CHAPTER ONE

1.1 BACKGROUND

Staphylococcus aureus and Salmonella Typhi are important medical microbes that cause diseases

in humans and have been associated with antibiotic resistance in recent years. Staphylococcus

aureus, a common human pathogenic microbe, can cause various diseases such as bacteremia,

osteomyelitis, and endocarditis, as well as skin and soft tissue infections (Guo. Y, et al 2020).

Due to an increase in antimicrobial resistance, Staphylococcus aureus was grouped into

Methicillin Sensitive Staphylococcus aureus (MSSA) and Methicillin-Resistant Staphylococcus

aureus (MRSA), the latter which now has a high infection rate is difficult to treat and shows

resistance to many kinds of antibiotics (Guo .Y, et la 2020 ). Salmonella Typhi causes typhoid, a

systemic febrile illness that can only be treated with antibiotics(Chatham, et al 2019). Recently,

the bacteria have been reported to show resistance to chloramphenicol, ampicillin,

fluoroquinolones, and third-generation cephalosporins, making them multidrug-resistant bacteria

while retaining their infectivity (Chatham, et la 2019).

The burden of reducing the effect of pathogenic microorganism on human health has been a

global and major public health challenge (Bhatia R, et al 2010). Multidrug-resistant strains of

bacteria are increasing at an alarming rate in developing countries due to the random use of

antimicrobial drugs to treat infection (Nitu, et la 2017).Several synthetic antimicrobial drugs

have been developed to counter the effect of these pathogens and their resistant strains, with few

of these antimicrobial compound shaving a significant effect on these pathogenic bacteria.

Therefore, the need to find novel and potent antimicrobial compound from various sources

becomes paramount due to the realities of the rapid worldwide dissemination of these resistant

strains of pathogenic bacteria and has forced researchers to explore new antibacterial substance

1
from medicinal plants (Davies J, et al 1996) ). Plants have shown immense potential as natural

sources of alternative antimicrobial agents with effective treatment ability against various

bacterial infections.The World Health Organisation (WHO) has recognised medicinal plants as

one of the best alternative sources of drugs to treat microbial infections(Manandher et al, 2019).

Plants have been discovered to possess secondary metabolites and have been usedas

antimicrobial due to these phytochemicals synthesised from various medicinal plants. Secondary

metabolites such as alkaloids, flavonoids, phenolic compound, terpenoids, steroids, quinones,

among many other metabolites, have been found in-vitro with high efficiency antimicrobial

properties against pathogenic bacteria (Djeussi et al, 2013).

The guava (Psidium guajava L.) tree, belonging to the Myrtaceae family, is a very unique and

traditional plant which is grown due to its diverse medicinal and nutritive properties. Guava has

been grown and utilized as an important fruit in tropical areas like India, Indonesia, Pakistan,

Bangladesh, and South America. Different parts of the guava tree, i.e., roots, leaves, bark, stem,

and fruits, have been employed for treating stomachache, diabetes, diarrhea, and other health

ailments in many countries. Guava leaves (Psidii guajavae folium; GL) are dark green, elliptical,

oval, and characterized by their obtuse-type apex. Guava leaves, along with the pulp and seeds,

are used to treat certain respiratory and gastrointestinal disorders, and to increase platelets in

patients suffering from dengue fever (Lazily N, et al 2015). GLs are also widely used for their

antispasmodic, cough sedative, anti-inflammatory, antidiarrheic, antihypertension, antiobesity,

and antidiabetic properties (Cohn H, et al 2007). Studies on animal models have also established

the role of GL isolates as potent antitumor, anticancer, and cytotoxic agents (Ashraf A, et al

2015, Jiang L, et al 2020). GLs are widely employed for treating diarrhea and digestive ailments,

while the fruit pulp is utilized to enhance the platelet count for treating dengue fever. The

2
potential of guava leaf extracts for diarrhea treatment was also studied ( DeWitt P.S et al 2013,

Mazamdar S et al 2015). The flavonoids present in guava leaf extract chiefly determine their

antibacterial activity, while quercetin, which is the most predominant flavonoid of guava leaves,

exhibits strong antidiarrheal activities. The antidiarrheal activity of quercetin is ascribed to its

relaxing effect on the intestinal muscle lining which prevents bowel contractions. Guava leaf

polysaccharides (GLPs) can be utilized as an antioxidant additive in food and for diabetes

treatment.9

The presence of a unique variety of bioactive polyphenolic compounds, like quercetin and other

flavonoids, and ferulic, caffeic, and gallic acids, present in guava leaves primarily determine

their bioactive and therapeutic properties (Chan.Y, et al 2007, Dewi P.S et al 2013). These

phenolic compounds are known as secondary metabolites which exhibit strong antioxidant and

immuno stimulant activities. This review aims is to investigate the in vitro antimicrobial effect of

graver leaves extract on staphylococcus aureus and sammonal typhi.

1.2 Problem Statement

Antibiotic resistance is a global concern, leading to an urgent need for alternative antimicrobial

agents. Evaluating natural extracts for their antimicrobial properties can provide potential

solutions to combat bacteria such as Salmonella typhi and Staphylococcus aureus.

1.3 Rationale

Salmonella typhi is responsible for causing typhoid fever, while Staphylococcus aureus is

known to cause various infections, including food poisoning and skin infections. The

effectiveness of traditional antibiotics is decreasing due to the emergence of antibiotic resistance

strains. Thus, there is a need to explore alternative sources of antimicrobial agents, such as

natural plant extracts, like ngover leaves, to combat these pathogenic bacteria.

3
1.4 Research Questions:

1. What are the antimicrobial properties of ngover leaf extract against Salmonella typhi?

2. What are the antimicrobial properties of ngover leaf extract against Staphylococcus aureus?

1.5 General Objective

The primary objective of this research is to evaluate the antimicrobial properties of ngover leaf

extract against Salmonella typhi and Staphylococcus aureus.

1.6 Specific Objectives

1. To assess the sensitivity of Salmonella typhi and Staphylococcus aureus on different

concentration of gauver .

2. To determine the if gauver leave has bacteriocicidal or bacteriostatic properties on salmonella

typhi and staphylococcus aureus

3. To compare the antimicrobial effectiveness of ngover leaf extract with standard antibiotics.

1.7 Significance of the Study

This research aims to contribute to the knowledge of natural alternatives to combat Salmonella

typhi and Staphylococcus aureus, which can be beneficial in several ways:

- Identifying ngover leaf extract as a potential antimicrobial agent can provide an alternative

treatment option for infections caused by these bacteria.

- The findings can support the development of new antimicrobial drugs or therapies derived from

natural sources.

- Help reduce dependency on antibiotics and minimize the development of antibiotic resistance.

1.8 Limitations of the Study

It's important to acknowledge the limitations of this research study, which may include:

4
 The study focuses only on the antimicrobial properties of guava leaf extract and does not

assess other potential biological effects.

 The research might use laboratory strains of Salmonella typhi and Staphylococcus aureus,

which may not fully represent the diversity and variability of these bacteria in real-world

settings.

 The study may face practical limitations, such as limited availability of guava leaves, time,

and resources allocated for the research.

1.9 Definition of terms

Pathogen: any organism that can cause disease

Agar: is a gelatinous material derived from algae, which is use an a culture medium to culture

bacterial or other pathogenic agent for the diagnosis or laboratory experiment purposes

Phytochemicals: this are natural product obtain from plants with potential biological effect

Antibacterial activity: have the ability to prevent the growth of microorganism

Bactericidal: has the ability to completely to completely killed pathogenic agent

Minimum inhibitory concentration: the lowest concentration of an antimicrobial that will

inhibit the visible growth of microorganism after overnight incubation

5
 CHAPTER TWO

LITERATURE REVIEW

2.1 OVRVIEW OF ANTIMICROBIAL AGENTS AND HERBAL MEDICINE

Initiation and development of a bacterial infection in an animal requires a three dimensional

interaction between bacterial agents, environment and immune status of the animal (Rahal et al,

2014a). A bacterial disease can be initiated only when pathogenic bacteria comes in contact with

a susceptible host in a disease favouring environment (Engering et al, 2012). The environment

plays a crucial role in modulating the virulence of the pathogens as well as reducing the host

defence (Martinez and Baquero, 2002; Rahal et al, 2014a) and thus increases the susceptibility of

the host towards various pathogens. A pathogenic agent can enter the animal body via, various

possible routes of exposure (Kumar et al, 2011;Dhama et al, 2014; Verma et al 2014a) but the

immune system of host can certainly phagocytise the pathogen (e.g by secreting chemical

factors) and thus checks the disease progress. Moreover, alterations in the microenvironment

such as abrasions, wounds, malnutrition, pathophysiological conditions may further facilitate the

disease development (Madenspacher et al, 2013).

For development of a disease, immune status of the human population is an important issue. A

susceptible host population can favour even a mild pathogen to produce an epidemic form of

disease while immunocompetent population can resist even highly pathogenic microbial strains.

The immune status of the animals depends on a number of environmental variables for its

fluctuations (Rahal et al, 2014a). animals with immunocompromised lungs usually show higher

incedences of pathophysiology due to leukotoxins and lipopolysaccharide exposures

6
(Panayotova-Pencheva and Alexandrov, 2010). Both these toxins act as chemotactic factors for

inflammatory cells and promote inflammation and severe damage in the lung tissue. In immature

animals, such outbreaks may take an acute form but with high mortality rates. Both these

mechanisms of initiation of inflammation as well as cellular damage are pharmacologically

understood and can be counteracted with the use of phytoconstituents of herbs and plants (Rahal

et al, 2009, 2014).

Even today, due to high cost of effective antibiotics and predicament of antibiotic resistance

microbial strains worldwide, about 60-85% of the population of developing world relies either on

herbal or on indigenous forms of Complementary and Alternative Medicine (CAM) medicines

for their various general health related issues and countering several disease/disorders (Kochnar,

1981; Okeke et al, 1999; Ernst, 2000; Archana et al ). Herbal plants have been used as a source

of valuable medication in virtually all cultures worldwide due to presence of important

antimicrobial principles, immunomodulatory activities, maintenance of general health, precious

therapeutic properties and healing potentials; thus ensure prevention and cure for several diseases

and disorders of humans and animals (Baquar, 1995; Rios and Receo, 2005; Mahima et al, 2012;

Rahal et al, 2014b). The healing properties of these medicinal plants and herbs were well

accepted by our ancestors and nowadays also being scientifically proven as well (Sharma et al,

2014). Extracts or phytoconstituents derived from various parts of disease provide therapeutic

modalities with broad spectrum antimicrobial activities against various pathogenic micoorganims

(Suck, 1989; Khan et al, 2006; Kumar et tal, 2013). In last few decades, there has been and

upsurge in demand and delivery of various herbal products for multifaceted health benefits. In

first world country like the United States, herbal remedies still continue as dietary supplements.

A variety of herbal therapies have stood the test of time because of their efficacy and potency in

7
the treatment of various bacterial infections with a few having scientific evidences of significant

usefulness (Blumenthal et al, 1998; Bisset and Wicht, 2001, Mahima et al, 2012; tiwaari et al

2012; Rahal et al, 2014).

Currently, the focus on herbal therapy is not only in third world developing countries but also in

developed countries. The studies regarding regarding their assessment of risks and benefits,

identifying pharmacologically active components/principles and biological activities; scientific

validation and revealing ethnomedical values are going on worldwide with regards to thosands of

herbs and their extracts, preparations and products which altogether would play vital role in

perpetuating, propagating, popularizing and promoting the wider usages of drugs/medications

based on herbs. Active research on various aspects of herbs is being carried out in Nigeria,

Rwanda, Maltese Islands, India, Cuba, Eastern part of east, Mediterranean and other countries

(Mahima et al, 2012; Odebiyi and Sofowora, 1978).

Medicinal plants and herbs, the ancient period magical chemotherapeutic drugs and natural

treasure to prevent or cure various diseases and ailments are very interesting and play crucial role

in safeguarding various health related issues (Famsworth et al, 1985; Ernst, 2000; Tiwari et al.,

2012,). Nowadays, herbal remedies, are also getting popularing in providing a viable and safer

alternative to prevent and treat cancers (Wargovich et al, 2001, Yarney et al, 2013). Being a

valuable treasure of natural medicines, these are gainging much attention with their demand

increasing day by day; owing to having several advantages over other medicinal systems

including in of allopathay. These possess multi-dimensional health benefits and show high utility

in alternative and complementary medicinal systems as effective prophylactic and therapeutic

regimens. Herbs also help in maintenance and boosting of general health condition, serve as

general tonics and have wider practical applications at global level. Though variations in

8
morphology, phytocontituents, active principles and genetic make occur but altogether the

wonder world of herbs forms a bunch of safer (show lesser or no side effects), cheaper/cost-

effective and easily available nature’s medicine (Dhar et al, 2006; Kumar et al, 2007; Mahima et

al., 2012). Such valuable therapeutic alternatives need to be explored fully, tested, standardized,

validated and finally be used optimally for the cause of mankind.

2.2 Salmonella typhi

Salmonella typhi is a Gram-negative bacterium that causes typhoid fever, a life-threatening

disease that affects millions of people worldwide, especially in developing countries. The

increasing incidence of antibiotic-resistant of Salmonella typhi has made the research for

alternative antimicrobial agents an urgent public health concern. Typhoid fever (enteric fever)

caused by Salmonella typhi is an endemic disease in the tropic and sub-tropic and has become a

major public health problem in developing countries of the world with an estimated annual

incidence of 540 per 100,000. The annual incidence of typhoid is estimated to be about 17

million cases worldwide (WHO, 2008). It is often encountered in tropical countries including

Nigeria where they constitute serious sources of morbidities and mortalities (Ibekwe et al.,

2008). Typhoid and paratyphoid fevers are infections caused by bacteria, which are transmitted

from faeces to ingestion. Clean water, hygiene and good sanitation prevent the spread of typhoid

and paratyphoid. Contaminated water is one of the pathways of transmission of the disease

(WHO, 2008). Typhoid and paratyphoid fevers are caused by the bacteria Salmonella typhi and

Salmonella paratyphi, respectively. Salmonella typhi is Gram-negative bacteria, which are

motile, though non-flagellate variants, occur. Capsules are not formed. They are intestinal

pathogens, which comprises of a species Salmonella typhi, which causes an enteric fever known

9
as typhoid fever (Philip, 2000). It is pathogenic to both man and mammals with associable

inflammatory reaction in the intestinal tract.

Typhoid fever is among the water-borne infections (Singh and Mcfeters, 1992) characteristic of

environments with poor sanitation and hygiene. It is a health problem that has been associated

with development (Jegathesan, 1984). Human infection with Samonella is mainly by the oral

route through ingestion of faecal contaminated food and water, unclean hands, flies and meat

from infected animals (Carol et al., 1989). Typhoid and paratyphoid germs are passed in the

faeces and urine of infected people. People become infected after eating food or drinking

beverages that have been handled by a person who is infected or by drinking water that has been

contaminated by sewage containing the bacteria. Once the bacteria enter the person’s body they

multiply and spread from the intestines, into the bloodstream (WHO, 2008). Even after recovery

from typhoid or paratyphoid, a small number of individuals (called carriers) continue to carry the

bacteria. These people can be a source of infection for others. The transmission of typhoid and

paratyphoid in less-industrialized countries may be due to contaminated food or water. In some

countries, shellfish taken from sewage-contaminated beds is an important route of infection.

Where water quality is high, and chlorinated water piped into the house is widely available,

transmission is more likely to occur via food contaminated by carriers handling food (WHO,

2008). Infection through contaminated surgical equipment and person-to-person contact in

hospital has also been reported (Carol et al., 1989). Salmonella typhi have somatic antigens and

glycolipid microcapsule the vi or virulence antigen. Phage typing can distinguish different strains

of the organism. Enteric fever caused by Salmonella typhi is often encountered in tropical

countries including Nigeria where they constitute serious sources of morbidities and mortalities

10
(Baver, 1995). It is a major public health problem in the developing countries of the world with

an estimated annual incidence of 540 per 100,000 (Ibekwe et al., 2008).

2.3 Staphylococcus aureus

The Gram-positive bacterium Staphylococcus aureus is considered a commensal bacterium

because roughly one third of the human population is colonized by it without developing disease

(1). Colonization occurs in the human nose, whereby the host nasal microbiota plays a major role

in promoting or inhibiting S. aureus colonization (2). Despite the fact that S. aureus is considered

a commensal, nasal carriage of S. aureus is linked to bacteremia (3). The bacterium may cause a

range of infections, from cellulitis and superficial skin disease to abscesses, bacteremia, sepsis,

endocarditis, and pneumonia (4). Moreover, S. aureus has been shown to adapt in its interaction

with humans by increasing resistance against methicillin and is currently a leading cause of

human bacterial disease worldwide. Methicillin-resistant S. aureus (MRSA) was identified in the

1960s as a nosocomial pathogen, when hospitalized patients showed distinct risk factors for

acquisition (5). Theprevalence of methicillin resistance among nosocomial S. aureus isolates

increased from 2.1% in 1975 to 35% in 1991 (6). MRSA epidemiology changed in the 1990s

when infections of healthy individuals outside hospitals were reported. These strains, with

increased virulence, were the first reports of community-acquired MRSA (7, 8). Now,

community-acquired MRSA has been reported as the leading cause of bacterial infections in the

bloodstream, skin, soft tissue, and lower respiratory tract in developed countries (9). As a

consequence, research interest in the pathophysiology of S. aureus has increased.

Combating S. aureus with (new) antibiotics is a challenge, since the bacterium has the

extraordinary ability to develop resistance against antibiotics and the discovery of new

11
antibiotics is declining (7). The use of medicinal plant extract like the guava leaf extract is the

strategy that can be used to solve many problems that we encounter with antibiotic resistance.

2.4. Guava leaf

The guava (Psidium guajava L.) tree (Figure 1), belonging to the Myrtaceae family, is a very

unique and traditional plant which is grown due to its diverse medicinal and nutritive properties.

Guava has been grown and utilized as an important fruit in tropical areas like India, Indonesia,

Pakistan, Bangladesh, and South America. Different parts of the guava tree, i.e., roots, leaves,

bark, stem, and fruits, have been employed for treating stomachache, diabetes, diarrhea, and

other health ailments in many countries. Guava leaves (Psidii guajavae folium; GL) are dark

green, elliptical, oval, and characterized by their obtuse-type apex. Guava leaves, along with the

pulp and seeds, are used to treat certain respiratory and gastrointestinal disorders, and to increase

platelets in patients suffering from dengue fever [7]. GLs are also widely used for their

antispasmodic, cough sedative, anti-inflammatory, antidiarrheic, antihypertension, antiobesity,

and antidiabetic properties [8]. Studies on animal models have also established the role of GL

isolates as potent antitumor, anticancer, and cytotoxic agents.

GLs are widely employed for treating diarrhea and digestive ailments, while the fruit pulp is

utilized to enhance the platelet count for treating dengue fever. The potential of guava leaf

extracts for diarrhea treatment was also studied [11,12]. The flavonoids present in guava leaf

extract chiefly determine their antibacterial activity, while quercetin, which is the most

predominant flavonoid of guava leaves, exhibits strong antidiarrheal activities. The antidiarrheal

activity of quercetin is ascribed to its relaxing effect on the intestinal muscle lining which

prevents bowel contractions. Guava leaf polysaccharides (GLPs) can be utilized as an

antioxidant additive in food and for diabetes treatment. The presence of a unique variety of

12
bioactive polyphenolic compounds, like quercetin and other flavonoids, and ferulic, caffeic, and

gallic acids, present in guava leaves primarily determine their bioactive and therapeutic

properties [8,13]. These phenolic compounds are known as secondary metabolites which exhibit

strong antioxidant and immunostimulant activities. This review aims to discuss the various

nutritional and bioactive compounds present in guava leaves and decipher the molecular basis of

their pharmacological and medicinal properties concerning human health, nutrition, and as

complementary medicine.

GLs are widely employed for treating diarrhea and digestive ailments, while the fruit pulp is

utilized to enhance the platelet count for treating dengue fever. The potential of guava leaf

extracts for diarrhea treatment was also studied [11,12]. The flavonoids present in guava leaf

extract chiefly determine their antibacterial activity, while quercetin, which is the most

predominant flavonoid of guava leaves, exhibits strong antidiarrheal activities. The antidiarrheal

activity of quercetin is ascribed to its relaxing effect on the intestinal muscle lining which

prevents bowel contractions. Guava leaf polysaccharides (GLPs) can be utilized as an

antioxidant additive in food and for diabetes treatment. The presence of a unique variety of

bioactive polyphenolic compounds, like quercetin and other flavonoids, and ferulic, caffeic, and

gallic acids, presenting uavaleaves primarily determine their bioactive and therapeutic properties

[8,13]. These phenolic compounds are known as secondary metabolites which exhibit strong

antioxidant and immunostimulant activities. This review aims to discuss the various nutritional

and bioactive compounds present in guava leaves and decipher the molecular basis of their

pharmacological and medicinal properties concerning human health, nutrition, and as

complementary medicine.

2.4 Chemical Composition

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2.4.1 Proteins

Guava leaves contains 9.73% protein on a dry weight basis [22]. Proteins are large biomolecules

composed of amino acids and act as building blocks of cells. Proteins play a major role in growth

and maintenance, enzyme regulation, and cell signaling, and also as biocatalysts [24]. Recently,

plant-based nutrients have gained potential because of the high demand for nutritionally rich

food, particularly protein. A great effort is now being made to find highly sustainable

nutritionally rich food sources [25]. Thomas et al. [23] reported 16.8 mg protein/100g and 8 mg

amino acids/100g in guava leaves as estimated according to Lowry’s and ninhydrin methods,

respectively. Jassal et al. [21] reported that guava leaves can be utilized as a novel and

sustainable dietary source as they are a rich source of proteins, carbohydrates, and dietary fibers.

2.4.2. Minerals and Vitamins

Guava leaves are the rich source of minerals, such as calcium, potassium, sulfur, sodium, iron,

boron, magnesium, manganese, and vitamins C and B. The higher concentrations of Mg, Na, S,

Mn, and B in GLs makes them a highly suitable choice for human nutrition and also as an animal

feed to prevent micronutrient deficiency [26]. Thomas et al. [23] reported the concentration of

minerals such as Ca, P, K, Fe, and Mg as 1660, 360, 1602, 13.50, and 440 mg per 100g of guava

leaf dry weight (DW), respectively. The concentration of vitamins C and B was 103.0 and 14.80

mg per 100g DW, respectively. Consumption of Ca- and P-rich GLs reduces the risk of

deficiency-related diseases like hypocalcemia, hypophosphatemia, and osteoporosis. The study

also reported that the concentration of

Ca,P,Mg,Fe,andvitaminBinGLswashigherthanthatinguavafruit. Thehighervitamin C content in

GLs may help in improving the immune system and maintain the health of blood vessels,

14
whereas vitamin B plays an important role in improving blood circulation, nerve relaxation, and

cognitive function stimulation.

2.5 Phytochemical Profile

2.5.1 Essential Oil Profile

GLs are a rich source of essential oils (Table 2). The major constituent of GL essential oil

includes 1,8-cineole and trans-caryophyllene [27]. Chen et al. [8] identified 50 compounds in GL

essential oil using gas chromatography (GC) and gas chromatography/mass spectrometry (GC–

MS), where they found β-caryophyllene, α-pinene, and 1,8-cineole to be the major ones. GL

essential oil from the Philippines was found to contain a different profile, with limonene, α-

pinene, β-caryophyllene, and longicyclene as major compounds [28]. Ecuadorian GL essential

oil contained a higher content of monoterpenes (limonene and α-pinene) whereas Tunisian guava

leaf oil displayed a higher content of veridiflorol and trans-caryophyllene [29,30]. Soliman et al.

[31] reported a larger amount of monoterpenes, contrary to the other studies, where

sesquiterpenes constituted the major compound in GL essential oil. El-Ahmady et al. [32]

reported 4 α-selin-7(11)-enol, α-selinene, β-caryophyllene, and β-caryophyllene oxide as the

major constituents of GL essential oil. In another study, sixty-four different compounds were

determined inessential oil extracted from GLs by gas chromatography–mass spectrometry (GC–

MS). Among them, caryophyllene (24.97%) was found to be predominantly present, which acts

as an antioxidant, anticancer, anti-inflammatory, and antimicrobial agent [21].

2.5.2 Phenolic Compounds

Guava leaves (GLs) are widely popular as a traditional source of medicine in Asian countries

due to their antihyperglycemic effect. As mentioned in the previous sections, they contain

superior quality bioactive polysaccharides, proteins, lipids, essential oils, vitamins, and minerals.

15
The various secondary metabolites present in GLs include phenolic acids, flavonoids,

triterpenoids, sesquiterpenes, glycosides, alkaloids, and saponins. Phenolic compounds (PCs)

serve as key bioactive compounds which provide antioxidant and hypoglycemic properties to

GLs. Generally, these PCs play a major role in managing various metabolic and physiological

activities in the human body.

2.6 Biological activities of Guava Leaf

2.6.1 Anticancer/ Antitumor Activity

Terpenoids and flavonoids present in GLs exhibit antitumor effects by regulating the immune

system, suppression of signal transfer and tumor cell adhesion, and an impediment to tumor

angiogenesis and cell proliferation [48]. Studies

suggestthattheseleavesexhibitapotentinhibitoryeffectagainstcancercelllineslikeMDAMB-231 and

Michigan Cancer Foundation-7 (MCF-7) for breast cancer, Henrietta Lacks (HeLa) for cervical

cancer, KB for nasopharyngeal cancer, LNCaP, DU 145, and prostate cancer-3 (PC-3) for

prostate cancer, and colorectal 320 double minutes (COLO320DM) for colon cancer [49].

2.6.2 Antidiabetic Activity

GLs have been widely used as ethno medicine for diabetes management [15]. Flavonoids and

polysaccharides of GLs have been reported for their antidiabetic potential in several studies.

Guaijaverin and avicularin flavonoids of GL extract were associated with

significantimprovementinthefunctionof β-cellsofpancreaticisletsandhepatocytemorphology in

diabetic mice [57]. Guaijaverin suppressed the activity of the blood glucose homeostasis enzyme

dipeptidyl-peptidase IV [58], while avicularin inhibited intracellular lipid aggregation by

impeding glucose uptake through GLUT-4 in vitro and revealed no distinct toxicity for 3T3-L1

16
adipose cells [59]. Luo et al. [14] extracted GL polysaccharides (GLPs) and further tested the

antidiabetic effects on streptozotocin-induced diabetic mice in combination with a high-fat diet.

2.6.3. Antidiarrhea Activity

Guava leaves (GLs) are considered to possess antidiarrheal properties, as reported

bymanyresearchers. Mazumdaretal.[12]reportedtheantidiarrhealpotentialofethanolic isolates of

GLs in Wistar rats. The authors reported that a dosage level of extracts at a concentration of 750

and 500 mg/kg had antidiarrheal potential in castor oil-fed rats. Besides this, Ojewole et al. [68]

reported similar activity using aqueous extracts of GLs in rodents. They reported that GL

extracts at doses of 52–410 mg/kg when administrated orally were found to combat diarrhea, and

also resulted in reduced intestinal transit and dilatory removal of unwanted gastric products.

2.6.4 Antimicrobial Activity

The incidence of systemic microbial infections such as septicemia, urinary tract infections,

meningitis, pneumonia, and gastritis affects the Foods 2021, 10, 752 11 of 20

entire human body and contributes significantly to global mortality. Food-borne diseases are

mostly caused by pathogens including Staphylococcus, Shigella, Salmonella, Bacillus,

Escherichiacoli, Clostridium, and Pseudomonas [69]. Guava Leaves (GLs), owing to the

presence of different organic and inorganic anti inflammatory compounds, are known to

possess anti microbial properties[71]. Guaessential oils display strong antimicrobial properties

against Pseudomonas aeruginosa, Escherichia coli, Streptococcus faecalis, Staphylococcus

aureus, and Bacillus subtilis [31].

2.7 Antibacterial mechanism of medicinal plants and herbs

17
The antibacterial activities of herbs are multi fold and depend upon the phytoconstiyuents,

concentration of phytoconstituents/phytochemicals and bioactive principles and their synergistic

as well as antagonistic actions (Archana et al, 2011, Mizaei-Aghsaghali, 2012; Mahima et al

2012, Midraullah et al, 2014). These phytochemicals include flavonoids, steroids, beta carotene,

anthocyanins, antraquinolones, glycosides, coumarins, alkaloids, saponins, tannins, phenolic

acds, gallics acid and others (Kwon et al., 2011, Sharma et al, 2014). Various mechanisms of

action and antibacterial abilities of herbal remedies relies on their several beneficial properties

via, immunomodulatory nature wiyh enhancement of both humoral and cell mediated immunity

as well ameliorating stress and immunosuppression, cytokine regulation, anti- inflammatory,

antioxidative and inhibiting oathogens by a variety of pathways (Archana et al, 2011, Mixaei-

Aghsaghali, 2012, Mahima et al, 2011, Rahal et al, 2014). Many herbs have anti- oxidative

potemtial by counteracting free radicals or metabolites produced during various biochemical

pathways (Barreto et al, 2007, Rufino et al, 2009, Rahal et al 2014c). A vast number of herbal

plants show anti- inflammatort action by selective inhibition of cyclooxygenase-2, 5-

lipoxygenase, glutathione S-tranferase and thus inhibiting the prostaglandin biosynthesis. Several

herbs decreases the production of infammatorty agents ( histamine, bradykinin, serotonin),

enhanceactivity of cortisol and augments blood circulation which help remove bacterial toxins

out of the body (Nahrstedt et al, 2006, Rahal et al, 2014c). Certain plants contain vitamin C and

carotenoids which have been found to augment both humoral and cell-mediated immune

infection , thereby reducing risk of infections by enhancing the antigenic surveillance of immune

the system (Bendich, 1989, Henson et al, 1991, Mahima et al, 2012). Immunomodulation of

most herbal extracts are governed by cytokine regulation as well as anti- inflammatory properties

(Wildeuer and Mayerhofer, 1994, Kumar et al, 2013c, Sharma et al, 2014). Certain plants

18
facilitate proliferationof CD4 T- helper and B type immune cells. Others have been found

beneficial in blocking bacterial adherence to different body cells thus help preventing the

infection. Steroidal alkaloids and steroidal lactones present in herbs enhance engulfing activity of

macrophages, increases the activity of white blood cells and other immune cells which in turn

enhances their antimicrobial potentials (Gunnung, 1999, Govindarajan et al, 2005; owais et al,

2005, tiwari et al, 2014d).

Alternative /traditional medicines are becoming progressively more popular and worth to

overcome many multi-resistant organisms like as seen with potent efficacy of herbal extracts

against multi-drug resistant Acinetobacter baumanni (Miyadaki et tal, 2019, Sharma et al, 2014).

Such activities of plant extracts are mainly observed due to the presence of saponin, tannin,

alkaloids anthraquinolone and cardiac glycosides.

2.8 Determination of antimicrobial susceptibility

According to studies carried by Adikwu , Oyiwona et al, 2022 shows that the aqueous and

methonalic extraction of the guava leaf extract produce different zone of inhibition at various

concentration ranging from 50 to 200mg/ml on an agar well diffusion plate in which 6mm

diameter hole bore on the plate after spreading it with our bacterial of interest and a 0.1ml of

different concentration of the extract was put in each whole on the plate and was allow to

incubate for one day at a temperature of 37 degree census. The following result where obtained

show that staphylococcus aureus produces a zone of inhibition that ranges from 10 mm to 11.9

mm while S . typhi 8.6 mm to 10.4 mm with aqueous extraction and 8.8 mm to 13.1 mm for S.

typhi while 10.9 mm to 19.0 mm for S. aureus with methanolic extract.

19
2.9 Determination of the MIC/MCB (mg/ml) of guava leaf extract

Their result further shows that the MIC/MCB aqeous concentration of S. aureus and S. typhi

where 6.25/12.5 and 12.5/25.0 and for methanolic extract it was 3.13/6.25 and 6.25/12.50

respectively. it was also observed that S. aureus produces a zone of inhibition of 21.7mm when

treated with 100mg/ml of amoxicillin while S. typhi produce a zone of inhibition 15.0 mm when

expose to the same concentration of Amox.

CHAPTER THREE

3.0 Introduction

this chapter presents the methodology used to evaluate the antimicrobial properties of guava leaf

extract on S. aureus and S. typhi. The chapter describes the experimental design, study area,

material used , procedures employed, and analysis methods.

3.1 Research Design

The research design utilized in this study was experimental. The study involved testing the

effects of different concentration of guava leaf extract on the growth of S. aureus and S. typhi.

The concentrated tested were 50, 100, and 200mg/ml.

3.2 Study Area

20
This research was carried out in the Biaka university Teaching laboratory which is found in

Buea, Fako division in the South West Region.

3.3 Materials and methods

3.3.1 Study Period

This study started from the 24 of October and will come to its completion on the 18 of march

2024.

3.3.2 Study Population

The test organisms for the study; pure isolates of Salmonella typhi and Staphylococcus aureus

were obtained from stool and mucous samples of patients attending Saint Veronica health centres

in Bokoko Buea. The isolates were confirmed at the Microbiology Laboratory using appropriate

diagnosistic test strip and stored in refrigerator at 4˚C.

3.3.3 Procedure:

Collection of Plant Materials

Leaf of Psidium guajava were collected from residential areas in Ndongo

Preparation of Extracts

Aqueous and methanol extracts of Psidium guajava leaves were prepared separately. The leaves

were washed in water and air dried on the desk in the laboratory for two weeks. They were

frequently turned to achieve uniform dryness. The dried leaves were then crushed and pulverized

by grinding using local mortar and pestle. 50 g powder of the leaves was soaked in 500 ml each

of distilled water and methanol respectively. The flasks were kept at room temperature for 3 days

with intermittent shaking. Filtration was done using Whatman filter paper. The extracts were

evaporated at 70˚C in water bath with shaker until dried extract samples were obtained. The pure

21
ethanolic extracts was consider to be 100% concentration and it was diluted into various

concentration of 75%, 50% and 25%

3.3.4 Preparation of the Agar culture media

S. aureus was selectively culture on mannitol salt agar (MSA) which contains high salt

concentration (7.5 – 10%) that inhibit the growth of most other bacteria, except for

staphylococcus aureus which tolerate it growth appearing yellow or golden color due to

fermentation of sugar mannitol in the MSA mediumand was further subculture in the same

culture media while S. typhi was selectively culture on MacConkey agar which contains bile salts

and crystal violet, which inhibit the growth of Gram – negative bacteria appearing pink in color

and was still subculture using the culture media

Preparation of the mannitol/ MacConkey agar

 111g of Mannitol and MacConkey agar powder was weight seperately using a sensitive

electronic scale balance

 Suspend 111g of Mannitol and MacConkey agar separately in 1000ml of distilled water

in the conical flask

 Boil the mixture to dissolve the medium completely

 Sterilize the mixture by autoclaving at 121 degree census for 15 minute.

 After sterilization remove the conical flask and pour the liquid into a well label petri- dish

and wait for both medium to solidify. Be sure that you are preparing the agar in a clean

enviroment to prevent ant contaminaton.

 Store two of each solidified medium in the incubator for 24h to check for any

contamination and stored the rest in the refrigerator.

22
3.3.5 Culture and antimicrobial susceptibility testing:

The following steps outline the culturing of S. aureus and S. typhi using their respective culture

media which has been solidified in the petri – dish.

 All materials and equipment used for culturing were Sterilize with a Bunsen bunner

 The isolated sample collected was dissolved in 2mls sterile distilled water in the test tube to

ensure the even distribution of the bacteria on the surface of the plate and to prevent

clumping of the bacterial.

 A sterile swap was gently deep into the dissolve dissolved sample in the test tube and excess

liquid was press against the walls of the test tube and it was inoculated on the patric dish ner

a flame to prevent any contamination.

 The strike plate was closed and incubated for 48hrs at a temperature of 37 degree census.

 After the incubation period the plates where observed for the presence of black colonies

which indicated the succefull growth of S. typhi while S. aureus appear as a yellow colonies

 Wells were created on the fresh agar plates using a sterile cork borer.

 A sterile loop was used to transfer a small amount of the bacterial from the agar plater into

fresh agar plate

 Each well was filled with 100 μl of the guava leaf extract at the desired concentration.

 The plates were incubated at 37°C for 24 hours.

 The diameter of the zones of inhibition was measured using a rular.

3.9 Data Analysis:

The data obtained from the antimicrobial susceptibility testing was analyzed using descriptive

statistics. The mean and standard deviation of the zone of inhibition were calculated for each

23
concentration of guava leaf extract. A comparative analysis was conducted to determine the

differences in antimicrobial activity between the different concentrations and bacterial strains.

3.10 Ethical Considerations:

Prior approval for this research was obtained from the relevant ethics committee. All laboratory

experiments were conducted following standard biosecurity and safety protocols to ensure the

safe handling of microorganisms.

CHAPTER FOUR

RESULTS

4.1 Sensitivity of guava leave extract on S. typhi and S. aureus

The antibacterial activity of ethanolic extract of guava leaves were evaluated against

staphylococcus aureus and salmonella typhi was evaluated using agar well diffusion method

different concentration of 100%, 75%, 50% and 25% and results of their zone of inhibition was

measured in millimeter suing a ruler. Results obtained from the experiment shows that the bac

24
terial where sensitive at all concentration of ethanolic extract of the guava leave. These results

are presented on table one and two below.

Table.1 Zone of inhibition S. aureus at different concentrations using agar well diffusion method

Staphylococcus aureus

S. No. CONCENTRATION (v/v) ZONE OF INHIBITIONS

(%) (mm)

1. 100% 19

2. 75% 17

3. 50% 15

4. 25% 9

Table 2: Zone of inhibition S. typhi at different concentrations using agar well diffusion method

Salmonella typhi

S. No. CONCENTRATION (v/v) ZONE OF INHIBITIONS

(%) (mm)

1. 100% 1.7

2. 75% 1.4

3. 50% 1.0

25
 4. 25% 0.0

9mm
4.1.1 The inhibitory effect of guava extracts on S. aureus using well diffusion

method
16mm
The sensitivity of S. aureus gradually increased with the increment of concentration of extract.

The highest inhibitory effect of ethanolic guava leave extract was observed at a concentration of

100% and 75% with an inhibitory zone of 2.8mm and 2.0mm respectively, moderate response

was observed of at a concentration of 50% which produce a zone of inhibition of 1.5mm while

the lowest concentration was observed at a concentration of 50% which produce an inhibitory

zone of 0.9mm. this is shown in table one and figure one

19mm

17mm

26
Fig 1: Zone of Inhibition S. aureus using agar at different concentrations well diffusion
15mm
method

4.1.2 4.1.1 The inhibitory effect of guava extracts on S. typhi using well
10mm
diffusion method

The sensitivity of S. aureus gradually increased with the increment of concentration of extract.
18mm
The highest inhibitory effect of ethanolic guava leave extract was observed at a concentration of

100% and 75% with an inhibitory zone of 1.7 mm and 1.4 mm respectively, moderate response

was observed of at a concentration of 50% which produce a zone of inhibition of 1.0mm while

the lowest concentration was observed at a concentration of 50% which produce an inhibitory

zone of 0.7mm. this is shown in table two and figure two

Fig 2: Zone of Inhibition S. typhi using agar at different concentrations well diffusion

method

The line graph below is an illustration of the level of sensitivity shown by S. aureus and S. typhi

on various concentration of guava extracts.

27
 ZONE OF INHIBITION (MM)
zo n e o f in h ib itio n S . ty p h i a n d S . a u ru s a t d ifferen t
eth a n o lic g u a v a ex tra ct
20
19
18 18
17
16 16
15
14
12
10 10
9
8
6
4
2
0 0
100 75 50 25

CONCENTRATION OF GUAVA LEAVE EXTRACT (V/V)

S. aureus S. typhi

Figure 3: An illustration of different zone of inhibition produce by different

guava concentration

4.2 MIC and MBC of guava ethanolic leaf extract against S. aureus and S.

typhi

The minimum inhibitory concentration (MIC) was determined for the ethanolic extract of guava

leaves against S.aureus and S. typhi by measuring the zone of inhibition. The MIC represents the

lowest concentration of the extract that completely inhibits bacterial growth. result obtained

shows that the ethanolic extract of guava leaves demonstrated a bacteriostatic effect against S.

aureus and S. typhi, as it inhibited bacterial growth without killing but as the concentration

increases the bacteriocidal effect was observed at a concentration of 50% for staphylococcus

aureus this is shown in figure three and four below. The result of the minimum inhibitory

concentration are display or summaries on table three below alongside with minimum

28
bacteriostatic and minimum bacteriocidal concentration of guava leaves extract on S. aureus and

S. typhi.

Table 3: MIC and MBC of guava ethanolic leaf extract against S. aureus and S. typhi

Guava leave MIC (V/V) Minimum Minimum

ethanolic extract Bacteriostatic bactericidal

concentration concentration concentration

S. aureus 25% 25% 50%

S. typhi 50% 50% NL

we will surely go back in the lab to conducted the bactericidal and

bacteriostatic effect since we can not provide proof on it. What do you
think

4.3 Combined effect of amoxicillin and guava leaves ethanolic extract on S.

aureus and S. typhi

In pharmacology, drug synergism happens when the effects of two or more different kinds of

drugs are amplified when they are administered jointly. The combine action of guava leave

extract and amoxicillin at the same concentration of 100% produces an inhibitory zone of 30mm

as compare to their individual effect of 10mm and 19mm respectively when there where tested

on staphylococcus aureus while the combine action of guava leave extract and amoxicillin at the

29
 Amox + guava Amox
Amox + guava
Amox: extract:
extract:

same concentration of 100% produces an

inhibitory zone of 31.5mm as compare to their

individual effect of 12.5mm and 18mm

respectively when there were tested on S. typhi.

These results are shown on table five and figure 5

and six below. Were diagram A represent the

combine effect of Amoxicillin and ethanolic guava leaves extract on S. aureus and diagram B

represent the combined effect off Amoxicillin and ethanolic guava leaves extract on S. typhi

Table five: The Combined effect of amoxicillin and guava leaves ethanolic extract on S.

aureus and S. typhi

(% v/v) Concentration S. aureus S. typhi

Amoxicillin (Drug A) 10mm 12.5mm

Guava extract (Drug B) 19 18mm

Combine effect (Drug A + B) 30mm 31.5mm

30
 Fig 4: The Combined effect of amoxicillin and guava leaves ethanolic extract

on S. aureus and S. typhi

ZONE OF INHIBITION (MM)

combine
Figure Figure B
A effect of Amoxicillin and ethanolic guava extract
35
30 31.5
30
25
20
19 18
15
10 12.5
10
5
0
DRUG A DRUG B DRUG A+B

DRUG CONCENTRATION (V/V)

S. aureus S. typhi

Figure 5: synergistic effect of Amox and ethanolic guava extract

CHAPTER FIVE

DISCUSSION, CONCLUSSION AND RECOMMENDATIONS

5.1 DISCUSSION

Result found in this study were supported and / or opposed in the data reported in the literature review in

chapter two. Nascimento et al., 2000 conducted a study which supported the finding of the present study

31
in which the guava leaves extracts was effective against staphylococcus aureus but contracted the results

obtained for this study. These results will be further discuss below.

5.1.1 Sensitivity of guava leave extract on S. typhi and S. aureus

According to studies conducted by Evbuomwan et al. (2013), the ethanolic extract of guava leaves

exhibited sensitivity to Staphylococcus aureus at concentrations ranging from 25% to 100%. The

inhibitory zone observed varied between 7mm and 14 mm. However, the extract was not

sensitive at concentrations below 25%. In contrast, a study by Bipul et al. (2013) found that

Salmonella typhi was not sensitive to the ethanolic extract of guava leaves at any concentration.

Interestingly, the results obtained for the sensitivity of guava leaf extract on Staphylococcus

aureus showed a greater inhibitory effect, with a range of 9 mm to 28 mm, compared to the

findings by Evbuomwan et al. (2013) at the same concentration. These variations could be

attributed to factors such as cultivation methods, geographical location, and the specific

secondary metabolites responsible for the antimicrobial activity of the guava plant. Additionally,

the volume of the extract used for susceptibility testing may also play a role in these differences

while results obtained from these studies shows that ethanolic extract of the guava leaves was

sensitive to salmonella typhi at a concentration of 50% to 100% which contracted results

obtained by Bipul et al. (2013), the variation in the results is because of the difference in the

microbial resistance level in the US as compare to others countries like Cameroon.

5.1.2 MIC and MBC of guava ethanolic leaf extract against S. aureus and S.

typhi

In the study of evaluating the ethanolic extract of guava leaves against Staphylococcus aureus, the

following results were obtained: The minimum inhibitory concentration (MIC) was observed at a

32
concentration of 25%of the ethanolic extract. The minimum bactericidal concentration (MBC) was

observed at a concentration of 50%. Interestingly, the ethanolic extract of guava exhibited a

**bacteriostatic effect at the same concentration of 25%. These findings align with a study by

Evbuomwan et al. (2013), which also reported a MIC at 25% and a minimum bacteriostatic concentration

at 50%. While result obtained on the evaluating the ethanolic extract of guava leaves against Salmonella

typhi completely contract results that were obtained by Bipul et al. (2013) which showed no effect of the

ethanolic guava leave extract on Salmonella typhi

5.1.3 Combined effect of amoxicillin and guava leaves ethanolic extract on S.

aureus and S. typhi

When guava leaf extract and amoxicillin were combined at a concentration of 100%, the

following inhibitory zones were observed: Staphylococcus aureus: Combined action: 3.5mm

Individual effects: Guava leaf extract (2 mm), Amoxicillin (2.8 mm) S. typhi: Combined action:

2.4 mm Individual effects: Guava leaf extract (1 mm), Amoxicillin (1.7 mm) this can be because

of the numerous mechanism purses by the guava leaves extract such as disruption of cell

membrane, enzyme inhibition, oxidative stress, cell wall alteration and quorum sensing

interference as compare to one mechanism of action purpose by amoxicillin which is inhibition

of cell wall synthesis. It's important to note that no past studies have been conducted to validate

this claim. Further research is needed to explore the antimicrobial properties of guava leaf extract

and its combination with amoxicillin.

5.2 CONCLUSSION

The ethanolic leave extract was susceptible to staphylococcus aureus and salmonella typhi with a

bactericidal effect and bacteriostatic effect respectively. Thus the results of this studies support

the traditional use of guava leave extract for the treatment of various infections caused by

33
Staphylococcus aureus and Salmonella typhi. In addition its concentration – dependent effects,

synergy with amoxicillin and potential clinical application make it a subject of interest for

researchers and healthcare practitioners.

5.3 RECOMMENDATION

 Further studies should Conducted on clinical trial to evaluate the safety, dosage and patient

outcomes.

 Promoting awareness among healthcare professionals about natural antimicrobials like guava

leaves extract

 Warn the population about antimicrobial resistance

34
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