An-Najah National University 

Faculty of Graduate Studies 

 
THE ANTIBACTERIAL EFFECT OF 

DIFFERENT PALESTINIAN OAK SPECIES 

ON MRSA 

 

 

By 

Renada Shafik Razmaq 

 

 

Supervisors 

Dr. Walid Basha 

Prof. Raed Alkowni 

 

 

This Thesis is Submitted in Partial Fulfillment of the Requirements for the Degree of 

Master of Biology, Faculty of Graduate Studies, An-Najah National University, Nablus - 

Palestine. 

2022 





III 

 

Dedication 

For those who made this journey worth walking, 

I dedicate this thesis to them. 
  



IV 

 

Acknowledgements 

As I give thanks to God, I would like to express my sincere appreciation to my 

supervisors, Dr. Walid Basha and Dr. Raed Alkowni, for the guidance and encouragement 

they provided me during the work on this thesis. All thanks to Abd-Alrazaq Zarour 

(Research Assistant) in the microbiology research lab, Ahmed Mousa, the supervisor of 

the central research laboratory in the faculty of medicine, Mohammed Nabeel (Lab 

technician) in the animal lab, and my dearest friend Hidaya Qash for their valuable 

assistance. 

All the love to those who light up my path and believe in me like no other, my husband 

and my precious kids, Khaled and Talia. 

There are not enough words to express my gratitude to my idols, Mom and Dad, my 

supporting teammates, my sisters, and brothers. 

All thanks and gratitude to my second family, who is always proud of me and supports 

me in every step I take. 

  





VI 

 

 List of Contents  

Dedication ................................................................................................................................... III 

Acknowledgements ..................................................................................................................... IV 

Declaration ................................................................................................................................. V 

List of Contents ........................................................................................................................... VI 

List of Tables .............................................................................................................................. IX 

List of Figures .............................................................................................................................. X 

List of Appendices ...................................................................................................................... XI 

Abstract ...................................................................................................................................... XII 

Chapter One: Introduction ............................................................................................................. 1 

1.1 Background ............................................................................................................................. 1 

1.2 Palestinian species ................................................................................................................... 3 

1.2.2   Quercus cerris .......................................................................................................... 5 

1.2.3 Quercus look ............................................................................................................ 5 

1.2.4 Quercus ithaburensis ................................................................................................. 6 

1.2.5 Quercus calliprinos ................................................................................................... 6 

1.2.6 Quercus coccifera ..................................................................................................... 7 

1.3 Phytochemicals and bioactive compounds of Quercus ........................................................... 9 

1.4 The effect of phytochemical compounds of Quercus on different      microorganisms ........ 11 

1.5 Traditional uses of Quercus .................................................................................................. 12 

1.5.1 Other uses of Quercus .............................................................................................. 13 

1.6 Biological activity studies of Quercus extracts ..................................................................... 13 

1.6.1 Anti-microbial effect ................................................................................................ 13 

1.6.2 Effect on wound healing ........................................................................................... 17 

1.6.3 Antioxidant effect .................................................................................................... 17 

1.6.4 Neuroprotective effect .............................................................................................. 18 

1.6.5 Anti-hepatotoxic effect ............................................................................................. 18 

1.6.6 Problems related to the gastrointestinal system ........................................................... 18 

1.6.7 Anti-viral effect ....................................................................................................... 19 

1.6.8  Tyrosinase inhibitory effect ..................................................................................... 20 

1.6.9  Anti-hyperlipidemic effect ....................................................................................... 21 

1.6.10  Anti-diabetic effect ............................................................................................... 21 

1.6.11 Anti-inflammatory effect ........................................................................................ 22 

1.6.12  Anti-alergic effect ................................................................................................. 23 

1.6.13  Vasorelaxant effect ............................................................................................... 23 



VII 

 

1.6.14 Hemostatic effect ................................................................................................... 23 

1.7  Emergence of MRSA ........................................................................................................... 24 

1.7.1 Evolution and genetic diversity ................................................................................. 24 

1.7.2 Community acquired MRSA (CA-MRSA) ................................................................. 26 

1.7.3 Hospital acquired MRSA (HA-MRSA) ...................................................................... 26 

1.8 Resistance to antibiotics on an intrinsic basis ....................................................................... 27 

1.8.1 The efflux system .................................................................................................... 27 

1.8.2  Permeability of the outer membrane ......................................................................... 28 

1.8.3  Overproduction of β-Lactamase enzyme ................................................................... 28 

1.9  Acquired antibiotic resistance .............................................................................................. 28 

1.9.1  Biofilm-mediated resistance .................................................................................... 28 

1.9.2  Mutations resistance ............................................................................................... 29 

1.9.3  Acquisition of resistance genes ................................................................................ 29 

1.9.4  The role of persister cells in antibiotic resistance ....................................................... 29 

1.10  Virulence Factors of MRSA ............................................................................................... 30 

1.10.1  Surface associated proteins .................................................................................... 30 

1.10.1.1  Staphylococcal protein-A .................................................................................... 30 

1.10.1.2  Clumping factors .......................................................................................................... 30 

1.10.1.3 Capsular polysaccharides .............................................................................................. 30 

1.10.2 Extracellular Toxins ............................................................................................... 31 

1.10.2.1 Staphylococcal hemolysins ........................................................................................... 31 

1.10.2.2  Staphylococcal enterotoxins ......................................................................................... 31 

1.10.2.3  Panton-Valentine Leukocidin ....................................................................................... 31 

1.10.2.4  Staphylococcal exfoliative toxins (ETs) ...................................................................... 31 

1.10.2.5  Toxic Shock Syndrome Toxin ..................................................................................... 32 

1.11  In vivo studies using a mouse model ................................................................................. 32 

1.12 Statement of the problem and rationale of the study ........................................................... 33 

1.13 Aim of the study .................................................................................................................. 33 

Chapter Two: Experimental Part .................................................................................................. 34 

2.1 Sample collection .................................................................................................................. 34 

2.2 Preparation of plants samples for extraction ......................................................................... 34 

2.3 Extraction method ................................................................................................................. 35 

2.4 In vitro antibacterial activity testing ..................................................................................... 35 

2.4.1 Preparation of growth media ..................................................................................... 35 

2.4.2  Preparation of bacterial suspension ........................................................................... 36 

2.4.3 Agar well diffusion method ...................................................................................... 36 



VIII 

 

2.4.4 Determination of the minimum inhibitory concentration (MIC) .................................... 36 

2.4.4.1 Preparation of extract samples ........................................................................................ 36 

2.4.4.2  Serial dilution of extracts ............................................................................................... 37 

2.4.5  Determination of minimal bactericidal concentration (MBC) ...................................... 38 

2.5 In Vivo antimicrobial activity testing using mice ................................................................. 38 

2.5.1 Preparing MRSA for experimental infection............................................................... 38 

2.5.2 Infecting laboratory mice ......................................................................................... 38 

2.5.3 Bacterial counts in infected mice tissue ...................................................................... 39 

2.6  Statistical analysis ................................................................................................................ 40 

Chapter Three: Results ................................................................................................................ 41 

3.1 In vitro antibacterial activity testing ..................................................................................... 41 

3.2  Determination of MIC .......................................................................................................... 41 

3.3  Determination of MBC ........................................................................................................ 44 

3.4  In vivo antibacterial activity in mice .................................................................................... 44 

Chapter Four: Discussion and Conclusions ................................................................................ 45 

4.1  Extraction and in vitro antibacterial activity testing ............................................................ 45 

4.2 In vivo antibacterial activity .................................................................................................. 47 

4.3 Limitations ............................................................................................................................ 47 

4.4  Conclusions .......................................................................................................................... 48 

4.5  Recommendations ................................................................................................................ 48 

List of Abbreviations .................................................................................................................. 49 

References ................................................................................................................................... 50 

Appendices .................................................................................................................................. 62 

 ب ........................................................................................................................................... الملخص 

 

 

 

 

 

 



IX 

 

List of Tables 

Table 2.1: Parts collected from each specie and the place they were collected from ................. 34 

Table 2.2: Amount of water lost in leaves of each Quercus specie............................................. 35 

Table 2.3: Test groups and extract dilution concentrations ........................................................ 39 

Table 3.1: Mean and standard deviation (M ± SD) MICs of three Quercus species  extracts tested 

against MRSA. Mean of 3 experiments .................................................................... 43 

Table 3.2: Means of bacterial counts from every group of infected mice tissue......................... 44 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



X 

 

List of Figures     

Figure 1.1: Field photos of Quercus .............................................................................................. 8 

Figure 1.2: Stilben derivative from Quercus species (39) ........................................................... 10 

Figure 1.3: Structures of tannins purified from Q. coccifera  (40). ............................................. 10 

Figure 1.4: Chemical structure of two polyphenolic compounds purified from Q. coccifera (20)

 ................................................................................................................................ 11 

Figure 1.5: Major genomic elements in methicillin-resistant Staphylococcus aureus (70) ........ 25 

Figure 3.1: Spectrophotometer readings and MIC points of different alcohol Quercus extracts. 42 

Figure 3.2: Spectrophotometer readings and MIC points of different aqueous Quercus extracts

 ................................................................................................................................ 42 

Figure 3.3: Minimum inhibitory concentrations (mg/ml) of three different Quercus extracts 

against MRSA ........................................................................................................ 43 

Figure 1.5 A: The role of active efflux systems in MRSA resistance (77) ................................. 62 

Figure 1.5 B: The scheme of decreased outmembrane permeability caused drug resistance of S. 

aureus (77) .............................................................................................................. 62 

Figure 1.5 C: The role of cellular enzymes in drug resistance of S. aureus (77) ........................ 63 

 
 

                          

 

 

 

 

 

 

 

 

 

 

 

 

 



XI 

 

List of Appendices 

Appendix A: Figures ................................................................................................................... 62 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



XII 

 

THE ANTIBACTERIAL EFFECT OF DIFFERENT PALESTINIAN 

OAK SPECIES ON MRSA 

By 

Renada Shafik Razmaq 

Supervisor 

Dr. Walid Basha 

Prof. Raed Al-Kowni 

 

Abstract 

Introduction:  The most common cause of nosocomial pneumonia is staphylococcus 

aureus, with substantial mortality and morbidity. The growing bacterial resistance and 

side effects of antimicrobial medications have led to significant attention being paid to 

traditional medicinal plants in the past few decades. Compounds, derived  from these 

plants,  can be used to treat microbial infections as an alternative treatment. 

Objective: The main aim of this study was to determine the potential antimicrobial effects 

of ethanol and aqueous extracts of  three different species of Quercus against MRSA 

isolates. 

Methods: Antibacterial activity of plant extracts was observed, using the agar well 

diffusion test.After observation, the researcher determined  the Minimum Inhibitory 

Concentration (MIC) by 2-fold dilution of plant extracts yielding a serial dilution of the 

original extract. To check if the desired compound entered the site of infection in vivo 

and reached  high levels to alter bacterial viability, mice were used as animal models of 

infection. 

Results: Ten extractions (water and ethanol) were prepared from the three oak species. 

However,  only seven showed MICs ranging between 1.56-12.5 mg/ml. In vivo testing 

showed an indication of bacterial inhibition ability of Q.coccifera extract (0.22 mg/ml) 

when added to the drinking water of a mice group. The bacterial count from a cultured 

swab from this group of infected mice was 160 CFU, the lowest count of all extracts. 

Conclusions: Ethanol extracts of oak have more efficient antibacterial activity than water 

extracts. Water extracts, when mixed with the drinking water of mice,  have a good 

antibacterial inhibitory ability. Future studies should be conducted to extract oak's active 



XIII 

 

ingredients using other solvents. Also, combining these extracts with other antibiotics 

might enhance their effect in vivo. 

Keywords: MRSA; Quercus;Palestine;  antibiotic resistance; plant extract;  MIC;animal 

model.



1 

 

Chapter One 

Introduction 

1.1 Background 

Palestine is one of the rich ecosystems as part of the Eastern Mediterranean region (1). 

Although it is a small country, and due to its varied geography and environment, it has a 

great variety of wild plants. More than 2600 plant species are covered in the hills and 

mountains of Palestine, of which more than 700 are noted for their use as medicinal herbs 

or botanical pesticides (2,3). 

Palestinian Quercus is one of the typical evergreen trees belonging to the Fagaceae 

family, which is considered an ecologically important woody plant family, as Quercus 

form the backbone of temperate forests in the Middle East (4).  

Quercus's various species have been preserved due to its historical and heritage 

significance in Palestine, where vast sizes of the Quercus trees are found near holy sites 

and worship places. Also, Quercus has been consumed as food but very limited; the 

acorns were roasted and eaten in times of great necessity (5). 

Like in many developing countries, herbal medicine is an integral part of Palestinian 

society and plays an essential role in today's public healthcare system (6). Many diseases 

in Palestine can be treated with herbal medicine, including intestinal diseases, urinary 

tract infections, infertility, and cutaneous abscesses. In addition, local Palestinian 

Quercus has been used to treat arthritis pain, ulcer, severe abdomen ache, bloody 

coughing, diabetes, and prostates issues. Besides, it helps treat urine problems, stomach 

cancer, and liver atrophy. It also helps lower blood pressure, giving energy to the body, 

purifying the blood, diarrhea, and breath irregularities (5). 

The occurrence of microbial infectious diseases is continuously growing, primarily 

because microbial drug resistance to antimicrobial agents is currently accessible (7). 

Furthermore, these microbes have developed resistance in three or more antimicrobial 

groups to at least one agent (8) therefore named Multidrug-resistant bacteria (MDRB). 

 



2 

 

The resistance of pathogenic microorganisms to antibiotics has led to extensive research 

on extracts and biologically active compounds isolated from plant species. As a result, 

there is a widespread use of medicinal plants for their antiviral, antibacterial, and 

antifungal properties in many parts of the world (9).   

In various parts of the world, Staphylococcus aureus is considered a major human 

pathogen in both community and health-related settings (10). It has a set of virulence 

factors and the capacity to develop resistance to most antibiotics. This ability is boosted 

by the continuous appearance of new clones, resulting in the emergence of methicillin-

resistant S. aureus as a "superbug," also, unnecessary clinical use of methicillin has led to 

the appearance of methicillin-resistant S. aureus (MRSA) (11). 

Plant-derived antimicrobials are promising sources of safe antimicrobials when 

contrasted with synthetic compounds because of their natural origin (12), and their 

phytochemicals may lead to new aspects of drug discovery (7). So There is an increasing 

interest nowadays in this alternative therapy for microbial infections (12). 

Many studies around the world were conducted on traditional medicinal plant extracts to 

prove their antibacterial activity against MRSA. For instance, a study conducted by Zuo 

et al. in 2008 in china on 19 extracts from Chinese herbs and all the presented plants 

showed promising anti-MRSA activity with MIC of 1.25–3.07 mg/ml (13). 

Another study by Aqil et al. in 2006 in India detected four medicinal plants with MICs 

ranged between (0.32 - 3.25) mg/ml, and phytochemical studies showed flavonoids and 

phenols to be the key active constituents in these plants (14). 

Mickymaray also mentioned in his study, traditional medicinal plants and their bioactive 

compounds against clinically important pathogens, that the Antimicrobial screening 

performed on various medicinal plants, including Quercus infectoria Olivier, showed 

MIC equal to 100–200 μg/mL against MRSA. Therefore, he concluded that the effects of 

conventional antimicrobials could be improved by crude extracts and plant-derived 

bioactive compounds. This can be cost-effective, have fewer side effects, and increase 

the quality of therapy (12). 

In addition to the above, a research described the tree Q. infectoria Olivier as a small tree 

that grows naturally in Greece, Asia Minor, and Iran. A gall-wasp, Adleria gallae-



3 

 

tinctoria, attacks the young branches of this tree to form galls. Women use the galls as a 

drinking remedy after childbirth in combination with other herbs to restore uterine 

elasticity. In addition, it is widely used as a mouth washing powder for toothache and 

gingivitis treatment in Indian traditional medicine. Q. infectoria galls are also used 

medicinally as astringents, antidiabetics, antitremorines, local anesthetics, antivirals, 

antibacterials, antifungals, larvicidal, and anti-inflammatory agents. The primary 

ingredients of Q. infectoria galls are tannin (50-70 %) and a small portion of free gallic 

acid and ellagic acid (15). 

The Quercus wood also has been found to have anti-bacterial properties against many 

bacteria like nosocomial Acinetobacter Baumannii (16), Escherichia coli (17), and many 

others. 

Additional study on Quercus fruits showed that the total phenolic content of Q. brantii 

fruit extract was 3.010 mg GAE/g DW. Furthermore, flavonols and flavonoids were 1.813 

mg/g and 0.654 mg/g, respectively. S. aureus and E. faecalis were significantly inhibited 

by the extract. In addition to its antioxidant properties, Q. brantii fruit extract also can act 

as a dietary supplement (18). 

1.2 Palestinian species 

Palestine is known for its rich plant variety represented by trees, shrubs, and herbs. For 

example, oak is considered a common evergreen tree that inhabits heights and plains of 

different places in Palestine, and sometimes it is found as dense forests that give shelter 

to a large number of beautiful plants and shade-demanding ferns, providing a habitat for 

a great diversity of different plants and animals (19). 

The genus Quercus (oak), which belongs to the Fagaceae family, consists of over 600 

species worldwide of evergreen trees and shrubs that might be monecious or deciduous 

(20).  

  There are many natural hybrid species in the genus Quercus, which makes taxonomy 

confusing. Three groups of oak trees were classified as subgenera: white oaks 

(Leucobalanus), red and black oaks (Erythrobalanus), and (Cyclobalanus). 

In Quercus species, leaves are alternately simple, deciduous, or evergreen, with lobed, 

toothed, or entire margins. The male flowers appear with or following the leaves as 



4 

 

pendant yellow catkins. On the same tree, female flowers bloom in one to more than two 

spikes; each flower has a husk of overlapping scales that expands to hold the fruit, called 

an acorn. There are sometimes glandular margins on the margins of leaves on white oaks, 

though they are not bristle-tipped. The seeds in their acorns have a sweet flavor and 

germinate within a few days after they fall. Their acorns mature in one season. During 

the second growing season, red and black oaks mature their bitter fruits, which are tipped 

with hairs and have hairy linings on the acorn shells. 

 From acorns, oaks are easily propagated and thrive in moderately moist rich soil or dry 

sandy soil. Many trees grow from stump sprouts again. The tree is hardy and long-lived 

but is not shade-tolerant and susceptible to oak wilt and leaf-eating organisms (21). 

Large areas of Palestinian forests are naturally dominated by six known species of 

Quercus, which are Q. boissieri, Q. cerris, Q. look, Q. ithaburensis, Q. calliprinos, and 

Q. coccifera (22). Only three species of them were used in this study; Q. ithaburensis, Q. 

calliprinos, and Q. coccifera. 

1.2.1 Quercus boissieri 

Quercus boissieri Reut. is the original name of this species, but later it was known as 

Quercus boissieri var. latifolia, but the current accepted name is Q. infectoria subsp. 

veneris. It is also known commonly as gall oak, dyer’s oak, and Aleppo oak (23). 

A Boissier oak is an evergreen deciduous tree that grows to a height of 10 m or more. A 

variety of alternate leaf arrangements are present. There are short petioles that carry the 

leaves. They have triangular points on their margins, rounded at the ends. A glabrous 

surface covers the upper surface of the leaves. Leaves are covered with hairs on their 

lower sides as they grow, but become glabrous as they mature. Various species of this 

species have hairs that fall off when touched by the fingers, which makes them easy to 

recognize. 

March-April are the peak months for the blooming of Quercus boissieri. Flowering 

occurs only once per year, the trees are monoecious, and male flowers are limp catkins. 

A small pair or single flower is arranged on a short pedicle by the female flowers. It 

produces elongated, cylindrical acorns that are 3-4 cm long, ovate, and usually found 

alone or in pairs. Compared to all other oak species in Palestine, the acorn is thinner and 



5 

 

longer. In most cases, the cupule covers less than a quarter of an acorn's length. A cupule 

is characterized by short, obtuse, tightly attached scales.  

There are forests and Maquis in the Mediterranean mountain range that are home to 

Quercus boissieri, which grows at an elevation of at least 800 m. Limey and basalt soils 

are both suitable for its growth. Many types of insects infect the trees with galls. The galls 

were used to produce a red dye, which is the origin of its former scientific name. A Swiss 

botanist who studied Middle East flora in the 19th century is credited with naming this 

plant in Latin after Pierre Edmond Boissier (24). 

1.2.2 Quercus cerris 

A large deciduous tree growing to 40 m tall, Quercus cerris L., is commonly known as 

Turkey oak. It has a trunk size between 1.5 m and 2 m, and a well-developed root system. 

The lifespan of this plant is between 120 and 150 years. Bark fissures are reddish-brown 

or orange and deeply furrowed in mauve-grey. Aside from shuttering or fuel wood, the 

wood is inferior to other common oak species. There are 7-9 pairs of triangular lobes on 

each leaf, which is dark green above and gray-felted below; the leaves are typically 9-12 

cm long and 3-5 cm wide. Usually swirled with long twisted whiskers, the egg-shaped 

buds on the tips of the twigs are hairy and egg-shaped. Flowering occurs between April-

May, and the flowers are monoecious and wind-pollinated. The acorns are without a stalk, 

about 2 cm in diameter and 2 cm long. There are dense bristles covering the acorn cup. 

There is an abundance of acorns on Turkey oak trees, and the acorns germinate readily 

and can be propagated easily (25). 

1.2.3 Quercus look 

Among mountainous deciduous oaks in the Middle East, Quercus look represents the 

southernmost species. It is an evergreen tree, that grows 4–10 m high. Without cutting 

the trees down, they will only have one trunk, but many trees now have several erect 

trunks, indicating they have been cut down previously. Usually wavy, with elongated, 

glossy, rough toothed, 5–11 cm long leaves. In comparison to Q. libani, the leaves have 

triangular teeth that are either regular or irregular, bigger and fewer than in Q. libani. 

Acorns develop during the summer and ripen in September when they bloom in spring. 

It doesn't take too long for European jays (Garrulus glandarius) and rodents to disperse 

the acorns by October, or else they are all gone. There are large acorns, with erect cupules 



6 

 

on top, and spreading outward or bending downward on the rest. Nuts are unique, as they 

are dark brown and flat or even slightly concave at the tip, regardless of whether they are 

exserted or not (26). 

1.2.4 Quercus ithaburensis 

 Also called the Mount Tabor oak, it grows in a woody park woodland with herbaceous 

vegetation between the trees, part of a complex of trees and shrubs species adapted to a 

dry Mediterranean environment. As a thermophilous tree, growing mostly at altitudes 

below 500 m, and deciduous tree to shed its leaves during the winter months, it is found 

mainly in the eastern Mediterranean (Turkey, Syria, Lebanon, Palestine, and Jordan). 

Q. ithaburensis develops an upright trunk that can reach 5-6 m and forms open 

formations. A broadly ovoid or globular crown is formed as the branches divaricate 

upward. Usually, 8-4 cm is the size and shape of the leaves; the buds are ovoid in shape 

and have upraised scales. The leaves vary in size and shape from one tree to the next but 

are mainly leathery and toothed. There are usually sexes in different inflorescences on the 

same main twig, and they have green flowers or inflorescences that are similar in color. 

Within a perianth of 4-6 lanceolate lobes, it forms staminate catkins of 5 cm in length. 

Two years later, the acorns mature. They can be found alone or in pairs. A total of five 

subvarieties were classified by Zohary (1966), each characterized by a different style of 

cupules and glands: var. calliprinoides, var.subcalva, var. subinclusa, var. dolicholpeis 

and var.ithaburensis. 

Tabor tree was planted as a supplementary food source in the past because of its huge and 

sweet acorns, and the cupules were largely used for dye industries until the beginning of 

the 20th century (27–29). 

1.2.5 Quercus calliprinos  

Also called Palestine oak because it’s the most common tree in the wild flora of Palestine. 

It is known as part of the genus Cerris. Quercus calliprinos is a medium-sized evergreen 

tree reaching 5-18 m tall and 1 m trunk diameter. It grows slowly, at less than 25cm each 

year, and can live for up to 700 years. It is also tolerant to hard environments and 

regenerates quickly after fires (30). 



7 

 

Sharp spines surround the edges of the shiny, stiff, dark green leaves, which are 3–5 cm 

long, and 1.5–3 cm wide (31). Flowers are found as male and female on the same tree. 

They bloom from March to April and fall soon after. Green male flowers grow as a catkin, 

a long cylindrical cluster of small flowers without petals, and female flowers grow as a 

nut called an acorn, borne in a cup-like structure known as a cupule which is 3-4 inches 

long and 2-3 inches in diameter (32). 

1.2.6 Quercus coccifera  

Also called kermes oak, it stays green all year long as an evergreen shrub. Although this 

tree has the potential to grow to a height of just 2 m, depending on where it is grown and 

how it is cared for, it can reach heights of up to 4 or 5 m and may become a small tree in 

the process. 

Its morphology indicates an impenetrable "wall" with extensive ramifications from its 

base. There is a pattern of green leaves that alternate between those that fall faster and 

those that do not fall at the same time. They have a wavy form and are hairless on both 

sides, with a smooth surface on both sides. To distinguish them, notice how the male 

blooms are significantly smaller than the female blossoms. Females are born on the same 

plant as males and might be solitary or in groups of two or three. Flowering occurs in 

April or later, and fruiting occurs in August of the year following flowering. 

Depending on its properties, this shrub can be used for various purposes. Its bark contains 

a high concentration of tannins and can be used to dye certain wool black. Wood has 

minimal value, although it can be used as fuel and to make charcoal. 

Because of their bitter flavor, they were also used to feed cattle and goats. Finally, it is 

important to remember the amazing protection that it may provide to impoverished areas, 

which is why it is critical to avoid causing their degeneration by frequent fires or extensive 

grazing. 

Q. coccifera is also used medicinally. Because of its high tannin content, it can be 

extracted as a cure for diarrhea and urinary incontinence by decoction of the bark. 

External use of the decoction can help alleviate hemorrhoids and chilblains. It is also an 

effective tonic and has anti-inflammatory, antibacterial, and febrifuge qualities (33). 



8 

 

Its common name, kermes, indicates the importance of the species in the past as the food 

plant of the Kermes insect, whose crushed bodies were used for the production of the 

original natural crimson dye (34,35).  

Figure 1.1   

Field photos of Quercus  

 

 A   

   B    

    C     

Quercus coccifera (A), Quercus ithaburensis (B), and Quercus calliprinos (C) 

 



9 

 

1.3 Phytochemicals and bioactive compounds of Quercus 

In addition to primary molecules such as carbohydrates, proteins, and lipids, secondary 

metabolites play a major role in the pharmacologic action of medicinal plants. 

Antimicrobial properties in plants are commonly attributed to secondary metabolites that 

functions naturally as defense mechanisms against pests, pathogens, and predators (36). 

A significant number of these metabolites have been discovered to be useful for 

developing safer antibacterial and antifungal chemicals (12). 

Quercus species have special compounds that have been isolated by researchers to 

investigate their biological activities and to validate the usage of genus species as 

medicinal treatments. Extracts were shown to have a variety of pharmacological actions, 

including antioxidant, antibacterial, anti-inflammatory, and cytotoxicity activity (37). 

 Numerous Known polyphenolic compounds were isolated from Quercus, which have 

been effectively used in several clinical studies. According to Okuda et al. (38), 

polyphenolic compounds have high biological and pharmacological properties. There are 

several biological effects of these compounds, including antisecretory and 

antiulcerogenic activities. 

Many Quercus species have phenolic secondary metabolites such as lignans, simple 

phenols, tannins, flavonoids, and coumarins. Also contains flavonols such as kaempferol, 

quercetin, and isorhamnetin as aglycon (appendix, figure.1.2), all of which can be found 

in Q. cerris, for example (39). 

Quercus species are among the most important tannin-containing plants. Several species 

of Quercus have high tannin content, for instance Quercuus rubur is characterized by 

approximately 10 % in ethanol (w/w) hexahydroxydiphenoyl esters, most of which are 

castalagins and vescalagins. Recent studies have identified several polyphenolic 

compounds in Algerian Q. coccifera leaves, including casuarictin, pedunculagin, 

tellimagrandin I and tellimagrandin II, vescalagin, castalagin, phillyraeoidin E, 

quercitrin,mongolicain A, acutissimin B, (+)-catechin, quercetin, and kaempferol 3-O-

(6′′-Ogalloyl)-â-D-glucopyranoside (40). 

 Epicatechin, gallocatechin, and epigallocatechin, flavan-3-ol monomers, and their 

derivatives, also trans-resveratrol 3-O-b-glucopyranoside which is a stilbene derivative 

(figure 1.3) have all been reported in some Quercus species, including Q. coocifera (39). 



10 

 

Figure 1.2 

Stilben derivative from Quercus species (39) 
 

 

The bark and wood of different Quercus species, like Q. coocifera, contain some lignans, 

such as, 5-methoxyisolariciresinol, lyoniresinol,  5,5-dimethylsecoisolariciresinol, 

matairesinol , olivil and their glycosylated derivatives (39). 

Figure 1.3 

Structures of tannins purified from Q. coccifera  (40) 
 

 

 

 



11 

 

Taib et al. reported that flavonoids (particularly flavan-3-ol), phenolic acids (especially 

gallic and ellagic acids and their derivatives), Only Q. coccifera was mentioned to contain 

two polyphenolic compounds shown in (figure 1.4) (20).  

Figure 1.4 

Chemical structure of two polyphenolic compounds purified from Q. coccifera (20) 
 

 

                            Polydatin                                Lyoniresinol-9-O-β-xylopyranoside 

 

It has been reported that a wide variety of simple phenolic acids and phenols, including 

ellagitannins and gallo, along with their conjugates epicatechin and catechin, have been 

isolated in many Quercus species, such as Q. coocifera . This type of compound 

dominates oak woods along with flavan-3-ols (39).                                    

1.4 The effect of phytochemical compounds of Quercus on different microorganisms 

Microorganisms are alleged to be absolutely toxic to phenolic compounds, but increased 

hydroxylation reactions lead to the destruction of bacterial cells. The presence of highly 

active flavonoids like quercetin reduces the thickness of lipid bilayers and increases their 

fluidity levels, thus facilitating the leakage of intracellular proteins and ions by S. aureus. 

As a cell-cell signaling antagonist, quercetin inhibits enteroaggregative biofilm formation 

by E. coli and P. aeruginosa at a concentration-dependent rate. Furthermore, it inhibits 

3-hydroxyacyl-ACP dehydrase in Helicobacter pylori  and eriodictyol, FAS-II, enoyl-

ACP-reductase, β-ketoacyl-ACP reductase, and β-hydroxy acyl-ACP dehydratases in 

Mycobacterium sp., which are responsible for fatty acid synthesis in bacterial cells. 

Moreover, quercetin inhibits the synthesis of peptidoglycan, another component of the 

bacterial cell wall. Furthermore, it inhibits ATP synthesis by constraining E. coli's F1FO 

ATPase system resulting in cell wall damage.    



12 

 

It is believed that catechins inhibit cell wall construction by attracting lipid bilayers of the 

membrane. These bilayers form hydrogen bonds and attract polar lipid head groups at 

membrane edges, thereby inactivating or inhibiting enzyme synthesis both intracellularly 

and extracellularly. In general, antimicrobial agents can target enzymes involved in fatty 

acid biosynthesis to block the growth of bacteria, and in particular the enzyme fatty acid 

synthase II (FAS-II) may be of significant importance. 

Molecular oxygen can be reduced partially to produce reactive oxygen species (ROS), 

which are capable of exerting antimicrobial activity, aiding the immune response against 

various diseases. Catechins' antimicrobial activity is thought to be related to the 

production of oxidative stress (ROS and RNS), which can alter membrane permeability 

and damage cell walls. Liposomes also suffer from catechin damage since they contain a 

high concentration of negatively charged lipids. Catechins were found to support 

potassium leakage and disturb membrane transport in a methicillin-resistant S. aureus 

isolate. 

Antibacterial poisons originate from bacteria like Vibrio cholerae, E. coli, S. aureus, 

Vibrio vulnificus, Bacillus anthracis, Neisseria gonorrhoeae, and Clostridium botulinum, 

which are neutralized by catchins and quercetins. Due to hyaluronidase-mediated 

degradation, bacteria produce bacterial hyaluronidases directly targeting host tissues and 

increasing their permeability of connective tissues. Streptococcus equisimilis and 

Streptococcus agalactiae have been shown to respond to quercetin in hyaluronic acid 

lyase inhibition experiments (12). 

1.5 Traditional uses of Quercus 

In previous times Quercus acorns were found to be rich in carbohydrates, amino acids, 

proteins, lipids, and sterols. They have high energy content and are easily digested (41). 

Also acorns were found to contain fat in low amount, starch in high amount and suggest 

they are a more nutritious food than cereals, with more than 50% starch content. 

Furthermore, acorn starch is a natural thickener and stabilizing agent due to its high paste 

consistency. Thus, acorns might provide food industry with a promising ingredient that 

has substantial commercial potential. Throughout Italy and Turkey, Quercus is dried and 

roasted and consumed as coffee. Likewise, livestock in Turkey and Algeria consume the 

acorns and leaves of Q. cerris var. cerris. 



13 

 

Turkey's cities of Adana and Osmaniye recorded the use of Q. infectoria subsp. boissieri 

acorns as a dye. Also Turkish rugs are dyed using the cups of Q. ithaburensis subsp. 

Macrolepis after being boiled in water (39). 

In addition to providing skin depigmentation prevention and skin aging treatments, 

Quercus extracts can also be used as a complementary remedy to acne therapy, as well as 

in pharmaceutical and cosmetic applications. As a form of hair care, the galls of different 

species of Quercus are used on the hair alone or combined with henna (42). 

Acorns and galls of Quercus have been one of the most important export materials in 

Anatolia since at least the middle of the first century BC. The galls of Quercus species 

are most commonly found on the branches, bark, leaves, buds, stems, flowers, even the 

roots and acorns of the plant species. Galls are comprised of a protective layer that is very 

hard surrounding a  membrane of proteins, fatty substances, and sugar. They were were 

used for tanning leather, dyeing textiles, inking, and coloring dyes at all times throughout 

history. Similar to the other parts of Quercus, gall has been used for medical purposes 

since ancient Greek times (39). 

1.5.1 Other uses of Quercus 

Quercus tannins nowadays are used in the leather and plastic manufacturing industries, 

in the glue-producing industry, in the ceramic industry, and in the oil business. Thus, their 

commercial value is increased as a result of this. Due to their strong antifungal properties 

and the crucial role that oak barrels play in wine maturation, Quercus polyphenols have 

been extensively researched (39).  

1.6 Biological activity studies of Quercus extracts 

1.6.1 Anti-microbial effect 

S. aureus and Enterococcus faecalis (Gram+ve bacteria), and E. coli and Pseudomonas 

aeruginosa (Gram-ve bacteria), as well as three species of yeast Candida parapsilosis, 

Candida albicans, and Candida krusei, were all tested for antimicrobial effects of Q. 

aucheri ethylacetate, n-butanol subextract, methanol extract and and water residue. 

All the tested microorganisms were most susceptible to the ethylacetate subextract extract. 

All tested fungi had a high activity against Candida parapsilosis with MIC 1.2 mg/ml; 

Candida albicans with MIC 2.4 mg/ml; Candida krusei  with MIC 4.9 mg/ml and S. aureus 



14 

 

(Gram +ve bacteria) with a MIC of 2.4 mg/ml. The subextract which was the most active was 

analyzed for phytochemical components, which led to the discovery of quercetin-3-O-a-L-

arabinopyranoside, quercetin-3-Ob-D-galactopyranoside, and (+)-catechin (39). 

Using broth microdilution method and measuring MIC and MBC, 70% ethanolic extract 

of Q. brantii acorn was evaluated for its antibacterial effects. Compared to vancomycin 

and nitrofurantoin, the extract significantly inhibited S. aureus and E. faecalis. Extracts 

had MIC values of (500 and 600) mg/ml, as well as MBC values of (1000 and 800) mg/ml 

against S. aureus and E faecalis, respectively. It was found that methanol extracts of Q. 

brantii seed hulls had significant anti-bacterial effects on E.coli and Proteus mirabilis. 

Shigella flexneri did not seem to be significantly affected by the extract, which had an 

effect equivalent to 25 g of cotrimoxazole against S. typhimurium (18). 

With a 96-well plate containing sterile extracts of Q. coccifera stems, the minimum 

inhibitory concentrations (MICs) of the extracts were determined against Bacillus subtilis 

(ATTC 6633), E. coli (ATTC 25922), Klebsiella pneumonia (ATCC 27736), P. 

aeruginosa (ATTC 27853), S. aureus (ATTC 29213), P. mirabilis (ATTC 12453), 

Bacillus cereus (ATTC 11778), S.epidermidis (12228) and Streptococcus agalactiae 

(ATCC 12386), using a microdilution method in 96-well plates with gentamicin as a 

positive control. In tests conducted with tested bacteria strains, aqueous and methanolic 

extracts were found to have MICs ranging from “0.5 to 256 µg/ml and 0.25 to 256 µg/ml, 

respectively. E. coli was the most resistant bacterium, while P. mirabilis and B. cereus 

were most sensitive” (43). 

An extract  of ethyl acetate from Q. acuta leaves was evaluated for antibacterial activities 

against Gram-positive, Gram-negative, and antibiotic-resistant strains acquired from 

hospitals, including P. aeruginosa with carbapenemase production, methicillin-resistant 

S. aureus (MRSA), E. coli, and vancomycin-resistant enterococci with extended spectrum 

lactamases. MRSA strains showed the best anti-bacterial activity against ethyl acetate 

extract. Additionally, the same extract also showed antibacterial activity against E. coli 

KCTC 1923, Micrococcus luteus ATCC 9341, S. aureus KCTC1928, Salmonella 

typhimrium KCTC 1925, and eight MRSA strains exhibiting MIC values of 125 to 500 

µg/ml (44). 



15 

 

On Q. infectoria gall extract, two fold serial micro dilution broth assays were performed 

to determine its antimicrobial activity against pathogenic Leptospira. An aqueous extract 

of Q. infectoria gall exhibited antimicrobial activity against  L. interrogans serovars with  

MIC of 0.125 mg/ml. MBC values ranged from 0.125 mg/ml for L. interrogans serovar 

Javanica, and 0.250 mg/ml for L. interrogans serovar Icterohaemorhagiae (45). 

Cell division and cell morphology were altered by extracts from Q. infectoria galls when 

they were used to inhibit the growth of gram positive and gram negative bacteria. MRSA 

and MSSA strains were significantly inhibited by water, 95% ethanol, ethyl acetate, and 

acetone extracts of the same plant. As for the minimum inhibitory concentrations (MIC), 

MRSA had MIC of 0.13 and MSSA 1.00 mg/ml. With MIC values of 0.625 mg/ml, 0.625 

mg/ml, 1.250 mg/ml, 0.313 mg/ml and 1.250 mg/ml, respectively. An ethanol extract of 

Q. infectoria galls showed antimicrobial activity against, E. coli, P. aeruginosa, S. 

typhimurium, S. aureus and C. albicans. Using this extract to disinfect eggshells from 

pathogenic microorganisms is an effective and natural disinfectant. Using 1% extract 

solution, eggshell microbial contamination was drastically reduced, including yeasts, 

molds, and Enterobacteriaceae. As a result of immersion in the extract for 60 minutes, 

both E. coli and S. aureus were completely inhibited (46). 

In a disc diffusion test, three formulas of a cream contained 10%, 20%, and 30% of Q. 

infectoria gall extract were tested for the antimicrobial activity of them against C. 

albicans. Anti-Candida activity has been observed for all extract cream formulations. 

Formulated extract creams containing 10%, 20%, and 30% extract showed MIC values 

of 1.09 mg/ml, 0.55 mg/ml, and 0.07 mg/ml, respectively. Inhibition zone diameters were 

significantly different between creams containing 10% and 20% extract (p = 0.0254). An 

extract cream with 30% fungucidal concentration (0.068 mg/ml) demonstrated the lowest 

MFC (47). 

Multi-drug resistant bacteria strains were tested using two methods: the disc diffusion method 

and the microdilution method to evaluate the antibacterial activity of ethanol and water 

extracts prepared from Q. infectoria galls. The extracts significantly inhibited both MRSA 

coagulase-negative and MSSA coagulase-positive. Acinetobacter species that are multidrug 

resistant showed a reduced inhibitory zone diameter, while K. pneumoniae and E. coli isolates 

with broad-spectrum beta lactamases showed no inhibition effect. According to the results of 



16 

 

the study, extracts are potentially good sources of antimicrobial substances, particularly 

against multidrug-resistant Gram positive bacteria (48). 

Q. infectoria galls extracts in methanol and aqueous medium were analyzed for their anti-

Candida activity against Candida parapsilosis, Candida glabrata, Candida albicans, 

Candida tropicalis, and C. krusei. The MICs were determined by the serial dilution 

method at concentrations ranging from 16 to 0.04 mg/ml. In both of their crude extracts, 

pyrogallol was the major component, and aqueous and methanol extracts both showed 

significant anti-Candida activity. An aqueous extract displayed good fungistatic activity 

against C. parapsilosis, C. krusei, and C. glabrata isolates. Almost all Candida species 

were found to be susceptible to fungicidal activity in methanol extract (49). 

 Extracts of Q. infectoria galls possessed antimicrobial properties in both aqueous and 

acetone solutions and MIC values between 0.0781 and 1.25 mg/mL. There was a 

significant difference between the MBC value of the water extract against S. aureus and 

S. typhimurium and the MIC value for these bacteria. In addition to having a higher MBC 

value than its MIC value, acetone extract also had a higher MBC value against S. aureus. 

A 0.25 mg/ml acetone extract had the same MIC and MBC values against S. typhimurium. 

As far as P. aeruginosa, S. epidermidis, S. typhimurium, and B. subtilis, , and were 

concerned, the extracts showed weak inhibitory effects, whereas E. coli did not show any 

inhibition zones (39). 

Q. crassifolia bark extract was investigated for its antibacterial properties and potential 

subacute oral toxicity against probiotics and pathogenic bacteria. In comparison to 

Lactobacillus bulgaricus and S. thermophylus, extract selectively inhibited E. coli. At  

250 and 500 mg/ml, aqueous crude extract and ethyl acetate soluble fraction did not 

significantly differ in diameters of growth inhibition on S. thermophilus and L. 

bulgaricus. In contrast, when ethyl acetate soluble fraction was applied at 500.1 and 750.3 

mg/ml, a significantly greater inhibition diameter was observed (p/0.05) in a dose-

dependent manner to E. coli. Among the subacute adverse effects, no adverse effects were 

observed at an 11 mg/kg/day dose, while the lowest adverse effects were reported at 33 

mg/kg/day for kidney damage (17). 

T. gondii, an intracellular parasite that can cause dangerous effects on babies and people 

with weak immunity, the outer bark extract of Q. crispula showed potent anti-toxoplasma 



17 

 

activity. As little as 10 µg/ml of extract resulted in complete inhibition (50). There has 

been a study investigating the larvicidal effects of the extract of Q. infectoria galls on 

Anopheles stephensi larvae which are the main vectors of urban malaria, India's endemic 

disease. A LC50 value of 116.92 ppm was found for ethanol-acetate extract, the most 

effective extract among the five extracts tested against larvae of the fourth instar. 

Gallotannin, nbutanol, acetone, and methanol also showed larvicidal activity against 

larvae of the fourth instar, respectively (39). 

Q. suber leaves and bark methanol extracts were tested on C. albicans growth using 

microbroth dilution parameters, coupled with their MICs.In both cases, the extracts 

showed antifungal activity. MICs ranged between 12.5 and 50 mg/ml for Candida 

albicans and Trichophyton rubrum in the presence of  bark methanol extract. Agar well 

diffusion method was used to test the antibacterial and antifungal properties of catechin 

derivative isolated from Qincana. Among the bacterial targets, S. pyogenes showed the 

best anti-bacterial activity with 80% inhibition, while C. glabrata showed the best anti-

fungal activity at 80.5% inhibition (39,51). 

1.6.2 Effect on wound healing 

In many countries around the world, Quercus species are used to treat wounds. Detailed 

investigations of their mechanisms of action have been conducted. It was found that ointment 

prepared with oak bark extract had wound healing, and antibacterial ,anti-inflammatory 

properties. A clinical study also demonstrated the efficacy of this ointment. Using albino 

mouse fibroblasts and cell lines of murine macrophage, the effect of aqueous and methanolic 

extracts of Q. coccifera stem parts on different stages of wound healing was investigated. In 

the presence of 200 µg/ml concentrations of methanol and aqueous extracts, proliferation of 

fibroblasts was significantly stimulated by 136.7% and 163.4%, respectively. There was a 

significant increase in wound closure rates between 18.5% and 52% after using an aqueous 

extract and 23.9% to 75.4% after using a methanol extract (39). 

1.6.3 Antioxidant effect 

To obtain extracts from Q.cerris wood samples, maceration, ultrasound assisted 

extraction, and accelerated solvent extraction techniques were used to determine anti-

oxidant activities. Extraction techniques were found to affect the relative antioxidation 

capacity index of the extracts obtained from thermo-treated wood in this rate : ultrasound 

assisted extract > macerate > accelerated solvent extract (52). 



18 

 

Furthermore, a DPPH assay was conducted on extracts from Q. coccifera stem parts to 

determine whether they have an anti-oxidant effect. IC50 values for methanolic extract 

were 58.7 x 2.42 µg/ml which exhibited more potent antioxidant activity (43). 

1.6.4 Neuroprotective effect 

In an effort to test whether Q. coccifera extracts have neuroprotective effects against 

acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), extracts from cups, and 

shelled acorns pieces of the plant were tested on the enzyme inhibitory effects of water 

and ethanol extracts. Between 50% and 74%, there were significant differences in ethanol 

extracts' cholinesterase inhibition activity on both enzymes (200 µg/ml). An AChE 

inhibitory test with galantamine at 100 µg/ml showed 93% inhibition and a BChE 

inhibitory test showed 88% inhibition (53). 

1.6.5 Anti-hepatotoxic effect 

Recent experiments analyzed the potential antihepatotoxic activity of the acorns of Q. 

liaotungensis on hepatic stellate cells (t-HSC/Cl-6) in humans. It has been found that the 

acorns are an excellent source of antioxidants and antihepatic fibrosis agents, with galloyl 

triterpenes being the most significant components of the acorns.  

A dose-dependent hepatoprotective effect was observed in an 80% methanol solution 

extracted from Q. robur leaves compared with the group of paracetamol-induced gastric 

damage. Also in comparison with ethanol-induced gastric damage group, the ethanol-

treated group experienced significant gastroprotection (39). 

1.6.6 Problems related to the gastrointestinal system 

There are several oak species used in folk medicine for problems in gastrointestinal 

system. In addition to alleviating ulcerative colitis, gastric lesions, Helicobacter pylori 

infection, and suppressing diarrhea, polar extracts and tannins isolated from them have 

been demonstrated to be effective. Q. ilex aqueous extract was evaluated for its 

gastrointestinal-physiological activity using phenol-red colorimetry and charcoal/gum 

arabic in water test meals. Researchers explored the extract's effects on gastrointestinal 

disorders by inducing colonic constipation and delayed stomach emptying with 

loperamide. Compared with the control group (70 %), both doses 150 mg/kg and 300 

mg/kg of extract reduced gastric emptying GE (66% and 60.8%, respectively) (54). 



19 

 

An experiment involving isolated polyphenols and Q. ilex root barks was conducted on 

rats to investigate the way that the isolated polyphenols affected on gastric lesions resulted 

from alcohol. Gastric lesions were significantly decreased after oral administration  of 

these extracts and tannic acid (47.7%-76%). According to the study, the anti-

lipoperoxidant properties of the compounds are responsible for the effect. Analyses were 

conducted on rats to determine whether the aqueous extract of Q. ilex root bark nor tannic 

acid were able to protect the gastro-intestinal system from ethanol-induced damage. 

Haemoglobin from ovines was precipitated in vitro by Q. ilex extract and tannic acid. 

After consuming orally administered tannic acid or extracts, gastric lesions decreased 

significantly (47.7%-76%). The gastric mucosal barrier can be strengthened by 

monomeric and polymeric polyphenols, according to the research (39). 

1.6.7 Anti-viral effect 

Q. infectoria extracts at 100 g/ml showed more than 90% inhibition of HCV protease 

against methanolic and water extracts. An analysis of linear regression was performed to 

determine the dose-dependent antiviral effects of Q. persica hydroalchoholic extract on 

the replication of  HSV-1  in a hamster kidney cells. Cell death is significantly correlated 

with extract concentration (p/0.01). As a result of its attachment to baby hamster kidney 

cells, Q. persica L has an IC50 value of 1.02 lg/ml for HSV-1 before and after attachment. 

Crude extracts of the seeds of Q. lusitanica were studied for their potential to inhibit the 

replication of Dengue virus type 2 (DEN-2). As demonstrated by the absence of 

cytopathic effects (CPE), methanolic extract of Q. lusitanica inhibited 10–1000 TCID50 

of virus at its maximum non-toxic concentration of 0.25 mg/ml. When administered at 

the low dose (15 mg/ml), Q. lusitanica inhibited 10 TCID50 of virus totally, but it only 

inhibited 50% and 25% at 100 TCID50 and 1000 TCID50, respectively. Infected C6/36 

cells were also tested for the effect of the extract of Q. lusitanica on the expression of 

NS1. After treatment with the extract, C6/36 cells infected with NS1 protein showed a 

reduction in expression. (H9N2) virus infection was studied in (MDCK) cells against 

(Influenza A/turkey/Wisconsin/1/1966) virus reference strain (AMN) as a standard anti-

influenza A antiviral drug, comparing the effects with Q. ilex wood extract as an antiviral 

agent. Q. ilex extract had a maximum nontoxic concentration of 100 g/ml. As compared 

to Q. ilex extract, Amantadin showed a  poor anti-viral effect against H9N2 virus at a 

dose of 50 TCID50 (39). 



20 

 

1.6.8  Tyrosinase inhibitory effect 

Tyrosinase was inhibited by alcohol extract of Q. coccifera bark with an IC50 value of 

75 µg/ml. Likewise, kojic acid exhibited an IC50 value of 51 µg/ml as a positive control. 

In contrast, (-)-8-chlorocatechin (IC50 value of 60 g/ml) is not as strong as kojic acid in 

inhibiting tyrosinase as transresveratrol-3-O-b-glucopyranoside, which exhibited an IC50 

value of 4 g/ml. There was a competing response (Ki 50 µg/ml) for (-)-8-chlorocatechin, 

while there was a noncompeting response (Ki 6.7 µg/ml) for polydatin (55). Q. infectoria 

galls extract 80% methanol did not cause any damage to B16/F10 cells (cells from 

melanoma-affected mouse skin tissue, this cell line exhibits spindle-shaped and 

epithelial-like morphology) up to 100 µg/ml. Cell viability at this concentration was 

reduced by 83% for B16/F10. A 50% decrease in cell viability was observed when kojic 

acid was used at the same concentration. Melanin content in cells decreased by 66% and 

37% when 50 and 100 µg/ml extracts were used as positive controls (39). 

Specifically, extract inhibits tyrosinase in a dose-dependent manner, with a maximum 

inhibition of 59% at 100 g/ml. Similar results were observed with kojic acid. In the dried 

extract, there were 287 mg gallic acid equivalents per gram of total phenols. A high 

phenolic content of the extract may be responsible for its melogenesis and tyrosinase 

inhibitory properties. It inhibited L-DOPA oxidation with Q. dentate leaves and stem 

extract at concentrations of 16 – 666 µg/ml, and it failed to inhibit mushroom tyrosinase 

activity at similar concentrations. When 50 µg/ml of extract was applied to B16 mouse 

melanoma cells, melanin synthesis was inhibited (> 50%) by Q. dentate (bark) (56). 

The decoction of Q. robur bark and the tannin fractions  from this extract were evaluated 

as a method for preventing basophilic cells from degranulating (b-hexosaminase activity) 

in response to allergens. Based on the results of this study, decoctions (0.058- 0.58 g/ml) 

and semolina fractions (12-100 g/ml) inhibited basophilic degranulation capacity in a 

dose-dependent way, comparable to azelastine, the positive control. The fraction did not 

exhibit any cytotoxic effects at the tested concentrations, indicating that the effect was 

not due to cytotoxicity. Human mast cells released late-phase allergy markers, IL-6, IL-

8, and TNF-a, after ingestion of the decoction or fraction D, which had an inhibition rate 

approximately equal to dexamethasone at high doses (100 mg/ml). Piperonylic acid ester 

isolated from oak galls also showed antihistamine effects in another study (57). 



21 

 

1.6.9  Anti-hyperlipidemic effect 

An extract of Q. infectoria gall that was administered at 1.5 g/kg methanol for 45 days 

markedly reduced total cholesterol, LDL levels and triglycerides, and its ability to lower 

lipids was comparable to or stronger than that of the atorvastatin drug (positive control). 

In addition, it reduced aortic valve and thoracic aortic plaque formation more effectively 

than either atorvastatin or orlistat (39). 

1.6.10  Anti-diabetic effect 

As compared to the positive control acarbose (IC50: 50 µg/ml), methanol bark extract of 

Q. coccifera significantly inhibited the activity of aglycosidase (IC50: 3 µg/ml). This 

extract was also tested for its inhibitory potential against a-glucosidase. Based on an IC50 

value of 44 µg/ml, (-)-8-chlorocatechin was the most effective a-glucosidase inhibitor of 

the isolated compounds (Ki of 30 µg/ml). An IC50 value of 50 was observed in the same 

test using acarbose as a positive control (55). Q. infectoria galls methanol extract showed 

a-glycosidase (amylase, sucrose, maltase, isomaltase) inhibitory activity against 3-O-

digalloyl-1,2,4,6-tetra-O-galloyl-bD-glucopyranoside. Acarbose showed lower a-

glycosidase inhibitory activity on isomaltase and sucrose, but showed higher inhibitory 

activity on 3-O-digalloyl-1,2,4,6-tetra-O-galloyl-b-D-glucopyranoside than acarbose. As 

a result of methanolic extraction of Q. gilva leaves, the activity of a-glycosidase was 

inhibited. An isolated compound named kaempferol-3-O-(600-trans-p-coumaroyl)-bD-

glucopyranoside with an IC50 value of 28.1 mM had the highest inhibitory effect on the 

a-glycosidases, followed by catechin and epicatechin, which have an IC50 of 168.6 mM 

and 920.6 mM, respectively (39). 

Q. dilatata fruit methanol extracts at 200 and 400 mg/kg, respectively, reduced blood 

glucose levels in alloxan-induced diabetic rats by 114 and 111%. The 200 mg/kg extract 

also ameliorated the deterioration of liver function markers, including ALT and total 

bilirubin, and renal function markers, including creatinine and serum urea, as well as 

changed serum triglycerides, LDL, cholesterol, HDL, and VLDL levels to those of non-

diabetic rats (58). 

In vitro and in vivo studies demonstrated an antidiabetic effect of different Quercus 

species. As well as inhibiting the activity of aglycosidase and b-glycosidase enzymes, 

Quercus extracts also ameliorated diabetic-associated issues through their antioxidant 



22 

 

properties. Furthermore, kaempferol and quercetin derivatives were evaluated for their 

antioxidative properties. In vitro and in vivo studies also showed an anti-diabetic effect 

of beverages prepared from some Quercus leaves used in folk medicine (55,59). 

1.6.11 Anti-inflammatory effect 

The inflammatory cytokines and chemokines were inhibited by extracts and isolated 

compounds from Q. mongolica acorns. Interleukin (IL)-13 release was most inhibited by 

the ethyl acetate fraction with an IC50 of 5.8 µg/ml. There was a notable decrease in the 

expression of monocyte chemoattractant protein-1 (MCP-1), IL-10, IL-13, and IL-6 

mRNA expression following the administration of pedunculagin (2,3-4,6-bis-(S)-

hexahydroxydiphenoylb-D-glucopyranoside) isolated from this extract. Furthermore, it 

decreased cyclooxygenase-2 (COX-2) and c-Jun N-terminal kinase (p38), ERK, and p38 

phosphorylation at concentration-dependent levels (60). NFjB (nuclear factor kappa-

light-chain enhancer of activated B cells) is a transcription factor involved in the 

inflammatory response. Q. infectoria extract demonstrated anti-inflammatory activity by 

downregulation of this pathway. Both high glucose and palmitate medium treated bone 

marrow-derived macrophages and high fat diet-treated macrophages expressed less Set7, 

p65, and inflammatory cytokines than vehicle controls. It was demonstrated that the in 

vivo diabetic environment also lowered inflammatory cytokines, IL-1ß and TNF-a, when 

macrophages and monocytes from diabetic mice were treated with extract solution (61). 

A study was conducted on the effects of infusions of Q. durifolia, Q. eduardii and Q. 

sideroxyla leaves on inflammation and anticarcinogenesis caused by 1,2-

dimethylhydrazine (DMH) in HT29 cells. There was a statistically significant reduction 

in mean tumor size and multiplicity (1.2 vs. 2.0) in adenocarcinomas compared with 

DMH groups, and a 2.2-fold increase in bcatenin protein levels (2.2-fold). 

Adenocarcinoma developed in 68.8% of the animals given only DMH. As compared with 

adenocarcinomas of DMH-treated groups (4.71 arbitrary units), Q. sideroxyla infusion 

significantly decreased total b-catenin protein expression (1.47 arbitrary units). The 

expression of b-catenin (3.54 arbitrary units) was reduced by 27% with Q. durifolia 

infusion. The treatment of HT-29 cells with Q. sideroxyla effectively decreased COX-2 

and IL-8 levels by modulating NF-jB expression (39). 



23 

 

In addition to inhibiting c-Jun N-terminal kinase, NF-jB, and ERK pathways, Polar 

Quercus extracts and isolated tannins exhibit anti-inflammatory activity in the presence 

of IL-1b, IL-13, IL-10, and IL-6 and TNF-a inflammatory cytokines. In addition, they 

inhibited COX-2 activity and suppressed histamine release as well. Inflammatory 

responses were also reduced by antioxidant mechanisms (60). 

1.6.12  Anti-alergic effect 

 Testing the effect of decoctions and tannin fractions obtained from Q. robur bark on the 

activation of allergen-specific basophilic cells using b-hexosaminidase. Decoctions 

(0.058 - 0.58 mg/ml) and their tannin fractions (12-100 mg/ml) inhibited basophil 

degranulation capacity dose-dependently and compared favorably to the positive control 

azelastine. As tested concentrations of fraction exhibited no cytotoxic effect on cells, it 

was not related to cytotoxicity. Mast cells of  human were inhibited by the decoction and 

fraction D, with a maximum inhibitory rate (100 mg/ml) comparable to dexamethasone 

levels at high doses. Another study showed that the anti-histamine properties of the 

isolated piperonylic acid ester from the galls of oak were based on substances within its 

structure (57). 

1.6.13  Vasorelaxant effect 

Inhibitors of nitric oxide synthase (Nʷ-nitro-L-arginine) abolished the vasorelaxing effect 

of Q. salicina leaf extract, which induced endothelium-dependent relaxations. A 

relaxation degree of 11.2%, 53.5%, and 102.00% was measured at concentrations of 10, 

30 and 100 µg/ml, respectively. Q. salicin extract had an ED50 of 23.7 µg/mL for 

vasorelaxing effects. In porcine coronary artery endothelial cells, ethanolic extract 

activates endothelial nitric oxide synthase (eNOS) in a dose-dependent manner, resulting 

in increased bioavailability of nitric oxide. It was concluded that the extract could 

possibly be regarded as a potential herbal cardiovascular protective medicine that may 

help to prevent cardiovascular diseases and endothelial dysfunction, which can lead to 

heart attacks (62). 

1.6.14 Hemostatic effect 

In standardized bone holes prepared in the calvaria of 5 Burgundy rabbits, two groups of 

cotton pellets were prepared. The first group of pellets were soaked in ferric sulfate 

solution and the second cotton pellets were soaked in alcohol extract of Q. persica fruit. 



24 

 

Then they were tested and compared for hemostatic effects. There was no significant 

difference in bleeding control between normal saline and Q. persica extract after the 

following of 4 and 5 minutes when compared with Q. persica extract (63). 

1.7  Emergence of MRSA 

Staphylococcus aureus is a gram-positive coccus (~0.6  μm in diameter), sometimes 

positive for the reduction of catalase and nitrate, and is an optional anaerobic that can 

spread without oxygen requirement (64) non-motile, non-spore former, and some strains 

are capsulated (65). Many skins and/or nares of most humans are colonized with it, and 

despite the disruption of tissue or impairment of immune function, it can enter other 

tissues. It has been found that S. aureus can cause osteomyelitis, pneumonia, skin and 

soft tissue infections (SSTIs), endocarditis, and septicemia, just to name a few of the 

severe clinical manifestations of the infection (66). 

With the emergence of hospital-based medicine, S. aureus has quickly become a major 

cause of healthcare-associated infections. Resistance developed just a few years 

following the introduction of penicillin and was started by the β-lactamase gene (blaZ) 

by the mid-1940s. 

The first semi-synthetic anti-staphylococcal penicillin’s were produced in 1960. 

Unfortunately, MRSA was discovered within a year after their first clinical application. 

Furthermore, genomic data suggest that methicillin resistance followed the first 

therapeutic use of anti-staphylococcal penicillins (67). 

1.7.1 Evolution and genetic diversity 

Genomes of bacteria are divided into core components, which hold genes present in all 

isolates that are related to cellular metabolism and replication, and accessory components, 

which MRSA and other pathogens have a lot of genetic variation within them like 

virulence mediators, immunological evasion, and antibiotic resistance (67). Figure (1.5) 

shows a representative map for the genome of the USA300 strain FPR3757. It displays 

the genomic content of MRSA as colored circular tracks as shown in the map key. 

Methicillin resistance is mediated by the mecA gene and acquired by horizontal transfer 

of the staphylococcal cassette chromosome mec (SCCmec) by a mobile genetic element 

(67). SCCmec is a DNA fragment ranging in size from 21 to 67 kb, depending on the type 



25 

 

of SCCmec (68). According to Zong et al. (69), SCCmec has two essential components: 

the mec gene complex and the ccr gene complex. The mec gene complex is made up of 

mecA, regulatory genes, and associated insertion sequences that have been classified into 

six classes (A, B, C1, C2, D, and E), as well as cassette chromosome recombinase (ccr) 

genes (ccrC or the pair of ccrA and ccrB) that encode recombinases that mediate the 

integration and excision of SCCmec into and from the chro SCCmec, also includes a few 

additional genes such as insertion sequences, transposons, and plasmids (69). 

Figure 1.5 

Major genomic elements in methicillin-resistant Staphylococcus aureus (70) 

 

The mecA gene encodes the penicillin-binding protein 2a (PBP2a), an enzyme in the 

bacterial cell wall that is responsible for crosslinking the peptidoglycans. PBP2a has a 

low β-lactam affinity, which results in resistance to this whole class of antibiotics (67). 

Therefore the therapeutic possibilities of MRSA strains are reduced and limited (71). 

MRSA infections could be community-acquired (CA-MRSA) or hospital-acquired (HA-

MRSA). There are strong variations in phenotypes and genetic history of these strains 

associated with infection. 

 



26 

 

1.7.2 Community acquired MRSA (CA-MRSA)  

CA-MRSA is a newer and more virulent strain that emerged among healthy and relatively 

young people that are more likely to contract skin and soft tissue infections. However, 

current epidemic-molecular investigations have revealed that CA-MRSA may affect a 

number of patients in health care settings. 

Generally, CA-MRSA is defined as any strain of MRSA isolated in an outpatient setting 

or within 48 hours of admission from a patient who has never been infected with MRSA 

or colonized, does not have permanent invasive medical devices, and has not experienced 

the following risk factors in the past year: hemodialysis, surgery, hospitalization, or long-

term care residence. It is considered HA-MRSA when the strain does not meet this 

definition (72). 

 CA-MRSA differs genetically from HA-MRSA that its strains typically possess a small 

type of SCCmec (types IV or V), and the majority of them are susceptible to non-β-lactam 

antimicrobials (73). Additionally, It carries (but not always) LukS-PV and LukF-PV 

encoding Panton-Valentine Leukocidin (PVL) genes associated with increased virulence 

(65,74). 

 S. aureus colonization main site is the front nares. But there are numerous organs where 

MRSA strains can be colonized, including the inguinal regions (23% of CA- MRSA 

strains) in children-patients, the rectum in young patients, and the throat in young cases 

(65). 

1.7.3 Hospital acquired MRSA (HA-MRSA) 

HA-MRSA strain is characterized by its epidemiological behavior in hospital settings 

where it is especially successful. In certain health care institutions, S. aureus can also 

cause epidemic levels of infections, usually over the threshold for S. aureus infections. 

The majority of this type of infection is caused by these dominant MRSA clones, with a 

very small number of clones actually being successful. Practically every geographical 

region has been affected by epidemics caused by successful clones. The reasons for a 

pathogen to succeed are many. They can survive in an environment that has a commensal 

component, they can resist a variety of antimicrobial agents, and they can have a variety 

of biomarkers for virulence. A broad range of HA-MRSA strains are responsible for the 

most prevalent infection types in industrialized nations like the United States, including 



27 

 

catheter-associated infections,ventilator-associated pneumonias, nosocomial infections, 

and surgical wound infections  (11). 

Genetically HA-MRSA strains carry type I, II, or III of SCCmec and rarely possess genes 

that encode PVL. HA-MRSA is associated with nosocomial infections such as 

endocarditis and is often resistant to non-β-lactam antimicrobials, especially 

aminoglycosides, macrolides, lincosamides, and fluoroquinolones (73). 

It is possible to transmit MRSA within hospitals through the air, droplets, direct contact 

(skin-to-skin), and indirect contact (via fomites). The clothing of healthcare workers 

could be contaminated with fomite, even if they are not aware of it. All inanimate objects 

in a health facility are potentially fomite sources (75). 

1.8 Resistance to antibiotics on an intrinsic basis 

There are three primary aspects to endogenous resistance (appendix figure 1.6): 

1.8.1 The efflux system 

As Ball and McMurry studied Escherichia coli resistance to tetracycline in 1980, they 

discovered the active efflux system of bacteria (76). The active efflux system was then 

studied, and its physiological occurrence in sensitive strains was confirmed through many 

experiments. Genes encoding the efflux system are activated and expressed when 

substrates in the environment are present for a significant period of time, resulting in a 

greatly enhanced ability to efflux drugs, resulting in drug resistance. Resistance to 

multiple drugs is influenced by active drug efflux systems. Staphylococcus aureus cell 

membranes contain three different types of multidrug-pumping proteins (77). 

A proton kinesin is a multidrug pumping protein. Instead of ATP hydrolysis, material 

exchange takes place via H+ gradients formed across the cell membrane rather than ATP 

hydrolysis. A reversible process is usually involved, meaning that H+ moves from the 

extracellular space to the intracellular space, while harmful substances like dyes and 

antibacterial drugs flow from the intracellular space to the extracellular space (appendix 

figure 1.6A) (77).  

 

 



28 

 

1.8.2  Permeability of the outer membrane 

Drug resistance occurs when the bacteria's energy metabolism is affected by lowered cell 

membrane permeability, resulting in decreased drug absorption. By decreasing membrane 

permeability, S. aureus resists aminoglycosides and eventually consumes less of them, 

resulting in resistance (appendix figure 1.6B) (77). 

1.8.3  Overproduction of β-Lactamase enzyme 

It is an enzyme that catalyzes the conversion of various amino acid antibiotics into their 

cyclic forms, it is encoded by chromosomal genes of bacteria and is transferrable from 

one species to another. A research showed that β-lactam antibiotics have a deadly effect 

on bacteria through two mechanisms: first one is by binding to penicillin-binding proteins 

(PBPs),which inhibits cell wall mucin synthesis, resulting in bacterial expansion and 

lysis; second, by triggering autolysis and death via the autolytic enzyme activity. 

Excessive secretion of β-lactamase by MRSA inhibits the effect of antibiotics through 

two mechanisms that leads to MRSA resistance. The first one is the hydrolysis 

mechanism, that is, β-lactamase hydrolyzes and inactivates β-lactam antibiotics; the 

second one is the mechanism of pinching, that is the binding of a large amount of β-

lactamase quickly to extracellular antibiotics, preventing the antibiotics from reaching the 

intracellular space. In turn, MRSA resistance to antibiotics results due to antibiotics being 

unable to reach the target site (appendix figure 1.6C) (77). 

1.9  Acquired antibiotic resistance 

1.9.1  Biofilm-mediated resistance 

Biofilms are extracellular complex structures consisting of microbial populations 

attached to the surface of substrates, which are surrounded by a highly hydrated 

extracellular polymer matrix created by the bacteria themselves, which provides a way 

for bacteria to adapt to their environment as a protective measure of survival (78,79). 

A biofilm is the most prominent bacterial structure in nature. Biofilms have numerous 

characteristics including strong adhesion and drug resistance, enabling bacteria to evade 

host immune responses and antibiotics. They can be a thousand times more resistant to 

antibacterial drugs than plankton. In clinical medicine, antibiotics, as well as chemical 

synthetic drugs, are used in clinical medicine with some toxic effects, but domestic and 

foreign anti-biofilm treatments mainly depend on the development of new antibacterial 



29 

 

drugs. Resistance to these conventional drugs is common among biofilm bacteria, and 

resistant strains have been increasing in recent years (77). 

1.9.2  Mutations resistance 

Mutations in DNA gyrase can result in drug resistance in Staphylococcus aureus, causing 

the outer membrane proteins to reduce, reducing drug accumulation in the bacteria (80).  

A modification of ribosomal RNA methylase is one of the mechanisms which are 

responsible for the resistance to clindamycin and erythromycin (77). 

1.9.3  Acquisition of resistance genes 

A plasmid-mediated resistance type called acquired resistance occurs as a result of 

inherited resistance. Bacteria can develop resistance to antibiotics through gene insertion 

and transduction through plasmids . 

 MRSA resistance is mainly attributed to plasmids, or plasmids capable of transmitting 

drug-resistant genes, which are capable of expanding the genome and transferring 

resistance genes between bacteria. By acquiring plasmids from Enterococcus, for 

example, MRSA can expand and enhance its resistance to drugs by obtaining drug-

resistant plasmids (80). 

1.9.4  The role of persister cells in antibiotic resistance 

In microbes, persister cells are a small subset of their genetic homologous counterparts, 

but they are genetically heterogeneous in their phenotypic traits (81). They grow slowly, 

remain dormant, and grow in the presence of high antibiotic concentrations. It has been 

suggested in early studies that bacterial retention is associated with temporary resistance 

to antibiotic stress, not with any genetic changes. As high-throughput sequencing 

technology develops, this assumption is being challenged. A small percentage of bacteria 

will resist external stimuli such as antibiotics by arresting growth and remaining inactive, 

but most bacteria will be killed immediately by these stimuli. It has been demonstrated 

that these persister cells can return to normal growth when the external pressure is 

removed (81). It is difficult to completely eliminate bacterial infections and prevent 

recurrent infections when persister cells are present. Despite antibiotic treatment, bacteria 

persister cells demonstrate antibiotic tolerance, slow growth, and the ability to reestablish 

infection after antibiotic administration. By reducing cell growth, metabolism, and even 



30 

 

becoming dormant, persisters can resist the killing effects of antibiotics. An isogenic 

subpopulation of bacteria with multidrug tolerance is known as bacterial persistence. It is 

important to understand that persisters are not mutants, but simply phenotypic variants. 

According to existing research results, bacterial persistence is a complex process and 

involves several signaling pathways, including toxins-antitoxin systems, energy 

metabolism reduction by cells physiologically reduced, protein and nucleic acid synthesis 

systems, DNA repair and protection systems, protease systems, trans-translation systems, 

external pumping systems, etc (77,81). 

1.10  Virulence Factors of MRSA 

1.10.1  Surface associated proteins 

1.10.1.1  Staphylococcal protein-A 

Staphylococcal protein A is a cell wall component that binds to circulating IgG, 

preventing opsonization of the complement system and protecting the microbe from 

phagocytosis. 

1.10.1.2  Clumping factors 

MRSA cell surface contains clumping factor molecules that are responsible for triggering 

the S. aureus adherence to the fibrinogen of the extracellular matrix of the host body. 

MRSA was identified by two unique Clf proteins (Clf A and Clf B). Clf A is present on 

the surface of MRSA at all stages of growth, and Clf B is mostly detected during the 

beginning of the exponential phase (65). 

1.10.1.3 Capsular polysaccharides 

The capsular polysaccharides are polysaccharide polymers found in MRSA's cell wall. 

They are produced by 76 – 90  % of clinical MRSA isolates, and 11 serologically different 

capsular polysaccharide types (CP 1- CP11) have been found, but the two major CP types 

are CP5 and CP8, which all clinical S. aureus strains have the biosynthetic pathways for 

making either of them (82). 

Capsular polysaccharides increase S. aureus pathogenicity by interfering with 

complement and antibody-mediated opsonization and impeding phagocytosis (65). 

 



31 

 

1.10.2 Extracellular Toxins 

1.10.2.1 Staphylococcal hemolysins 

MRSA strains have been found to express alpha, beta, gamma, and delta toxins, although 

the levels of synthesis vary amongst strains. Within these toxins, Alpha toxin is produced 

by the majority of pathogenic MRSA strains and is thought to be a primary virulence 

factor. MRSA enterotoxins are primarily implicated in both human and animal food 

poisoning (65). 

1.10.2.2  Staphylococcal enterotoxins 

Staphylococcal enterotoxins are pyrogenic exotoxins that are superantigens of 

Staphylococcus aureus. Enterotoxin types A, B, C, D, and E are the primary virulence 

agents implicated in human food poisoning, particularly SEA. Bacterial enterotoxins 

operate as superantigens (SAgs), stimulating the production of IL-4 and IL-10 genes, 

resulting in the activation of TH2 cells and the suppression of pathogen clearance. 

MRSA-produced Panton-Valentine leucocidin can cross the blood-brain barrier, causing 

severe damage to the cell membrane of human polymorphonuclear cells (65). 

1.10.2.3  Panton-Valentine Leukocidin 

One of the strongest staphylococcal exotoxins is Panton-Valentine leukocidin, it is a two-

component pore-forming toxin that acts mainly on neutrophils, and it is found in high 

concentrations in S. aureus strains obtained from necrotizing infections (83). PVL is 

regulated by two kinds of secretory proteins, F and S. The genes lukS-PV and lukF-PV, 

which are found in the staphylococcal chromosome, encode for PVL production. These 

genes are transferred by PVL-phages, which infect the PVL-negative strains and release 

toxins. 

PVL destroys the plasma membrane of polymorphonuclear cells in humans, increases the 

release of oxygen metabolites from polymorphonuclear cells, the release of interleukin 8, 

the synthesis of lysozymes, and the release of histamine from human basophils (65). 

1.10.2.4  Staphylococcal exfoliative toxins (ETs) 

Exfoliative toxins (also known as "epidermolytic" toxins) are specific serine proteases 

that recognize and cleave desmosomal cadherins (desmoglein-1 protein ) only in the skin's 

superficial layers resulting in the sloughing of the epidermis, Infected infants and 



32 

 

neonates are mainly affected by staphylococcal scalded skin syndrome (SSSS) due to this 

bacteria (65,84).  

1.10.2.5  Toxic Shock Syndrome Toxin 

Toxic shock syndrome toxin (TSST) is one of the most potent S. aureus superantigens 

implicated in toxic shock syndrome (TSS) in humans. The condition developed as a result 

of the release of TSST, which stimulates the synthesis of TNF-, IL-1, and IL-2. Toxic 

shock syndrome is a lethal condition with a high morbidity and mortality rate. TSST 

typically affects women during menstruation, particularly on the second and third days. 

During this time, S. aureus thrives in the vagina and produces toxins. Fever, headache, 

vomiting, and diarrhea are all symptoms of the condition, which is characterized by both 

systemic and gastrointestinal issues (65). 

1.11  In vivo studies using a mouse model  

The purpose of animal experiments with infectious agents is to demonstrate the molecular 

bases of pathogenesis. Over the past forty years, researchers have used the mouse as a 

model for human infectious diseases because they are susceptible to various diseases 

caused by bacteria like MRSA, just like humans. This helped in releasing a multitude of 

reagents that have allowed rapid advances in the field of infectious disease (85).  

Infection models can be constructed using mice because of a number of their 

characteristics. There are several advantages to them, including their small size, smaller 

footprint, lower cost, rapid reproduction, and similarities to humans with respect to their 

immune, nervous, cardiovascular, and endocrine systems (86).  

An epidemic like MRSA spread needs to be studied closely in order to be able to control 

it. So mouse models have been extensively used in studies regarding S. aureus infections 

like infections of soft tissues and skin that can be generated by injecting staphylococci 

subcutaneously. In addition to understanding the role of virulence factors during 

infection, mice have also been used to investigate the role of specific host pathways and 

factors in the response against Staphylococcus aureus. In the past few years, several 

important clinical diseases have been modeled in mice (86,87). 

An article published by Tseng et al. described and examined skin, subcutaneous, and 

muscle pathologies from a subcutaneous infection of a mouse model. The article also 



33 

 

indicated that animal model is crucial for understanding how MRSA interacts with 

immune responses, resulting in a more severe infection (88). 

1.12 Statement of the problem and rationale of the study 

Due to the potential of methicillin-resistant Staphylococcus aureus (MRSA) to spread in 

hospitals and the population, its growing emergence of it has become a major global 

burden and a major challenge for infection control. A study published by Kaibni et al. in 

2009 attempted to examine MRSA colonization in nasal swabs collected at the time of 

hospital admission from 843 patients without a history of hospitalization and 72 

healthcare staff selected in Palestine for comparison. In 2.0  % of patients and 13.9  % of 

healthcare staff, MRSA was observed. As a result of this study, in healthcare staff, MRSA 

transport was relatively large compared to that recorded in many other countries (89). 

The emergence of MRSA and the increasing existence of a population reservoir for 

methicillin-resistant strains threaten potential healthcare regulation of antimicrobial 

resistance; additional antimicrobial resistance could evolve (89) especially due to the lack 

of effective antimicrobial treatment.  

This study will contribute to solving this problem by discovering new, effective, and safe 

plant-derived antimicrobial that will prevent further spread of MRSA. 

1.13 Aim of the study 

This study aimed to test the antimicrobial activity of crude extracts from three of the most 

distributed species of Quercus in Palestine, including  Quercus calliprinos, Quercus 

ithaburensis, and Quercus coccifera, on existing MRSA isolates and determine their MIC 

and MBC to be then tested in vivo using mice as an animal model. 

 

 

 



34 

 

Chapter Two 

Experimental Part 

2.1 Sample collection 

Fresh leaves of the three Quercus species were collected from Jenin city, North West 

bank, as shown in table (2.1), in the period between May and December 2021. Fruits were 

then collected from the same places except for Q. coccifera’s tree which didn’t have any 

fruit at that time of the year so only leaves were collected. 

The identification of the plant samples was authenticated at An-Najah National 

University, College of Science, Department of Biology. 

Table 2.1 

Parts collected from each specie and the place they were collected from 

Quercus species Plant part collected Collection place 

Quercus calliprinos leaves and fruits Sir village 

Quercus ithaburensis leaves and fruits Sir village 

Quercus coccifera Only leaves Siris village 
 

2.2 Preparation of plants samples for extraction 

Quercus leaves were dried at room temperature without exposing them to direct sunlight 

in a shaded area to bring down the initial large moisture content for two weeks. Table 

(2.2) shows the amount of water lost from the collected Quercus leaves.  

Fruits (acorn  +  cupule) of Q.  calliprinos and Q.  ithaburensis were dried in an oven at 

50°C for 48 hours, then they were ground to powder by an electric grinder. 

  



35 

 

Table 2.2 

Amount of water lost in leaves of each Quercus specie  

Quercus species Fresh weight Dried weight Water lost 

Quercus calliprinos 0.6 kg 0.4 kg 0.2 kg 

Quercus ithaburensis 0.56 kg 0.35 kg 0.21  kg 

Quercus coccifera 0.55 kg 0.4 kg 0.15 kg 

 

2.3 Extraction method 

Two extracts were prepared every time they were needed from each Quercus species: 

aqueous extracts and alcoholic extracts, at room temperature by infusion method.  

Aqueous extracts were prepared by soaking dried leaves (50  g/800 ml) and fruit powder 

(70  g/400  ml) in distilled water for 48 hours in a covered flask at 4°C. Alcohol extracts 

were prepared by soaking dried leaves (50  g/800 ml) and fruit powder (70  g/400  ml) in 

100% ethanol for 48 hours in a covered flask at 4°C. 

After 48 hours, aqueous and alcohol extracts were filtered by a mesh strainer to attain a 

clear filtrate. Aqueous filtrate was placed in the lyophilizer to be freeze-dried for three 

days to get a fine powder extract. Alcohol filtrate was placed in a flask in the hood for 

five weeks to evaporate ethanol and get a fine powder. These concentrated, powdered 

extracts were labeled and stored in a sealed flask at 4°C for further use (90–93).  

2.4 In vitro antibacterial activity testing 

2.4.1 Preparation of growth media 

In order to prepare Mueller-Hinton agar (MHA), the manufacturer's instructions were 

followed. With stirring, 38  g of MHA powder was suspended in 1 liter distilled water and 

allowed to boil until everything was dissolved. After autoclaving the media for 15 minutes 

at 121°C, it was placed onto sterile Petri dishes and allowed to cool to room temperature 

before use. 

  



36 

 

2.4.2  Preparation of bacterial suspension 

Available clinical isolate of MRSA was sub-cultured on MHA 24 hours before use. Then 

a sterile sample was taken gently from the colony surface of the culture and transferred 

to a sterile tube that contains 5 mL of sterile normal saline. The optical density of the 

suspension was measured by spectrophotometer at ƛ = 620 nm, where normal saline was 

used as blank. In order to obtain a bacterial suspension with a 1.5 x 10^8 colony forming 

units (CFU/mL), the turbidity of the bacterial suspension was adjusted to 0.5 McFarland 

turbidity standard (94). 

2.4.3 Agar well diffusion method 

The bacterial inoculum was uniformly spread using a sterile cotton swab on the entire 

surface of the sterile 12 MHA plates (previously prepared). Then, three wells (holes) with 

a diameter of 7mm were punched aseptically with a sterile tip in every plate. Two wells 

for two different volumes of the extract (35 µL, 70 µL) dissolved in its original solvent 

that it was soaked in at the beginning (distilled water or absolute ethanol), and the third 

well for the solvent control (distilled water or absolute ethanol). 

The test was performed two times only for leaves extracts, every time with a set of six 

MHA plates with different extract concentrations (0.1  g/ml, 0.2  g/ml). The plates were 

incubated for 24 hours at 36ºC under aerobic conditions. After incubation, inhibition of 

the bacterial growth around wells was observed (95,96). 

2.4.4 Determination of the minimum inhibitory concentration (MIC) 

2.4.4.1 Preparation of extract samples 

Every Quercus sample (leaf/fruit) was prepared by dissolving 0.1  g of extract powder in 

1ml of their solvent (distilled water/ethanol) in a micro centrifuge tube. Vortex was used 

for mixing to ensure better dissolving of the extract. Then micro tubes were ultra-

centrifuged (15000 rpm) for 7 minutes. Next, the supernatant was transferred to a new 

tube, and the pellet was discarded.  

  



37 

 

2.4.4.2  Serial dilution of extracts  

Seventy sterile micro tubes were prepared on racks for serial dilution to be done (ten 

micro tubes for each Quercus specie). 250 µL from the stock solution prepared previously 

for each Quercus specie was transferred to the first micro tube, and mixed with 250 µL 

of 2X Mueller Hinton Broth (MHB) solvent to get a total volume of 500 µL. Then serial 

dilution was done by taking 250 µL from the first tube and transferring it to the second 

micro tube already containing 250 µL of the solvent and mixing it to obtain (2 fold) serial 

dilution with a total volume of 500 µL. After that another 250 µL or half of the total 

volume were taken from the second micro tube and transferred to the  third and so on till 

the 10th micro tube. 

A sterile 96 micro-well plate was labelled, and then 100 µL from 2X MHB media were 

filled in all plate wells using a multichannel pipette. Then, 200 µL from each tube in the 

serial dilution done earlier was transferred and divided on the first two adjacent rows of 

the 96 micro-well plate until all dilutions were transferred (100 µL in the test wells and 

100 µL in the extract control wells next to the test wells) except columns (11+12). Then, 

50 µL from MRSA suspension was added to all wells except the extract control rows next 

to the test wells and the 12th column. 

Wells in column number 11 contained 200 µL of 2X MHB media and 50 µL MRSA 

suspension without plant extract, which served as positive growth control. Wells in 

column number 12 contained only the growth media (200 µL of 2X MHB media + 50 µL 

1X MHB media) served as the negative growth control. While extract control wells 

contained only the extract solution and 2X MHB media to ensure that there was no 

contamination. 

The same steps were performed for all Quercus species extracts. The absorbance of each 

well before incubation was determined using an ELISA reader (BioTek Instruments) at 

the wavelength of 450 nm. The plates were then incubated for 24 hours at 35 ̊ C. Turbidity 

in the wells as a result of bacterial growth was detected by reading the absorbance of the 

wells again by the ELISA reader (97). All MIC tests were performed in triplicates. 

The absorbance of every extract control well (ne