Chapter 2

Review of Literature

This chapter of the study addresses previous related studies that are relevant to the topic. It

provides literature about the Hibiscus rosa-sinensis flower extract antibacterial activity. The synergistic

effect of herbal extracts with different antibiotics and its significance in fighting antimicrobial resistance

have also been proven in previous studies, which will be discussed in this chapter. Furthermore,

amoxicillin, the chosen antibiotic to be utilized in this study, and Escherichia coli, the pathogen to be

reacted with, will also be presented.

The studies collected and reviewed are presented under the following headings:

1. Hibiscus flower extract

2. Amoxicillin

3. Antibacterial activity of Hibiscus flower extract against ESBL Escherichia coli

4. Synergistic activity of Hibiscus flower extract against ESBL Escherichia coli

Hibiscus flower extract

Hibiscus rosa-sinensis, one of the malvaceous ornament plants that is commonly grown in the

Philippines. The H. rosa-sinensis has secondary metabolites that are responsible for plant defenses.

Those include compounds such as phenolics, flavonoids, saponin, and quinine (Sobhy and Elaleem,

2017).

Phenolics and Flavonoids

 According to the study of Trung et al. 2020, flavonoids and phenolics found in the leaves and

petals extract of H. rosa-sinensis is suggestive to be involved in affecting varying bacterial structure when

studied against H. pylori. The researcher observed that flavonoids and phenolics reduced the bacterial

membrane fluidity and permeability, inactivates microbial adhesions, disrupts the cell envelope engaged

in protein transport, and interfere with the proton motive force and intracellular acidification which

disturb the H+, K+ - ATPase that is required for the microbial production of ATP. The researchers assessed

the antibacterial effect of specific flavonoids isolated from the H. rosa extract. Protocatechuic acid (PCA)

which inhibits the growth of 15 strains of H. pylori, luteolin (LUT) disrupts the cell wall structure by

increasing the permeability of cytoplasmic membrane causing cell lysis, naringenin (NRG) was found to

inhibit biofilm formation of H. pylori by altering its motility, lastly myricetin (MCT) was reported to have

growth inhibitory and bactericidal effect against the multi-drug resistant B. cepacia.

Saponin

In the study of Lokesh, Manasvi, and Praveena in (2016), saponin compounds from Abutilon

indicum leaves were proven to exhibit an antibacterial and antioxidant activity against gram negative

pathogens such as S. aureus and E. coli. A crude saponin extract has a significant 1.11 mg/ml MIC value

and 2.5 mg/ml antioxidant activity which is similar to a standard antibiotic.

Quinine

A 2020 study conducted by Antika, Triana, Ernawati has proven quinine to be a potential

antimicrobial to both gram-negative and gram-positive strains of bacteria that is due to the lessened

growth of Staphylococcus aureus and Escherichia coli when tested towards quinine.

Amoxicillin
 One of the most often prescribed antibiotics in the setting of primary care is amoxicillin.

Amoxicillin is a penicillin-type antibiotic. It kills bacteria and prevents its growth inside of the body.

Amoxicillin is used to treat a wide range of bacterial infections, including pneumonia, gonorrhea, ear

infections, bladder infections, Salmonella or E. coli contamination (Durbin, 2022). Amoxicillin belongs to

the group of antibiotics known as beta-lactams. Beta-lactam's mechanism of action works by attaching to

penicillin-binding proteins and other high molecular weight penicillin binding proteins through a

competitive mechanism, which prevent the formation of cell walls by inhibiting the transpeptidation

process. This causes the bacterial cell wall's autolytic enzymes to become active. This procedure causes

the cell wall to lyse, which kills the bacterial cell. Bactericidal killing is the term used to describe this kind

of action (Akhavan et al., 2022). Amoxicillin is administered orally through tablets, capsules, and powder

for suspension. The dosage for infections caused by bacteria is, in adults, adolescents, and children

weighing 40 kilograms (kg) or more, should take 250 to 500 milligrams (mg) every eight hours, or 500 to

875 mg every twelve hours. In children and babies older than 3 months of age and weighing less than 40

kg, the doctor will calculate the dosage depending on the body weight. 20 to 40 milligrams (mg) per

kilogram (kg) of body weight per day, split and administered every eight hours, or 25 to 45 mg per kg per

day, divided and administered every twelve hours, is the typical dose. In infants, 3 months and younger,

the doctor will select the dosage based on the body weight (Mayoclinic, 2022).

Antibacterial activity of Hibiscus flower extract against ESBL Escherichia coli

Escherichia coli is from the Enterobacteriaceae family and is a commensal of the gastrointestinal

tract. It is the most prevalent and increasingly involved in broad spectrum of intestinal and extra-

intestinal infections as an opportunistic pathogen (Denamur et al, 2021). E. coli is a fecal indicator

bacterium, and it is commonly to be found in the Filipino households when feces or raw sewage has
entered the water supply (Philippine Statistics Authority [PSA], 2022). It can cause diarrhea, and it can

infect the urinary tract, respiratory tract, and other illnesses (Centers for Disease Control and Prevention

[CDC], 2022). Moreover, presence of this bacterium and its resistance to antibiotics are common in some

areas in Cavite and its nearby cities. A study was conducted in Imus, Cavite where the researchers found

that 1 out of 5 selected street-food sauce samples had a Most Probable Number (MPN) of more than

1100/100 ml indicating high concentrations of gram-negative rods E. coli (Arce et al, 2019). Also,

researchers found that inappropriate disposal of trash 13 has caused negative effects to the quality of

water in Cavite. In the study, it was concluded that the Sunny Brooke Dam located in General Trias,

Cavite contains high fecal coliform which is indicated through the MPN that is greater than 1100 (Acosta

et al, 2019). A study was also done among public markets in Laguna, Philippines. Out of the 19 public

markets, 63% were positive for the presence of E. coli in pork chops. Resistance was also found in

tetracycline (96%), and doxycycline (93%), followed by cephalothin (89%), trimethoprim (89%), ampicillin

(78%), chloramphenicol (70%) and ciprofloxacin (48%). All isolates were resistant that concludes a 100%

multidrug-resistant bacterial population (UPLB Journals, 2021).

A study conducted by Chimurkar, Maraskolhe and Kamble in 2020, states that the components of

H. rosasinensis such as flavonoids, alkaloids, triterpenoids, and tannins has shown antibacterial activity

to pathogens like E. coli, P. aeruginosa, and Salmonella species. In Singh’s study (2019), the antibacterial

and antiviral properties of Hibiscus rosasinensis flower extracts in methanol and ethanol from western

Himalaya's antioxidant activities were investigated. The antibacterial activities of the plant's flower

extracts against common and clinical isolates of pathogenic bacteria were tested using the disc diffusion

method. Maximum antibiotic activity against a standard isolate of Pseudomonas aeruginosa was

observed as 11.4 1.7 mm and 13.60 2.1 mm, respectively, in the methanolic and ethanolic extracts. More

antibacterial activity was displayed by the ethanolic extract than the methanolic extract. As a result, the
therapeutic potential of the antioxidant was assessed by free radical scavenging activity by DPPH (2,2-

diphenyl-1-picryl-hydrazylhydrate) assay with an IC50 (half maximal inhibitory concentration) value of

19.54. Plants may be used in medicine formulation due to their antioxidant and antibacterial properties.

Synergistic activity of Hibiscus flower extract against ESBL Escherichia coli

A prime modern problem now is the growth and evolution of superbugs that is worrisome and

puts the pharmaceutical world in turmoil and panic. According to the World Health Organization,

antibiotics are increasingly becoming ineffective as drug-resistance spreads globally. This growing threat

must be resolved as it makes fighting of common and lethal pathogen a difficult fight to win (WHO,

2021). Herbal products or phytochemicals are emerging as a promising therapeutic strategy against

infectious diseases. The function and mechanism of these phytochemicals can be categorized as

membrane targeting, enzyme inhibition, and biofilm inhibition activities (Singh et al., 2022). Continued

research and application of this mechanism on plants and its parts will provide data that could prove

vital in the race against antimicrobial resistance. Antimicrobial combinations between plant extracts and

antibiotics can enhance the activity of the antimicrobials, lower the concentration of antibiotic agents,

and thus reduce unwanted side-effects. An example of a synergistic plant extract and antibiotic is the

antibacterial activity of leaves and bark extracts of Azadirachta indica in combination of aminoglycosides

and carbapenems against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. The

bark extract combined with amikacin, imipenem, gentamicin shown to have synergistic activity

(Nwanekwu, 2021). An in vitro evaluation was conducted among the antibacterial effects of rose petals

and leaves extract when combined with antibiotics on E. coli. The researcher used different types of

antibiotics such as ampicillin, cefotaxime, cefuroxime, cotrimoxazole, gentamycin, and piperacillin. The

experiments displayed to have higher synergy when combined with rose petal extract than the leaves.
This synergistic effect is associated with the complex effect of the plant extract to extracellular, soluble

protein, and cell wall of bacteria (Chimurkar et al., 2020). The different parts of the Hibiscus rosa-sinensis

have been studied in-depth in many cultures and research has proven the most of its parts have

incredible curing and could assist and synergize with some antibiotics. Some examples of these beneficial

gains and the parts associated with; Hibiscus roots had been tested to treat carbuncles and made into

eye-drop, the Hibiscus flower was also studied to have been able to treat bladder infections and can

even be used as an antimicrobial (Megan, 2020). AL-Oqaili et al. in 2015, performed a study determining

the synergistic effect of H. rosa-sinensis with the antibiotic amoxicillin against 5 pathogenic bacterial

isolates. Antibacterial action was measured using agar-well diffusion and MIC was used to determine its

synergy. The combination of H. rosa-sinensis extract and amoxicillin was recorded to be 33 mm to 62

mm, yielding higher activity when compared to the MIC of amoxicillin and H. rosasinensis extract (10

mm – 27 mm) alone. The flavonoid content of the H. rosa-sinensis flower called naringenin was found to

be able to synergize with antibiotics such as ampicillin, methicillin, tetracycline, and vancomycin against

MRSA (Hieu Tran Trung, et al., 2022). Naringenin and its derivatives have been out to have immense

antimicrobial properties and synergistic effect with antibiotics to fight off MRSA. However, this is poorly

understood and requires further studies (Duda-Madej et al., 2022).

Despite the increasing interest in the antimicrobial properties of plants, there is a discernible

lack of knowledge concerning the direct application of these findings into clinically applicable solutions.

While numerous studies highlight the in vitro efficacy of plant extracts against various pathogens, the

leap from laboratory experiments to practical clinical applications remains minimal. Clinical trials

exploring the effectiveness, safety, and optimal dosages of plant-derived antimicrobials, are lacking.

Bridging this gap is essential for the development of evidence-based therapeutic interventions, ensuring

the successful integration of plant-derived antimicrobials into mainstream clinical practices.

 There is also a lack of knowledge on plants' antimicrobial ability against drug-resistant

pathogens. Most of the research focuses on the broad antibacterial qualities of plants, but less is known

about how these natural components could be effective against bacteria and other microbes that have

developed resistance to prescription medications. To create alternative therapies for infections caused

by drug-resistant organisms and provide optimism in the face of the increasing issues posed by antibiotic

resistance, it is important that this research gap be closed. Researching the processes and effectiveness

of substances produced from plants against drug-resistant strains can open opportunities for the

creation of innovative and long-lasting treatments for resistant infectious illnesses. Such research is

essential for utilizing the full potential of natural compounds in addressing the growing challenges of

antimicrobial resistance and promoting the development of alternative treatments in clinical settings.