Antibacterial Effect of Ethanolic Extract of Guava Leaves
Antibacterial Effect of Ethanolic Extract of Guava Leaves
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).
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 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
have been developed to counter the effect of these pathogens and their resistant strains, with few
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
among many other metabolites, have been found in-vitro with high efficiency antimicrobial
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
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
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
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
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?
The primary objective of this research is to evaluate the antimicrobial properties of ngover leaf
concentration of gauver .
3. To compare the antimicrobial effectiveness of ngover leaf extract with standard antibiotics.
This research aims to contribute to the knowledge of natural alternatives to combat Salmonella
- Identifying ngover leaf extract as a potential antimicrobial agent can provide an alternative
- 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.
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
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,
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
5
CHAPTER TWO
LITERATURE REVIEW
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
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
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
understood and can be counteracted with the use of phytoconstituents of herbs and plants (Rahal
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
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
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
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,
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
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
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
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,
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
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
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
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,
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
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
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
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.
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
and antidiabetic properties [8]. Studies on animal models have also established the role of GL
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
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
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
complementary medicine.
13
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.
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
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
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-
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]
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
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,
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
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
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].
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.
significantimprovementinthefunctionof β-cellsofpancreaticisletsandhepatocytemorphology in
diabetic mice [57]. Guaijaverin suppressed the activity of the blood glucose homeostasis enzyme
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
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.
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
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
17
The antibacterial activities of herbs are multi fold and depend upon the phytoconstiyuents,
2012, Midraullah et al, 2014). These phytochemicals include flavonoids, steroids, beta carotene,
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
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
pathways (Barreto et al, 2007, Rufino et al, 2009, Rahal et al 2014c). A vast number of herbal
lipoxygenase, glutathione S-tranferase and thus inhibiting the prostaglandin biosynthesis. Several
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,
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,
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.
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
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,
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.
20
This research was carried out in the Biaka university Teaching laboratory which is found in
This study started from the 24 of October and will come to its completion on the 18 of march
2024.
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
3.3.3 Procedure:
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
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
111g of Mannitol and MacConkey agar powder was weight seperately using a sensitive
Suspend 111g of Mannitol and MacConkey agar separately in 1000ml of distilled water
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
Store two of each solidified medium in the incubator for 24h to check for any
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
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
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
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
Each well was filled with 100 μl of the guava leaf extract at the desired concentration.
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.
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
CHAPTER FOUR
RESULTS
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
Table.1 Zone of inhibition S. aureus at different concentrations using agar well diffusion method
Staphylococcus aureus
(%) (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
(%) (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
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
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
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
S. aureus S. typhi
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
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:
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.
30
Fig 4: The Combined effect of amoxicillin and guava leaves ethanolic extract
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
S. aureus S. typhi
CHAPTER FIVE
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.
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
obtained by Bipul et al. (2013), the variation in the results is because of the difference in the
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
**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
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
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
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
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
34
Reference
Staphylococcus aureus. Front Cell Infect Microbiol. 2020 Mar 17;10 (March): 1–11.
Chatham-Stephens K, Medalla F, Hughes M, Appiah GD, Aubert RD, Caidi H, et al. Emergence
- United States, 2016-2018. MMWR Morb Mortal Wkly Rep. 2019 Jan 11;68(1):11–3.
Girlich D, Bonnin RA, Dortet L, Naas T. Genetics of Acquired Antibiotic Resistance Genes in
Bhatia R, Narain JP. The growing challenge of antimicrobial resistance in the South-East Asia
https://doi.org/10.4103/0971-5916.73313.
2017;9:1429–34. https://doi.org/10.25258/phyto.v9i11.11187.
https://doi.org/10.1128/mmbr.00016-10.
35
Manandhar S, Luitel S, Dahal RK. In Vitro Antimicrobial Activity of Some Medicinal Plants
https://doi.org/10.1155/2019/1895340.
Djeussi DE, Noumedem JAK, Seukep JA, Fankam AG, Voukeng IK, Tankeo SB, et al.
13-16
Laily N., Kusumaningtyas R.W., Sukarti I., Rini M.R.D.K. The potency of guava Psidium
Chen H.Y., Yen G.C. Antioxidant activity and free radical-scavenging capacity of extracts from
Ashraf A., Sarfraz R.A., Rashid M.A., Mahmood A., Shahid M., Noor N. Chemical
composition, antioxidant, antitumor, anticancer and cytotoxic effects of Psidium guajava leaf
Jiang L., Lu J., Qin Y., Jiang W., Wang Y. Antitumor effect of guava leaves on lung cancer: A
36
Dewi P.S., Sutjiatmo A.B., Nurdiansyah A. Antidiarrheal activity of water extracts of guava
leaves (Psidium guajava L.) and water extracts of green tea leaves (Camellia sinensis L.)
combination in Swiss Webster mice. Acta Pharm. Indones. 2013;38:67–70. [Google Scholar]
Mazumdar S., Akter R., Talukder D. Antidiabetic and antidiarrhoeal effects on ethanolic extract
of Psidium guajava (L.) Bat. leaves in Wister rats. Asian Pac. J. Trop. Biomed. 2015;5:10–14.
Farag R.S., Abdel-Latif M.S., Abd El Baky H.H., Tawfeek L.S. Phytochemical screening and
antioxidant activity of some medicinal plants’ crude juices. Biotechnol. Rep. 2020;28:e00536.
37