Basic Education Department

Senior High School (SHS)

900 San Marcelino St., Ermita Manila,
Philippines
Tel No.: 5242011 loc.203/134

DETERMINATION OF ANTIMICROBIAL ACTIVITY OF EXTRACTED

HIBISCUS ROSA SINENSIS LEAVES

Submitted to:

Mr. Evan John Daynus

Submitted by:

Marc Eugene Belen

Shiela Mae Liwanag

Tracy Abigail Quilang

Jia Anne Sajonia

Christine Samiana

Crishia Belle Valencia

March, 2019
 CHAPTER 1

THE PROBLEM AND ITS BACKGROUND

INTRODUCTION

Countries in South-East Asia Region have had a long history in utilizing of complementary

medicine for treatment and prevention in a particular diseases, especially in remote areas (WHO

South-East Asia, 2019). About 80% of world’s population still depend on herbal medicine

estimated by the World Health Organization (Akintonwa et al, 2009). In the past years, public

interest in natural products specifically herbal medicines has expanded dramatically in developing

countries and industrialized countries because of extreme poverty and lack of access to synthetic

drugs. Despite of the great improvement in latest synthetic medicine in the recent years, plants still

make a significant contribution to health services (Sharma et al, 2010).

Herbal medicine has been the oldest procedure of treating human diseases and infections.

Many natural products become treatment because of their anti-inflammatory, antibacterial,

antifungal and antiviral activities and represent a possible source of affordable and effective herbal

medicine (Seyyednejad et al, 2010). The use of herbal medicine has been broadly executed since

ancient times and have contributed remarkably to human struggles in fighting against diseases.

Earliest recorded of herbal medicines have seen 5000 years ago in Greek, Roman, Indian, Chinese,

Egyptian and Syrian (Kumar, 2017). One of the examples of how herbal medicine evolved was

through traditional Chinese medicine, The Devine Farmer’s Classic of Herbalism was discovered
about 2000 years ago which orderly collected the information on herbs and has been developed in

different herbal pharmacopoeias and numerous monographs on individual herbs exist (Benzi &

Watchel, 2011).

A natural product is a composite of deduced natural sources and has many purposes beyond

what humans have discovered and recorded for the past years. With all the historical researches

with natural products as medicinal agents, studies showed that it is the primary source in drug

discovery because it only consists of terrestrial plants, animal products, marine organisms, and

microorganisms (Ji, et. Al., 2009). Without the natural products there would not be that much of

medicines or treatments created. These were considered as the practical therapeutic agents for

more than a thousand years. As of today, more and more discoveries were known and it has

evolved to becoming a complex active chemical substance used to create modern medicines (Dias,

2012).

Hibiscus Rosa sinensis (Gumamela) have different colors but the most popular is the red

flowers that are known to be used for medical purposes such as teas and extracts that help relieve

conditions like an upset stomach, high blood pressure, bacterial infections and fever. Some

research suggested that Hibiscus rosa sinesis showed a great potential for treating cancer because

of its anticancer property like anthocyanin (Goldman, 2017).

According to another study, various parts of Hibiscus Rosa sinensis like flowers, leaves

and roots have been known to possess medicinal properties, it was used internally in the

treatment of excessive and painful menstruation, illnesses, bronchial catarrh, and hair growth. Its

leaves and flowers were pulverized to become a paste in order to be used as anti-inflammatories
such as cancerous swellings and mumps. There are commercial products of Hibiscus Rosa

sinensis nowadays like essential oils, shampoo, teas and other ointments (National Library Board

Singapore, 2019).

This research study tested and determined the antimicrobial activity of Hibiscus rosa

sinensis leaves. This will be done with three common pathogens: Escherichia coli, Staphylococcus

aureus, and Pseudomas aeruginosa; subjected to agar-disk diffusion.

STATEMENT OF THE PROBLEM

Diseases and infections caused primarily by microbes have been a great public health

concern worldwide. As researchers attempt to discover alternative antimicrobial agents, it is a great

challenge to seek for agents that are both effective and readily available.

This study attempts to determine the antimicrobial activity of extracted Hibiscus rosa

sinensis leaves.

Specifically, it seeks to answer the following questions:

1. The difference of the antimicrobial activity of Hibiscus rosa sinensis liquid extract if it is

compared to an antibiotic, such as amoxicillin.

2. What type of bacteria are to be tested in inhibition?

2.1. Escherichia coli

2.2. Staphylococcus aureus

 2.3. Pseudomas aeruginosa

3. Which among the three groups of bacteria has the biggest zone of inhibitions?

RESEARCH OBJECTIVES

The primary goal of this experiment is to investigate the effectiveness and credibility of

Hibiscus rosa sinensis leaves extract as an anti-microbial product and expound the idea of this

product to be used as an alternative antimicrobial agent.

Approach:

1. To determine the antimicrobial activity of the extracted Hibiscus rosa sinensis extract to

common pathogens through agar disc diffusion assay based from the related researches

2. To identify which of the three types of bacteria: Escherichia coli, Staphylococcus aureus,

and Pseudomas aeruginosa, is the most sensitive to the plant extract (greatest zone of

inhibition).
 CONCEPTUAL FRAMEWORK

Collection of leaves

Extraction of Hibiscus rosa sinensis

leaves

Liquid Filtrate

Escherichia coli Staphylococcus aureus Pseudomas aeruginosa

Determining the Antimicrobial

activity of liquid extract

Figure 1

Conceptual Paradigm of the Study

The paradigm illustrates the effect of the independent variable to the dependent variable.

This pertains to the possible effect of Hibiscus rosa sinensis leaves as a liquid filtrate. To be tested

in three different bacteria which are Escherichia coli, Staphylococcus aureus and Pseudomas

aeruginosa, and to determine the antimicrobial activity of Hibiscus rosa sinensis.

SCOPE AND LIMITATIONS

Agar disk-diffusion method is compatible for the type of observation needed to measure

the zone of inhibition. The zone of inhibition was effective to measure the area of bacterial growth

that the leaves essential oil destroyed or mitigated, which had been measured in centimeters. The

experiment is limited to using leaves, since more materials have been collected compared to the

other parts of the plant like the flower. The Hibiscus rosa sinensis leaves have been sourced from

one specific location; San Andres, Manila. This was necessary to ensure that different plants

profile will not interfere as a confounding variable to the study. The extract will be limited to its

liquid filtrate and its concentration will not be specified. On the other hand, the isolates used in

this research study are common pathogens: Escherichia coli, Staphylococcus aureus, and

Pseudomas aeruginosa. These pathogens are usually a part of the human microbiota, but they are

also usually pathogenic, sometimes causing skin or gastrointestinal infections, making them

effective subjects of the experiment.

SIGNIFICANCE OF THE STUDY

This research study is expected to produce an effective alternative microbacterial agent to

give the community a natural approach if the commercial ones didn’t work. Furthermore, this

could be more importance to the medical field as it may bring clarification to the effectiveness of

Hibiscus rosa sinensis essential oil on pathogenic bacteria. Finally, if this research study is a
success, this may be used as a basis or as a referential information to be used in conducting another

study similar to this.

DEFINITION OF TERMS

Antibiotics- a medicine (such as penicillin or its derivatives) that inhibits the growth of or

destroys microorganisms.

Anti-inflammatory property-quality of a drug to reduce inflammation on the skin.

Antifungal property- ability of a drug to prevent fungal growth on the skin.

Antimicrobial- a property of a substance to inhibit microbial growth.

Bacteria-a member of a large group of unicellular microorganisms which have cell walls but

lack organelles and an organized nucleus, including some which can cause disease.

Ethanolic- relating to, containing, or derived from ethyl alcohol: alcoholic sense.

Microbiota- community of microorganisms found on other multicellular organisms.

Pathogen-a bacterium, virus, or other microorganism that can cause disease.

 CHAPTER 2

REVIEW OF RELATED LITERATURE AND STUDIES

Importance of Herbal medicines

Herbal medicines are used since the beginning by different people with different cultural

traditions. Nowadays, people see herbs as spices that are needed for cooking, some have forgotten

that it’s more than just a food. Herbs have various uses, it is known for its ability to become a

medical treatment, because of that there are increasing interest in biomolecular effects of herbs.

Biomolecular effects are the measurable and observable changes that occur in cells, animals or

humans, under controlled conditions of treatment with an herb. According to the study, herbs are

antioxidants, anti inflammatory and can be used for cardiovascular diseases, cancer, diabetes, skin

disorders and neurodegenerative disease. Currently, there are many commercial herbal medicines

yet it’s not certified natural, it’s mixed with different chemicals. As herbs can vary with season,

growing conditions, preparation and storage. It’s all about the quality, consistency and control in

order to have the promotion of herbal medicines (Benzie & Wachtel-Galor, 2011).

According to Noor Ahmed Khan and Syed Aftab Iqbal, herbal products are being used

again throughout the world as it is recognized for being safer than synthetics to the human

environment. Some developing countries have established into using plant drugs just like 25% of

it are contributed to the total drugs in the United States and 80% in China and India who gave

much more important in the usage of medicinal plants. Despite the continuous production of

modern medicine, a huge population of the world still prefer to use drugs from plants. Usage of

plants as a source of medicine has increased for it can cure a variety of human ailments. In some
countries, it is very convenient to use plant drugs for it is more affordable and modern drugs are

usually cannot be afforded by 80% of the population. It is also indicated that many countries have

spent 45-50% of their money on drugs and health care. In order to solve this crisis, it is possible

that more plant drugs will be accessible in the future (Khan N. and Iqbal S., 2011).

According to Dr. Mahtab Alam Khan (2016), there will be an increased emphasis on the

use of plant materials as source of medicines than synthetic drugs because of the growth of the

population, insufficient supply of drugs, expensive treatments, several side effects of drugs and

resistance to currently used drugs for infectious diseases. He said that medicinal plants are

considered as a rich resources of ingredients which can be used in drug development either

pharmacopoeial, non-pharmacopoeial or synthetic drugs. Other than that, it is also vital for human

cultures around the world. Herbalists believes that herbs can be very useful in the treatment of

many conditions, but without the unwanted side effects that are often seen in conventional

pharmaceutical treatments. Herbal medicines or medicinal plants are not only used for treatment

of diseases but it can be also a tea, toothpaste, essential oils, and lotions (myDr, 2010).

Herbal medicines in the Philippines

In the Philippines, it’s not really that different with the other countries when it comes to herbs.

Some are used for cooking and some are used for its medicinal purpose. Most of the time Filipinos

know what are the uses of plants around them. In the Philippines there are 32 possible plants for

wounds, 28 for fever, 25 for diarrhea, 21 for intestinal parasites, and 19 for coughs listed. Filipinos

have this trait wherein they use plants as their “first aid”. For example, when someone has a wound

the first thing they’ll think of is the plant bayabas. These practices are mostly done in provinces.
Herbal medicines in the Philippines are not really ignored, instead more and more are being

approved by the government (Tiglao, 2014).

The rich knowledge about the use of traditional medicinal plants and herbal medications have

been seen in many ethnic minorities in our country, the Negritos from Guimaras Island is still

recognized for their outstanding knowledge about traditional medicine. The excessive maternal

mortality rate in the Philippines in the past years improve social and health conditions of women

in Guimaras Island, with the total 49 medicinal plants are still used in treating women concerning

reproductive health related. The 49 plant taxa utilized in four classification relating to their

reproductive health problem. 49% leaves, 38% stems and 6% barks are the most usually used

plants because of their availability (Ong & Kim, 2015).

A study tested edible mushrooms in the Philippines for its antimicrobial activity on

common pathogens. The mushrooms: Lentinus edodes, Pleurotus sajor-caju, and Auricularia

polytricha, were screened for their phytochemicals and found carbohydrates, glycosides, proteins,

amino acids, phenolic compounds, gums, mucilage, fats, and fixed oils and saponins. These

constituents are potentially the determinants of the antimicrobial activity of the mushrooms

(Campos, P.E., et al, 2012).

Activity of Hibiscus rosa sinensis Extract as Antimicrobial agent.

Gumamela (Hibiscus rosa sinensis) is commonly used as an ornament. Its flowers have

varying colors that attracts most people through its pleasing physical features, but Hibiscus rosa

sinensis plant has more functions than just its appearance. Aside from being known for its sweet

fragrance, it also yields a rich level of antimicrobial agent (Barraquia, A., et. al., 2017).
 Hibiscus rosa sinensis flowers extract exhibit high levels of antioxidant and antimicrobial

activities. Aqueous extracts showed that Hibiscus rosa sinensis flavonoid levels, are high,

determining its equally high level of antioxidant properties. Another significant finding of this

study; aqueous and ethanolic extracts of Hibiscus rosa sinensis inhibited the growth of common

microorganisms Salmonella typhimurium and Staphylococcus aureus. Its extract is a source of

many possible medicinal properties such as antioxidant, antidiabetic, antiulcer and anti-

inflammatory properties, which can help to our modern treatment of many diseases. Other studies

conducted the use of Hibiscus rosa sinensis flowers and leaves as a medicinal plant in animals

(Khristi, V., PatelV. H., 2016).

The pharmacological activity of Hibiscus rosa sinensis dried leaves as ethanol extract

which tested on mice and rats through intraperitoneal injection, which showed medicinal properties

such as analgesic activity, anti- inflammatory, and antipyretic activity. Water and ethanol

extraction administered to guinea pig exhibited an antispasmodic activity and anti- fungal activity

in Rhizoctonia solani which is a pathogenic fungus (Jadhav V.M., et al, 2009).

Hibiscus rosa sinensis, leaves and flowers have exhibited medicinal properties in line with

its components: flavonoids, thiamine, riboflavin, niacin and ascorbic acid. Its leaves and flowers

aid both in hair growth and ulcers. The infusion of its leaves were used as expectorant to bronchitis.

Its flower buds are pounded into a paste, then applied to external swellings. Its leaves also wound-

healing activities, as it’s pounded form cured abrasions, although it worked at a slower rate than

commercially available antiseptics (Dela Cruz, M. C., et. al., 2013).

 Hibiscus rosa sinensis flowers and leaves contain ingredients that identify its antibacterial

activity and may be used in treating pathogenic contaminations or infections caused particularly

by isolates (P. aeruginosa, Serratia, Micrococ-cus, Enterobacter and Salmonella). The watery

extracts of Hibiscus rosa sinensis were tested against isolates and the results showed that the crude

extracts of them with boiling water inhibited more of the isolates than the cold watery extracts

(crude and diluted) (Khudhr Al-Alak S., et. al., 2015).

Several articles have shown that the aqueous ethanolic extract of Hibiscus rosa sinensis

was used for constipation and diarrhea. It was also used as a traditional medicine wherein the

leaves of the plant are used in fatigue and skin disease. Its roots were given for those who have

menorrhagia when it’s powdered, but with fresh root juice it’s given for those who have gonorrhea.

The flowers are used in diabetes, epilepsy, bronchial catarrh and leprosy. Other studies in

Ayurvedic medicine in India showed that the petal of the plant was used as a demulcent refrigerant

drink in fever. The plant posseses anti-phologistic, anti-diarrhetic and anticomplementary activity

according to Pekamwar S. S., Kalyankar T.M., Jadhav A.C. (2013).

There are recent studies about the phytochemical constituent and the antibacterial activity

of the methanolic extract of leaf and flowers that proves that Hibiscus rosa-sinensis extracts has

potential antibacterial. All extracts obtained from the Hibiscus rosa sinensis showed antibacterial

activity against the human pathogens such as E. coli, B. subtillis, P. aeruginosa, S.

aureus, Streptococcus sp. Salmonella sp. (Udita, T., et.,al., 2015).

 The results of the experiment conducted for an article entitled “A survey on Hibiscus rosa

sinensis, Alcea rosea L. and Malva Neglecta Wallr as Antibacterial Agents” showed that the four

concentration extracts of plants mentioned had inhibitory effects at varying concentrations against

these bacteria: Pseudomonas aeruginosa, Listeria Monocytogenes, Staphylococcus epidermidis,

Staphylococcus aureus, Klebsiella pneumonia, Salmonella typhi etc. The most resistant strain was

Escherichia coli. The highest inhibitory zone was exhibited by ethanolic extract of M. neglecta

against Staphylococcus epidermidis (22mm inhibition zone) and Staphylococcus aureus (20 mm

inhibition zone). The Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal

Concentration (MBC) values against Staphylococcus epidermidis were equivalent. This study

shown antibacterial activity in both gram-positive and gram—negative bacteria, considering the

zone of inhibition it is more effective in gram-positive bacteria than the gram-negative bacteria.

Staphylococcus aureus were one of the more susceptible to the extracted H. rosa than normal

antibiotics while E. coli showed resistance to all extracted plants. These discoveries proposed that

these local plants have great antibacterial properties that can be utilized for disease control and

treatment and could likewise be as new source for antimicrobials discover and contamination

treatment (Seyyednejad et. al., 2010).

According to a study the extracts from different parts of the Hibiscus rosa-sinensis have

shown great qualities for the treatment of fungal or bacterial infections (Ruban, P., Gajalakshmi,

K., 2012). Flowers belonging to Hibiscus and Cassia were investigated by their antioxidant

activity and antibacterial properties. The results of the experiment show that both of the flower

extracts encompass high amount of antioxidant compounds and exhibit significant antioxidant

activities. The hibiscus flower extracts exhibited inhibition to common food-borne pathogens like
Salmonella t. and Staphylococcus a. This identifies its antimicrobial property (Mak, Y. M., et al.,

2012).

Several articles have shown that the aqueous ethanolic extract of Hibiscus rosa sinensis

was used for constipation and diarrhea. It was also used as a traditional medicine wherein the

leaves of the plant are used in fatigue and skin disease. Its roots were given for those who have

menorrhagia when it’s powdered, but with fresh root juice it’s given for those who have gonorrhea.

The flowers are used in diabetes, epilepsy, bronchial catarrh and leprosy. Other studies in

Ayurvedic medicine in India showed that the petal of the plant was used as a demulcent refrigerant

drink in fever. The plant posseses anti-phologistic, anti-diarrhetic and anticomplementary activity

according to Pekamwar S. S., Kalyankar T.M., Jadhav A.C. (2013).

Bacteria Species

Escherichia coli or most commonly known as E. coli is a fast-growing bacterium that was

discovered by the German microbiologist and pediatrician, Theodor Escherich. Escherichia coli

have been considered to be a gram negative bacterium that can be found in lower intestine of

homeothermic species, most its strain are harmless and part of the normal flora of gastrointestinal

tract and producing vitamin k2 which benefit the host or preventing the building up of pathogenic

bacteria in the intestine but certain serotype can lead to food poisoning in human. This bacteria

grow easily and its genetics are easy to manipulate and it is considered as the leading studied model

organism in biotechnology (Abello, 2009).

Pseudomonas aeruginosa is common gram- negative that considered as an oppornistic

pathogen that found in soil, plants and water. It can cause diseases and usually occur in hospital,
when expose to water and it can lead to major infection especially in people with weak immune
system. This bacteria is very adaptive and it is resistant to large number of antimicrobial agents
and can develop secondary resistance genes from more organisms which makes it difficult to
eliminate when resistant (Wu, W. et al, 2015).

Staphylococcus aureus have been considered to be a gram positive bacteria that are in a

form of cocci-shaped and arranged in clusters. It is normally considered as normal flora on the

epidermis and mucous membranes and it is commonly found in humans. Staphylococcus aureus

are considered being the major bacterial human pathogen and widely recognized as leading

bacterial infection in human. Infection resulted from Staphylococcus aureus remain challenging

because it can develop a drug resistant from antibiotics like Methicillin-Resistant Staphylococcus

aureus caused by mutation in its strains (Taylor & Unakal, 2018).

SYNTHESIS

Based on related literatures stated that herbal medicine are still being used in various ways by

the people because not only that it is more affordable to the population but also it is much more

safer to use for its natural approach. that’s why a huge population of the world still prefer to use

drugs from plants. Certain countries like China and India give much more importance to herbal

medicine rather than the synthetic ones because of its large and still growing population, causing

the insufficiency of the supply of synthetic drugs and expensive process. Herbalists also believes

that herbs can be very useful and effective to many conditions, but without the unwanted side

effects.
 Hibiscus rosa sinensis contains medicinal properties that aid in bacterial or fungal infections.

These studies directly tested the antibacterial activity of the plant on common pathogens such as

Salmonella tp., Staphylococcus aureus, Escherichia coli. The results showed that Hibiscus rosa

sinensis inhibits the growth of bacteria justifying its antibacterial property. The aqueous extract

of the Hibiscus rosa sinensis has high flavonoid levels determining its equally high level of

antioxidant properties indicating that Hibiscus rosa sinensis can be a medicinal properties such as

antioxidant, antibacterial, antidiabetic, antiulcer and anti-inflammatory properties which can be

used in preventing diseases.

On the other hand, the study suggest that the phytochemicals found in the mushrooms are

responsible for its antimicrobial activity. One of the said phytochemicals are phenolic compounds

that is an effective inhibitor of bacteria, is also found in Hibiscus rosa sinensis.

All points stated by the studies and literature related to the study made a significant evidence

that Hibiscus rosa sinensis has a great potential as an antimicrobial. Some of the studies prove that

the plant has been a great contributor to herbal medicine as it has been used for a long time for its

antioxidant, antidiabetic, antiulcer and anti-inflammatory properties. While some of the studies

clearly point out that Hibiscus rosa sinensis has phytochemicals, particularly phenols that displays

inhibition to a great amount of pathogens that greatly affect its ability to mitigate bacterial growth.

These studies and literatures provide a strong backbone to this study’s goal since all of their

findings suggest that Hibiscus rosa sinensis leaves essential oil is an effective antimicrobial.
 CHAPTER 3

METHODOLOGY

1. MATERIALS

1.1. The plant materials

The fresh plant samples of Hibiscus rosa sinensis leaves were bought from Bureua

of Plant Industry San Andres, Manila during the month of February 2019.

1.2. Bacterial species

The three bacterial species used in this research study were brought from Adamson

University Technology in Research and Development Center. The gram-positive

specimens are Staphylococcus aureus and the gram-negative species are Pseudomas

aeruginosa and Escherichia coli.

2. METHODS

2.1. Sterilization of Hibiscus rosa sinensis leaves

Hibiscus rosa sinensis leaves were sterilized with the mixture of 70mL of 70%

Ethyl Alcohol, 30 mL distilled water and soaked the leaves for one (1) minute to the

mixture, after that the samples were air dried until the leaves were brittle enough to be

pulverized. The leaves were pulverized as fine as possible using a blender from the

Adamson University Biology Laboratory.

2.3. Extraction preparation

The pulverized Hibiscus rosa sinensis leaves were extracted with a ratio of 1:3 of

900mL of 95% ethanol, 300g of pulverized of Hibiscus rosa sinensis dried. The plant

samples were soaked in the Ethanol solution and were later filtered with Whatmann filter

paper no. 1, a funnel and Erlenmeyer flask.

2.4. Bacteria growth conditions

The three test bacteria, the gram-positive specimens are Staphylococcus aureus,

Pseudomas aeruginosa and the gram-negative species is Escherichia coli were inoculated

and grown in test tube with nutrient broth for 24 hours in room temperature.

2.5. Antimicrobial activity of the extract

Kirby-Bauer method or agar disk diffusion method is necessary in quantifying the zone

of inhibition. This process involves Mueller-Hinton Agar (MHA) and three types of

bacteria: Staphylococcus aureus, Escherichia coli, and Pseudomas aeruginosa, cultured in

petri dishes through cotton swabbing. The petri dishes are labeled in four quadrants

beforehand, to distinguish the type of treatment of the paper discs that are placed on top of

the bacteria culture. There are three experimental set ups of liquid filtrate that have three

discs with the measurement of 30 µl of Hibiscus rosa sinesis liquid extract, then another

positive control group with 30 µl of amoxicillin and negative control group with the 30 µl

of distilled water. The set ups are then placed in an area with room temperature for 24

hours. The area that shows how much pathogens are destroyed: the zone of inhibition, is

measured in centimeters. The greater the value recorded, the higher the antimicrobial
 activity. After recording the zone of inhibitions, all data are subjected to further analysis

and interpretation.

3. Descriptive Analysis of the Antimicrobial Activity

Through the Screening for the Antibacterial Activity of Medicinal plants by Delia

Ontengco, the numbers recorded were presented along with its interpretation. As the

laboratory activity shows that with a disk diameter of 6 cm, a zone of inhibition greater

than the said diameter indicates sensitivity to the plant extract, while a zone of inhibition

within 6 cm suggests resistance to the plant extract. All recorded zone of inhibition from

the experiment will be analyzed and interpreted with the aforementioned laboratory activity

as a guide.
 CHAPTER 4

PRESENTATION, ANALYSIS AND INTERPRETATION OF DATA

Screening the Hibiscus rosa sinensis liquid extract for its antimicrobial activity suggests

that aside from quantifying its activity by measuring its zone of inhibition, further descriptive

interpretation is necessary to determine the qualitative classification of its activity. In this chapter,

the zone of inhibition of all set ups: Negative control (distilled water), positive control

(Amoxicillin), and the three experimental set ups with three bacteria; will be presented along with

its descriptive interpretation based on the Screening for the Antibacterial Activity of Medicinal

plants by Delia Ontengco; a laboratory guide that sets numerical data to descriptions.

Zone of Inhibition

Bacteria Negative
Positive
Mean Zone Descriptive control
1 2 3 control
of Inhibition Interpretation (distilled
(Amoxicillin)
water)
Staphylococcus
14 cm 12 cm 10 cm 12 cm Sensitive 12 cm 0 cm
aureus
Escherichia coli 8 cm 7 cm 7 cm 7.33 cm Sensitive 20 cm 0 cm
Pseudomas
7 cm 8 cm 9 cm 8 cm Sensitive 11 cm 0 cm
aeruginosa

Table 1: Antimicrobial activity of Hibiscus rosa sinesis extract against pathogenic bacteria.

The numbers recorded were presented to determine the antimicrobial activity of Hibiscus rosa

sinensis liquid extract and comparing to amoxicillin and the three bacterial species, the gram-

positive specimens is Staphylococcus aureus and the gram-negative species are Pseudomas

aeruginosa and Escherichia coli. The antibiotic as a positive control shows a greatest zone of

inhibition having a 12 cm for Staphylococcus aureus, 20 cm for Escherichia coli 11 cm Pseudomas

aeruginosa. This shows that the zone of inhibition of amoxicillin is greater than the zone of

inhibitions of the Hibiscus rosa sinensis liquid extract.

The disk diameter used in the experiment is 6 cm, a zone of inhibition greater than the said

diameter indicates sensitivity to the plant extract, while a zone of inhibition within 6 cm suggests

resistance to the plant extract. Based on the experiment, it shows that the three pathogens are

sensitive to Hibiscus rosa sinensis leaves liquid extract, but it has the most effect on the gram-

positive bacteria, Staphylococcus aureus, with a mean zone of inhibition of 12cm. While the two

gram- negative bacteria, Escherichia coli has a mean zone of inhibition of 7.33cm and the

Pseudomas aeruginosa has 8cm. This study shows that the Gumamela (Hibiscus rosa sinensis)

leaves liquid extract exhibits effective antimicrobial activity to the three bacteria but had a greatest

antimicrobial activity to gram-positive bacteria compared to the two gram- negative bacteria.

There was a significant difference between this research study’s findings to previous related

studies. Crude extracts of Hibiscus rosa sinensis with boiling water inhibited more isolates than

cold watery extracts. (Khudhr A-Alak, S., et. al., 2015) This comparison shows that the type of

extract is a factor affecting the antimicrobial activity. On another research study, the inhibitory

effects of plant extracts differed at varying concentrations. (Seyyednejad, et. al., 2010) This

exhibits that the level or amount of concentration is a factor in the antimicrobial activity of

Hibiscus rosa sinensis.

 CHAPTER 5

DETERMINATION OF ANTIMICROBIAL ACTIVITY OF EXTRACTED

HIBISCUS ROSA SINENSIS LEAVES

SUMMARY

In the past years, public interest in natural products specifically herbal medicines has

expanded dramatically in developing countries and industrialized countries because of extreme

poverty and lack of access to synthetic drugs. Herbal medicines is considered as the oldest

procedure for treating human infections and diseases represent a possible source of affordable and

effective herbal medicine. Hibiscus rosa sinensis (Gumamela) flowers extract exhibit high levels

of antioxidant and antimicrobial activities. The pharmacological activity of Hibiscus rosa sinensis

dried leaves as ethanol extract which was tested on mice and rats through intraperitoneal injection,

which showed medicinal properties such as analgesic activity, anti- inflammatory, and antipyretic

activity.

This research study shows the antibacterial effects of Gumamela (Hibiscus rosa sinensis)

leaves extract on three common pathogens: Escherichia coli, Staphylococcus aureus, and

Pseudomas aeruginosa; subjected to agar-disk diffusion. Nutrient broth were used for the bacterial

culture and test. The plant leaves were collected from the same place, each leaf were sterilized

with distilled water and ethanol with a concentration of 95%. The leaves were prepared in liquid

form and further filtered. The discs containing the plant’s extract were emplaced on the bacterial

culture and incubated for 24 hours. After the experiment, the data gathered were analyzed and

interpreted. Descriptive Analysis of the Antimicrobial Activity is the technique used in this study

to interpret the zones of inhibition. The numbers recorded were presented along with its
interpretation. With a disk diameter of 6 cm, a zone of inhibition greater than the said diameter

indicates sensitivity to the plant extract, while a zone of inhibition within 6 cm suggests resistance

to the plant extract. Based on the experiment, it shows that the three pathogens are sensitive to

Hibiscus rosa sinensis leaves extract, but it has the most effect on Staphylococcus aureus. Its mean

zone of inhibition is 12cm, while Escherichia coli has a mean zone of inhibition of 7.33cm and

the Pseudomas aeruginosa has 8cm. This study shows that the Gumamela (Hibiscus rosa sinensis)

leaves are effective as an antimicrobial agent.

CONCLUSION AND RESULTS

As the title of this study states; the Determination of Antimicrobial Activity of the Extracted

Hibiscus rosa sinensis, the extracted liquid from the plant was subjected to antimicrobial

sensitivity test through Kirby-Bauer method or agar disk diffusion method and it shows that the

three bacteria are sensitive to Hibiscus rosa sinensis leaves liquid extract. The extract exhibits

effective antimicrobial to the three bacteria but had a greatest antimicrobial activity in gram-

positive bacteria such Staphylococcus aureus than the two gram- negative bacteria such

Escherichia coli and Pseudomas aeruginosa. Hibiscus rosa sinensis has a promising future

because of its being abundance in the Philippines as it only grows in a tropical country and its

promising medicinal properties especially in potential as antimicrobial agents that can use against

pathogenic bacteria. For further development of this antimicrobial agent may help in developing

antibiotic to pathogens that are resistant to antibiotic that are available in the present.
RECOMMENDATION

The parameters of this study is limited to testing the antimicrobial activity of the Hibiscus

rosa sinensis liquid extract by itself. For further improvements and analysis, future researchers

may refer to this study and follow these recommendations;

1. The researchers recommend to use different concentrations to test the sensitivity of

antimicrobial activity of a plant extract.

2. The researchers recommend to use other bacteria that are not used in this study to know

further effects of the plant extract.

3. The researchers recommend to use 99% ethanol for soaking the plant materials for higher

concentration.

4. Test the Hibiscus rosa sinensis for other properties such as hair growing properties.

5. Application of the data presented in this study for commercial use.

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