 
Shapiro–Wilk test. For normally distributed and homogeneous data, one-way 
analysis of variance (ANOVA) was applied. When the data were not normally 
distributed but homogeneous, the Kruskal–Wallis test was used. Comparisons across 
three time points were performed using repeated-measures ANOVA, with the 
Friedman test employed as the nonparametric alternative. 
 
 
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