ANTIBACTERIAL ACTIIVTY of MAKAHIYA (Mimosa pudica) LEAF ETHANOLIC

EXTRACT AGAINST SELECTED BACTERIA.

Thesis
Presented to the Faculty of
College of Arts and Science
Department of Natural Sciences
Lyceum-Northwestern University
Dagupan City

In Partial Fulfilment
Of the Requirements for the Degree
Bachelor of Science in Biology

By

Ramesh Mano Bhargavi

Gancheeri Vamsi Krishna
 CHAPTER I

INTRODUCTION

Rationale and Background of the Study

Plants are well known to have a characteristics and traits in producing substances

that could help in the wellness of human. Substances like saponins, tannins, flavonoids

are produced by the plants in order to defend themselves to whatever may harm them

especially insects. (Khan, 2011)

According to the Medical Health Guide, bacteria have been one of the major

problems worldwide that causes more than 10000 diseases. which in turn is alarming in

the community and may have been the primary cause that may lead to the extinction of

humanity.

According to R. Cox (2001), the major bacterial infections are caused by common

pathogenic microorganisms namely Escherichia coli, Pseudomonas aeruginosa and

Staphylococcus aureus. The most common infections caused by these bacteria are

impetigo, cellulitis, folliculitis, and skin abscesses.

Many plants species used traditionally have potential antibacterial and antiviral

properties and this has raised the optimistic thinking of scientists about the future of

antibacterial agents. Mimosa plant is used for the treatment of various ailments, among

its part leaves are commonly used. (Cabi, 2015).

 Since ancient time, people had been using herbal medicines. With the advent of

modern medicine and the development of new drugs, people had switched to the use of

commercial medicines. These medicines had been manufactured from the flora and fauna

that abound. Since this plant is very common in the researcher’s area, this aroused their

interest to make a study on the medicinal prospects of this plant.

Hence, in this study, the phytochemical screening and the antimicrobial properties

were conducted in the hope that the initial findings of this study will serve as a basis for

future investigations on the medicinal prospects of the plant understudy, and for the

discovery of new therapeutic agents from this plant.

Conceptual Framework

The study aims to determine antibacterial activity of Makahiya leaves extract

against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus.

Sudhakaran (2016), studied the phytochemical content of the plant and found out that

alkaloids, flavonoid C-glycosides, sterols, terpenoids, tannins, and fatty acids are present

in the plant. The presence of these phytochemical in Makahiya is the central concept to

which this research revolves. In the study of Cowan (1999) he identified that plant rich in

secondary metabolites like terpenoids, tannins, and alkaloids have been found to have

antibacterial properties in vitro.

Marjorie Murphy Cowan (1999): Plant Products as Antimicrobial Agents, Clinical

Microbiology Reviews Vol. 12 Issue 4 pp.564-582

Research Paradigm

Independent Variables Dependent Variables

 Makahiya leaves crude Zone of inhibition of the

extract following bacteria

 Test Bacteria
 Escherichia coli  Escherichia coli
 Pseudomonas
aeruginosa  Pseudomonas
 Staphylococcus
aureus aeruginosa
 Controls
 Staphylococcus aureus
 (+) Ciproflaxin
 (-) NSS

Intervening Variable

 pH
 Temperature

Figure 1: Research Paradigm

Figure 1. Shows the relationship of the variables which were used in the study. The

independent variables are the makahiya leaves (Mimosa pudica) extract, the test bacteria

which are Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus these

bacteria were chosen by the researcher since they commonly causes infectious disease
(Cox et al.). Ciprofloxacin is the positive control used in the study since it is the drug of

choice to treat the aforementioned bacterial isolates, furthermore, Normal Saline Solution

or (NSS) was used as a negative control since it does not give any effect on the bacteria.

The pH and the temperature on the other hand was labeled as intervening variable since

it is a variable where the researcher has no control. The zone of inhibition was used to

measure the effect of the extract on the growth of the bacteria.

Statement of the Problem

The study aims to find out the antibacterial activity of makahiya leaves (Mimosa

pudica) ethanolic extract against E.coli, Pseudomonas aeruginosa and Staphylococcus

aureus. It specifically sought to answer the following specific questions:

1. What is the phytochemical content of the Makahiya leaves ethanolic extract?

2. What are the zones of inhibition exhibited by the extract against?

a. Escherichia coli

b. Pseudomonas aeruginosa

c. Staphylococcus aureus

3. Is there any significant difference between the zones of inhibition of the

makahiya leaves (Mimosa pudica) ethanolic extract and the positive control

used against the test bacteria?

Hypothesis of the Study

Ho: There is no significant difference between the zones of inhibition of the makahiya

leaves (Mimosa pudica) ethanolic extract and the positive control Ciproflaxcin against the

test bacteria.

Ha: There is a significant difference between the zones of inhibition of the makahiya

leaves (Mimosa pudica) ethanolic extract and the positive control Ciproflaxcin against the

test bacteria.

Significance of the Study:

This study would of great help to the following entities:

Community: the result of the study can be a baseline of information about capability of

the plant which can be disseminated to the community for the protection and cultivation

of the said plant.

Future Researchers: the result gathered from the study can be a local baseline data of

further study regarding the plant.

Students: the research output from the study can help the students to further understand

the importance of the plant as to its medicinal value.

Pharmaceutical Industries: this study can help the to produce a cheaper medicine out

of the makahiya leaves.

Scope and Delimitations of the Study

This study dealt with the determination of the antibacterial activity of Makahiya

leaves using ethanol as a solvent for extraction. The study used three bacterial isolates

which are Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus which

the researchers obtained from Virgen – Milagrosa University Foundation, San Carlos City

Pangasinan. The Phytochemical screening of the plant was done at the Virgen –

Milagrosa University Foundation, San Carlos City Pangasinan, while the determination of

the plants antibacterial property was done at the Medicine Laboratory of Lyceum-

Northwestern University. The research experiment was done April 2017.

This research however is delimited to the following first, the research will not

perform and identify the minimum inhibitory concentration of the extract, and second, it

will not go further in identifying the active component within the extracts that had caused

the antibacterial activity.

Definitions of Terms:

The following terms were defined conceptually in search of the study.

Antibacterial - is an agent that inhibits bacterial growth or kills bacteria.

The term often used synonymously with the term antibiotic.

Escherichia coli – is a bacterium, that thrives in the digestive tracts of humans and animals.

Some strains of Escherichia coli can also cause urinary tract infections.
Ethanol – is a colorless volatile flammable liquid that is known to be the intoxicating agent

in liquors, it is also used as solvent and in fuel.

Phytochemical - Chemical compounds occurring naturally in plants.

Pseudomonas aeruginosa is member of the Gamma Proteo-bacteria class of

Bacteria. It is a Gram-negative, aerobic rod belonging to the bacterial family

Pseudomonadaceae.

Staphylococcus aureus -Boils. The most common type of Staphylococcus infection is

the boil, a pocket of pus that develops in a hair follicle or oil gland.

Zone of Inhibition – it is the area on an agar plate where the growth of a organism is

prevented by an antibiotic usually placed on the agar surface.

 CHAPTER II

REVIEW OFRELATED LITERATURE

Mimosa pudica is a species introduced from tropical America, but is now a

pantropical weed. It belongs to the family Mimosaseae. It is a common weed widely

distributed in the Philippines. ( www.stuartxchange.com) Mimosa pudicafrom Latin: pudica

"shy, bashful or shrinking"; also called sensitive plant, sleepy plant and the touch-me-not,

is a creeping annual or perennial herb often grown for its curiosity value: the compound

leaves fold inward and droop when touched or shaken, to protect them from predators,

re-opening minutes later.(Nodado et. al. 2014 ).

Figure 2. Mimosa pudica

M. pudica plant is a diffusely spreading, half-woody herb, have short prickly branches and

hairs glandular. Leaves are pinnate, sensitive to touch. Flowers axillary, globose head,

lilac pink in color. Stem is erect, slender, prickly and well branched. Petals crenate

towards base. Pods 1.5 to 2.5 cm long, closely prickly on the sutures and falcate.

(International Journal of Pharmaceutical Sciences and Drug Research 2013)

 The study of Mohan et. al. (2015) identified the different phytochemical found in the

flowers and fruits of the plant and according to their result it contains active constituent

like an alkaloid mimosine, mucilage, tannins, non- protein amino acid (mimosin), flavonoid

C- glycosides, sterols, terpenoids, tannins and fatty acids. Phytochemical screening of

leaf from different solvent was reported. The methanolic extract of leaves of M. pudica

showed the presence of bioactive components like terpenoids, flavonoids, glycosides,

alkaloids, quinines, phenols, tannins, saponins. In hexane, carbohydrates, steroids, and

saponins were present. Alkaloids, glycosides, carbohydrates, steroids, flavonoids and

saponins were also present in chloroform fraction. While in ethyl acetate extract of the M.

pudica leaf, alkaloids, glycosides, carbohydrates and flavonoids were the secondary

metabolite present. (Ahmad et. al. 2012)

On the other hand, the roots of the plant are indicative of the presence flavonoids,

phytosterol, alkaloids, amino acids, tannins, glycoside, and fatty acids. Chromatographic

procedures revealed that petroleum ether fraction majorly contains flavonoids,

phytosterol, alkaloids, and amino acids. Acetone fraction has confirmed the presence of

flavonoids. The chloroform fraction showed the presence of alkaloids. The essential oils

and fatty acids were majorly contained in the benzene extract. (Pande and Pathak 2009)

M. pudica is a multipurpose weed, used as vegetable, spice, a source of cooking

and cosmetic oil and as a medicinal plant. All parts of the plant are considered to possess

medicinal properties. (Azmi et. al., 2011). It draws attention to the research world because

of its wide medicinal activities like antidiabetic, antitoxin, antihepatotoxin, antioxidant,

wound healing and effects.( Mathew et al 2016). M. pudica is a plant with exceptionally

rich medical applications. According to Godofredo Stuart, M. pudica was included in the

lists of Philippines medicinal plants, its roots was used as diuretic, also used for dysentery

and dysmenorrhea. Decoction or infusion of leaves used in asthma; expectorant. Used

for hypertension, menorrhagia, glandular swelling, sore throat and hoarseness.

The entire plant can be boiled and its decoction can be used as anti-asthmatic agent

which means that the decoction of the whole plant has an anti-inflammatory effect, this

claim by Pande and Pathak was strengthened by the work of R Rathore et. al. (2012)

where the result of their study showed an anti-inflammatory effect of M. pudica seeds

extracts in mice, the extract was able to sustain the phase of inflammation (9 and 12

hours). It further explained that a single oral dose of M. pudica (200 and 400 mg/kg) was

more effective in reducing edema volume than the positive control indomethacin.

According to Pandey and Hussain (2015), that the susceptibility of various microbial

agents to different concentrations of Mimosa pudica indicates that plant is a potential

source for antimicrobial compound. In the study of Sadashiv S. O. (2015) entitled “An in-

vitro study on antibacterial activity of Mimosa pudica, Cynodondactylon and Impatiens

balsamina”, the results revealed that alcoholic leaf extrac of Mimosa pudica showed very

high antibacterial activity against three pathogens. Nguyen et al (2015), the result of tests

for in-vitro antibacterial activity indicates that the ethanol extract showed significant

activity against E.coli, S. aureus, B. subtilis and S. typhii. It can be noticed that the three

studies supports each other’s result. It is even evident that the extract can both inhibit the

growth of a gram negative and a gram positive bacteria. Aside from its antibacterial and

anti-inflammatory activity the plant has been reported to have a wound healing property.
In the study of Dnyaneshwar et al. (2009) Treatment of wound with ointment containing

2% (w/w) the methanolic and 2% (w/w) the total aqueous extract exhibited significant

(P < 0.001) wound healing activity. The methanolic and total aqueous extracts were

analyzed for total phenols content equivalent to Gallic acid. The content of total phenols

was 11% (w/w) and 17% (w/w) in methanolic and total aqueous extract respectively. The

methanolic extract exhibited good wound healing activity probably due to amount of

phenolic constituents present in the extracts.

Other Biological activities

Apart from antibacterial properties, M. Pudica was reported to have a number

of other properties like antioxidant. According to the result obtain from the study of Patro

et al. (2016) total phenolic, flavonoid contents and in-vitro antioxidant potential were

evaluated from various extracts of M. pudica leaves. Again, in-vivo antioxidant evaluation

from brain homogenate on oxidative stress markers as TBARS, SOD, CAT and GSH from

rat was investigated. From this study findings revealed that M. pudica possesses both in-

vitro and in-vivo antioxidant activity due to presence of phenolics and flavonoids .These

results revealed that the ethyl acetate extract of M. pudica exhibits both in vitro antioxidant

activity against DPPH and in vivo antioxidant activity by modulating brain enzymes in the

rat. This could be further correlated with its potential to neuroprotective activity due to the

presence of flavonoids and phenolic contents in the extract.

Literature on Bacteria

Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus

are among the bacteria that causes several different infections in the body. According to
the article published by Clark (2015), and according to the article Escherichia coli is

referred to as the most-studied free-living organism because of their genes which can be

easily modified (Jewets et. al. 2009). Escherichia coli is the causative agent of

gastrointestinal infection such as Urinary tract infection and Diarrhea. On the other hand,

Pseudomonas aeruginosa become increasingly recognized as an emerging

opportunistic pathogen of clinical relevance. Several different epidemiological studies

track its occurrence as a nosocomial pathogen and indicate that antibiotic resistance

is increasing in clinical isolates. (K. Todar, Ph D., 2012). Lastly, Staphylococcus aureus

isolated from an abscess or boil or other skin lesion, it is usually due to its

secondary invasion of a wound rather than the primary cause of disease. S.

aureus may similarly be isolated from abscesses, breast abscesses or mastitis,

dermatitis or skin infections and genital tract infections. (A. Mandal, MD, 2017)

Ciprofloxacin is a fluoroquinolone (flor-o-KWIN-o-lone) antibiotic that fights bacteria

in the body. Ciprofloxacin is used to treat different types of bacterial infections.

Ciprofloxacin should be used only for infections that cannot be treated with a safer

antibiotic. Ciprofloxacin may also be used for purposes not listed in this medication guide.

(Medguide.com)
 CHAPTER III

RESEARCH METHODOLOGY

Research Design

The study employed an experimental research method using post-test control,

which was a systematic and specific approach to research where the researcher

manipulates one or two variables and controls and measures any change in the

variable.

The bacteria namely Escherichia coli, Pseudomonas aeruginosa and

Staphylococcus aureus were tested against Makahiya leaves (Mimosa pudica) extract

to determine its effectiveness as an antibacterial agent. Antibacterial susceptibility

testing of pure cultures of Escherichia coli, Pseudomonas aeruginosa and

Staphylococcus aureus were used in the experiment as test organisms to determine

the antibacterial activity of the plant of study. To determine the activity of the plant. Zones

of inhibition was measured on each of the replicates in each trials and was compared to

a standard used in antibacterial assay.

Moreover, the study used three trials in each of the treatment and each trials were

replicated three times to check the validity.

Sources of Data

The leaves of Makahiya (Mimosa pudica) plant were collected inside the vicintiy of

Lyceum- Northwestern University, specifically in the botanical garden of Natural Sciences

Department. Ciprofloxacin which is used as positive control in the study and Normal
Saline Solution (NSS) which was used as negative control in the study was bought from

St. Joseph Drug Store, Ethanol a solvent used in maceration was bought in Libra Medical

Supply.

Furthermore, the Mueller Hinton Agar (MHA) used to culture the bacteria, pure

culture of Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus, glass

ware and apparatus that is used in the research was obtained from the Medicine

Laboratory of Lyceum-Northwestern University.

Instrumentation and Data Collection

Collection of plant materials:

500gms. of fresh leaves were handpicked in the Natural Sciences Botanical

Garden of Lyceum-Northwestern University, the leaves were no longer quantified as to

its age. Fresh leaves were brought to the Medicine Laboratory of Lyceum-Northwestern

University where the researcher washed with tap running water to remove the adhering

dirt in the leaves.

The leaves were sun dried for 5 days and was placed in a clean container before

it is processed for maceration.

Preparation of Materials:

Petri dishes, Erlenmeyer flask, stirring rods, loops and other materials which was

used in the study was subjected for autoclaving at 121 psi for 20 minutes for sterilization

before usage.
Preparation of extracts:

250gms of the dried leaves were pulverized into a course powder using cleaned

mortar and pestle. 100 grams of the leaves were separated from the 250 grams for the

phytochemical analysis.

The 100gms of leaves was soaked in 100ml of 95% ethanol and was placed in a

sterilized dark colored bottle and was brought to Virgen Milagrosa University Foundation

Pharmacy Department for phytochemical analysis.

The other 150grams were soaked in 150 ml. of 95% of ethanol and was placed in

an Erlenmeyer flask, the flask was covered with foil and was left for 24 hours maceration.

After maceration, the ethanolic extract were then subjected to water bath technique, a

technique used to remove the ethanol in the extract and to retain the crude. The pure

crude extract were then placed in a sterile container which will be used for the antibacterial

test.

Preparation of Sensitivity Disks:

The preparation of the sensitivity disc were prepared 24 hour before planting the

bacteria. The researchers brought the 500mg of Ciprofloxacin capsule from St. Joseph

Drug store. 50ml-distilled water were brought from Luzon Medical Center Pharmacy for

the dilution of the positive control. 50mg of Ampicillin was separated from the 500mg

capsule by the help of pharmacist in pharmacy laboratory. Separated Ciprofloxacin were

dissolved in 50ml distilled sterile water. The control was set aside while the researcher

prepared for the sensitivity disk.

 Whattman paper no. 1 was obtained from the Medicine Laboratory of Lyceum-

Northwestern University. The Whattman paper was cut into 5mm each using a puncher

which now becomes the disk. The disk was placed in a sterile container before using.

The disk were now then soaked into the sterilized container of the extract, the

positive and the negative control. The disk were soaked for 24 hours before impregnation

in the media.

Preparation of culture media

Antibacterial activity was carried out using disk diffusion method. An Artificial

culture media was prepared by adding 38 grams of Mueller Hinton Agar (MHA) into 1 liter

of distilled water. MHA is the standard base medium used for testing microorganism since

the condition of this medium is managed by commercial media. The agar was dissolved

in water in an Erlenmeyer flask by continuous stirring, after which the solution was placed

in Autoclave for sterilization.

The prepared media was then resulting the solution and was dispersed in sterile

petri dishes, let it cooled and hardened before the bacteria were streaked.

Streaking of the bacteria

After solidifying the Agar, the researcher prepared the bacteria from the

Refrigerator. Sprit lamp, Inoculating Loop, Alcohol, Tissue Paper were ready and surgical

sterile mask were used to protect from the cross infection to the researchers, sterile

surgical gloves were used. Lit up the sprit lamp and open the cap of test tube and moved

towards the flamed sprit lamp, Inoculating loop were heat in the flame of the sprit lamp
till it converted into red orange colour. Removed from the flame deepen into the bacteria

containing test tube and gently rub horizontally in the Agar plate. After finishing streaking

the bacteria the researcher placed the disc, simultaneously on the prepared agar plates.

There in total 15 Petri Disc per bacteria at one time and was incubated at 37°C ± 2 for 24

hours. The researcher done three replicate and three trials all in all.

Determination of the Antibacterial Activity

After 24 hours of incubation, the antimicrobial activity was determined by

measuring the zone of inhibition around each paper disk. Three separate trials were

conducted for plant extract against each organism. The inhibitions were measured using

a Vernier Caliper to the nearest mm for accuracy and precision. Data of the experiments

were represented by three replicates from each experiment.

Disposal of Bacteria

When the researchers are completely done with the experiment, they

decontaminate the plates that were used. The researcher decontaminate the

experimental materials using liquid disinfectant (Lysol). The researcher poured an ample

amount of disinfectant in the surface of the agar before the agar was discarded. The agar

was placed in a plastic bag and was sealed and thrown into the garbage.

Tools for Data Analysis

 In this study the researcher made use of the following statistical tool in order to

come up with the correct answer to the problem which was raised. First, to determine the

zone of inhibition of the researchers made us of mean. Furthermore, the results of the

mean will also be used to answer the significant difference between the zones of inhibition

exhibited by Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus in

extract as well as in the control, the researcher used One way analysis of Variance

(ANOVA) under F-test at 0.01 level of significance. A Post- hoc test Scheffe’s shall be

used to determine which among the hypothesis shall be accepted.

To identify the inhibitory activity of the extracts, the researchers made use of the

standard measurement for the zone of inhibition of the bacteria from Clinical and

Laboratory Standards Institute. [CLSI, January 2012]

Where:

≥ 20 – Susceptible 15-19 – Intermediate ≤ 14 – Resistant

For the computation, we will be using the following formulas:

∑𝑥
𝑀𝑒𝑎𝑛 = 𝑁

where:

X –raw data

N-total number of trials/replicates

The correction factor (CF), given by

CF== (ΣTk)2

The total sum of squares (SST)

SSB= ΣSSk- CF

The between sum of squares (SSB)

SSB= Σ(Tk)2 – CF

The within sum of squares (SSW)

SSW= SST-SSB

The Degrees of freedom

Between dF= n-1

Within dF= p-n

Mean sum of the squares is:

Between MSS= SSB/dFb

Within MSS = SSW/dFm

The formula for the computated F-value is:

Fc = MSSb/MSSm

Where:
n= total number of treatment

p= number of groups

m= the number of trials

 Chapter IV

Results and Discussion

Table 1

Phytochemical analysis of makahiya leaves (mimosa pudica) Ethanolic Extract

Secondary Metabolites Presence

Alkaloids Present

Unsaturated Sterols Present

Flavonoids Present

Tannins Present

Phenolic Acids Present

Saponin Glycosides Absent

Table 1 shows the phytochemical constituents present on the plant leaf ethanolic extract.

The presence of alkaloids, unsaturated sterols, Flavonoids, Tannins, Phenolic Acids and

Saponin Glycosides indicates that the extract is capable of inhibiting the growth of

bacteria. The secondary metabolites different confirmatory test were implied to the extract

to confirm the presence of the abovementioned compound in the plant’s leaf.

Table 2.1

Zone Of Inhibition of the makahiya leaves (mimosa pudica) Ethanolic Extract

Against Escherichia coli

Treatment T1 T2 T3 Mean Inhibitory

Activity

100% 21 19.67 19.33 20 Susceptible

 Positive 30 32 32 31.33 Susceptible

Negative 0 0 0 0 Resistant

Zone of diameter Interpretation for Escherica coli according to Clinical and

Laboratory Standards Institute (CLSI)
D≥ 20 – Susceptible 15-19 – Intermediate D≤ 14 – Resistant

The table shows the zone of inhibition of the extract to the bacteria Escherica coli thus

the extract inhibited 20 mm which has an equivalence of susceptible base on the

standards set by Clinical and Laboratory Standards Institute (CLSI). The results shows

that the extract has antibacterial activity on the bacteria Escherica coli. This result can be

further strengthened by the result of the phytochemical content found in the plant which

was stated in Table 1.

Table 2.2

Zone Of Inhibition of the makahiya leaves (mimosa pudica) Ethanolic Extract

against Pseudomonas aeruginosa

Treatment T1 T2 T3 Mean Inhibitory

Activity

100% 21.33 20.56 19.33 20.41 Susceptible

Positive 30 32 32 31.33 Susceptible

Negative 0 0 0 0 Resistant

Zone of diameter Interpretation for Psuedomonas aerogenosa according to Clinical

and Laboratory Standards Institute (CLSI)

D≥ 20 – Susceptible 15-19 – Intermediate D≤ 14 – Resistant

The table shows the zone of inhibition of the extract to the bacteria Psuedomonas

aurogenosa thus the extract inhibited 20 mm which has an equivalence of susceptible

base on the standards set by Clinical and Laboratory Standards Institute (CLSI). The

results shows that the extract has antibacterial activity on the bacteria Psuedomonas

aurogenosa.

Table 2.3

Zone Of Inhibition of the makahiya leaves (mimosa pudica) Ethanolic Extract

against Staphylococcus aureus

Treatment T1 T2 T3 Mean Inhibitory

Activity

100% 22 19.67 18.67 20.11 Susceptible

Positive 30 32 32 31.33 Susceptible

Negative 0 0 0 0 Resistant

Zone of diameter Interpretation for Staphylococcus aureus according to Clinical

and Laboratory Standards Institute (CLSI)
≥ 20 – Susceptible 15-19 – Intermediate ≤ 14 – Resistant

The table shows the zone of inhibition of the extract to the bacteria Staphylococcus

aureus thus the extract inhibited 20 mm which has an equivalence of susceptible base on

the standards set by Clinical and Laboratory Standards Institute (CLSI). The results

shows that the extract has antibacterial activity on the bacteria Staphylococcus aureus.

Table 3.1

Analysis Of Variance of the Antibacterial Activity of makahiya leaves (mimosa

pudica) Ethanolic Extract against Echerichia coli

 Source of Degree of Sum of Mean of F observed Tabular F
variance freedom squares squares

Treatment 2 1855.22 927.61 8.03 10.42

between
group

Error 6 693.21 115.54

within
group

total 8 2548.43 1043.15

8.03<10.42|Fc<Fb|; null accepted

The table shows the Analysis of variance to test the significance of the extract with that

of the control. The results shows that the computed value is less than the tabular value,

such that the null hypothesis, There is no significant difference between the zone of

inhibition of the extract and the positive control against the selected bacteria is accepted.

Thus, rejecting Alternative hypothesis stating there is a significant difference. This

indicates that the extract is good as the control since it shows no significant differences

in the activity of the extract and the control in inhibiting the Echerichia coli.

Table 3.2

Analysis Of Variance of the Antibacterial Activity of makahiya leaves (mimosa

pudica) Ethanolic Extract Against Pseudomonas aeruginosa

Source of Degree of Sum of Mean of F observed Tabular F

variance freedom squares squares

Treatment 2 1887.37 943.68 7.95 10.42

between
group
 Error 6 712.32 118.72
within
group

total 8 2599.69 1062.4

7.95<10.42|Fc<Fb|; null accepted

The results shows that the extract shows no significant difference in the zone of

inhibition of the extract and the control against Pseudomonas aeruginosa. The computed

value was tested under 0.01 level of significance whereas the computed value is less

than the tabular value under 0.01 level of significance. This means that the extract exhibits

good antibacterial activity as that of the control used. Thus, rejecting the alternative

hypothesis which states that there is a significant differences in inhibiting the bacteria.

Table 3.3

Analysis Of Variance of the Antibacterial Activity of makahiya leaves (mimosa

pudica) Ethanolic Extract against Staphylococcus aureus

Source of Degree of Sum of Mean of F observed Tabular F

variance freedom squares squares

Treatment 2 1863.23 931.62 8.01 10.42

between
group

Error 6 697.76 116.29

within
group

Total 8 2560.99 1047.91

8.01<10.42|Fc<Fb|; null accepted

 The table shows that the computed value is less than the tabular value under 0.01

level of significance. Thus, the alternative hypothesis, there is a significant differences in

the zone of inhibition of the extract and the control against the bacteria Staphylococcus

aureus is rejected. This means that there is no significance of the extract and the control

in inhibiting the bacteria. The results show that the extract has a good antibacterial activity

as that of the control.

Chapter V

Summary, Findings, Conclusions and Recommendation

Summary

Makahiya is a native plant in the Philippines and it is well known to have many

medicinal uses. In traditional ways of curing diseases the Makahiya leaves and other

parts were boiled and its filtrate was used to cure fever and other illnesses. Such that the
increasing rate of communicable diseases were most of it are caused by bacteria

Escherica coli, Psuedomonas aurogenosa and Staphylococcus aureus, Organizations all

over the world fled to seek information on how to lessen and even so, kill these bacteria.

And so came lots and lots of antibiotics were most are expensive. This is the reason for

the researchers opted to conduct a study on the antibacterial activity of Makahiya leaves

(SN) ethanolic extract against Escherica coli, Psuedomonas aurogenosa and

Staphylococcus aureus.

This research is an experimental type, whereas three treatments with three

corresponding replications was made to see if the extract’s activity. Prior to the

experimentation Phase of the study, the researchers asked for the certification of the plant

from PENRO-Dagupan indicating that the plant used by the researchers is the real

Makahiya plant. The collected leaves were soaked in 95% ethanol and some of the

collected leaves were subjected into phytochemical analysis to see the presence of the

secondary metabolites. The test was made in the Virgen Milagrosa University Foundation,

San Carlos City, Pangasinan While the other parts off experiment were done in the

Medicine Laboratory of Lyceum-Northwestern University. After which, the leaves were

subjected to water bath technique to get the crude extract. Whattman’s paper of no. 5

was soaked in the extract and put in the petri dish with an exact amount of agar, then the

impregnation of the bacterial isolates selected were done with the use of inoculating loop.

After which, the researchers measured the Zone of inhibition by the extract. Mean was

used in this study to get the accurate measurement of the zone of inhibition yielded by

the extract against the three bacteria.

 The results were further Analyzed using F-test and ANOVA under 0.01 level of

significance to see the significant difference in the zone of inhibition of the extract and the

control used. Based on the measurement of the three treatments, the extract yielded a

mean value of 20mm and based on the set standard of Clinical and Laboratory Standard

Institute, the 20mm for Escherica coli is susceptible, 20.41mm for Psuedomonas

aurogenosa which has the highest among the three bacteria and 20.11mm for

Staphylococcus aureus. The computed F for the three bacteria is less than the tabular F,

which indicates that there is no significant differences in the Zone of inhibition of the

extract and the control against the selected bacteria.

Findings:

The following were proven in the study:

1. That the plant extract contains the following secondary metabolites: alkaloids,

unsaturated sterols, Flavonoids, Tannins, Phenolic Acids and Saponin Glycosides.

2. That the positive control used Ciprofloxacin is a good antibacterial drug against

Escherichia coli, Pseudomonas aerogenosa and Staphylococcus aureus.

3. The extract yielded a mean value of 20mm for Escherichia coli, 20.41mm for

Psuedomonas aurogenosa which has the highest among the three bacteria and

20.11mm for Staphylococcus aureus the results were compared to the standard

measurement set by the Clinical and Laboratory standards Institue (CLSI).

4. The results of the Zone of Inhibition was able to meet the standards. The results

also shows that there is no significant differences in the zone of inhibition of the

makahiya leaves and the control used against Escherichia coli wherein the
 computed F=8.03 is less than the tabular F=10.42. The results also shows that

there is no significant differences in the zone of inhibition of the makahiya leaves

and the control used against Pseudomonas aerogenosa wherein the computed

F=7.95 is less than the tabular F=10.42. The results also shows that there is no

significant differences in the zone of inhibition of the makahiya leaves and the

control used against Staphylococcus aureus wherein the computed F=8.01is less

than the tabular F=10.42.

Conclusions

Based on the findings of the study, the researcher concluded the following:

1. The Phytochemicals present in the leaf are Alkaloids, Unsaturated sterols,

Flavonoids, Tannins, Phenolic Acids and Saponin Glycosides.

2. The zones of inhibition of the extract against Escherica coli is 20 mm,

Psuedomonas aurogenosa is 20.41mm and Staphylococcus aureus is 20.11mm.

3. There is no significant difference in the Zone of inhibition of the extract and the

control used against Escherica coli, Psuedomonas aurogenosa, Staphylococcus

aureus.

Recommendation

Based on the conclusion of the study, the researchers recommends the following:

1. Further studies must be done on the other parts of the Makahiya plant.

2. Future researchers are recommended to use other solvents such as water and

methanol.
3. Further test using concentrations to see the strength of the extract until the least

concentration possible.

4. Formulation of drug / or other antibacterial product can be done in this study.

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 APPENDIX A

Sample Computation from Table 2

Table 2

The zone of inhibition of makahiya leaves (mimosa pudica) against Escherica coli

Treatment Trial 1 Trial 2 Trial 3 Mean

R1 22 21 21

R2 21 20 20

R3 20 18 17

Positive 30 32 32

Negative 0 0 0 0

21 19.67 19.33 20

Psuedomonas aurogenosa

Treatment Trial 1 Trial 2 Trial 3 Mean

R1 22 21 21

R2 22 21 20

R3 20 19 17

Positive 30 32 32

Negative 0 0 0 0

21.33 20.56 19.33 20.41

 Staphylococcus aureus

Treatment Trial 1 Trial 2 Trial 3 Mean

R1 22 21 20

R2 22 19 20

R3 22 19 16

Positive 30 32 32

Negative 0 0 0 0

22 19.67 18.67 20.11

 APPENDIX B

Sample Computation of ANNOVA

 APPENDIX C

Certificates ( plant authentication, Phytochemical result, letters any document

used in the study)

 Appendix D

Documentation