KYAMBOGO UNIVERSITY

FACULTY OF SCIENCE

DEPARTMENT OF CHEMISTRY

ASSESSMENT OF THE PHYTOCHEMICAL COMPOUNDS AND

ANTIBACTERIAL POTENTIAL OF Portulaca oleracea PLANT LEAVES
ON SELECTED BACTERIA

BY

KIMULI LILLIAN

18/U/CTD/8086/PD

A RESEARCH REPORT SUBMITTED IN PARTIAL FULFILMENT OF

THE REQUIREMENTS FOR THE AWARD OF A BACHELOR’S DEGREE
OF SCIENCE TECHNOLOGY-CHEMISTRY OF KYAMBOGO
UNIVERSITY

MAY 2022
 DECLARATION
I, KIMULI LILLIAN, hereby declare that the work Presented in this report is exclusively a true
and original account of my research study under the topic ““Assessment of the

phytochemical compounds and antibacterial potential of Portulaca oleracea

plant leaves on selected bacteria” It has never been submitted to any other university or
institution of higher learning for any academic award.

Name: KIMULI LILLIAN.

RegNo.:18/U/8086/CTD/PD

Signature………………..

Date…………………………..

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 APPROVAL
This research report by KIMULI LILLIAN (18/U/CTD/8086/PD) under the topic “Assessment
of the phytochemical compounds and antibacterial potential of Portulaca oleracea plant
leaves on selected bacteria” was developed with my consultation and it is hereby approved for
submission to the Department of Chemistry, Kyambogo University.

Sign: (Supervisor)

…………………………………………

Dr. HANNINGTON TWINOMUHWEZI

Department of Chemistry,

Kyambogo University

Date: .......................................

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 DEDICATION
This piece of work is dedicated to my parents Mrs. Atine Sylvia and Mr Atine George for the
selfless sacrifice and support they have rendered to shape me into the woman that I am today. It
is because of them that I have come this far. For indeed, they have always believed in my
abilities and capabilities for which reason, I am deeply proud of.

iv
 ACKNOWLEDGEMENT
Verily, I am grateful to the Almighty God for my life, education, health and all the unlimited
bounties that He has bestowed upon me. I also convey my sincere appreciations to my supervisor
Dr. Hannington Twinomuhwezi for the close supervision and utmost guidance he gave me
throughout the entire course of the research study. He dedicated much of his time to ensure that I
produce the best out of my research. I equally credit the work of Mr. Isaac, Mr. Olado Simon
Peter for the technical support and assistance, they accorded me in the Kyambogo University
Laboratories. I am also pleased to acknowledge the significant contributions of my family
members for the moral and financial support they have generally given me throughout my
academic life as well as my fellow researchers; Odongo Tonny, Muhindo Jeniffer, Isaac and
Brenda. It is them that enabled the success and completion of this research through embracing
team work and laying a support platform.
May, the Almighty God bless them all.

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 TABLE OF CONTENTS
DECLARATION .............................................................................................................................ii
APPROVAL ...................................................................................................................................iii
DEDICATION ................................................................................................................................ iv
ACKNOWLEDGEMENT ...............................................................................................................v
LIST OF TABLES ........................................................................................................................ viii
LIST OF FIGURES ........................................................................................................................ ix
LIST OF ABBREVIATIONS AND ACRONYMS ........................................................................x
DEFINITIONS OF OPERATIONAL TERMS .............................................................................. xi
ABSTRACT ................................................................................................................................... xii
CHAPTER ONE: INTRODUCTION ............................................................................................. 1
1.1 Background of study ............................................................................................................. 1
1.2 Problem Statement ................................................................................................................ 2
1.3 General objective................................................................................................................... 2
1.3.1 Specific objectives .................................................................................................... 2
1.4 Scope of the study ................................................................................................................. 2
1.5 Justification ........................................................................................................................... 3
CHAPTER TWO: LITERATURE REVIEW ................................................................................. 4
2.1 Purslane (Portulaca oleracea) ................................................................................................ 4
2.1.1 Origin .............................................................................................................................. 4
2.1.2 Classification .................................................................................................................. 4
2.1.3 Propagation and distribution ........................................................................................... 5
2.1.4 Botanical Description of Portulaca oleracea ................................................................. 6
2.1.5 Benefits of Portulaca oleracea....................................................................................... 6
2.2 Photochemistry ...................................................................................................................... 7
2.2.1 Phenolic Compound ....................................................................................................... 8
2.2.2 Flavonoid Compound ..................................................................................................... 9
2.2.3 Alkaloids ....................................................................................................................... 10
2.2.4 Terpenoids .................................................................................................................... 10
2.2.5 Tannins ......................................................................................................................... 11
2.2.6 Other chemical constituents.......................................................................................... 12

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 2.3 Antimicrobial Agents .......................................................................................................... 13
2.3.1 Anti-microbial Activity of Portulaca oleracea ............................................................ 13
2.3.2 Pharmacological activities of Portulaca oleracea........................................................ 14
CHAPTER THREE: MATERIALS AND METHODS ............................................................... 16
3.1 Materials .............................................................................................................................. 16
3.2 Sample collection and preparation ...................................................................................... 16
3.3 Preparation of the aqueous crude extract ............................................................................ 16
3.4 Phytochemical analysis of aqueous crude extract ............................................................... 16
3.4.1 Test for Saponins .......................................................................................................... 16
3.4.2 Test for Alkaloids ......................................................................................................... 16
3.4.3 Test for anthraquinones ................................................................................................ 17
3.4.4 Test for Tannins ............................................................................................................ 17
3.4.5 Test for Quinones ......................................................................................................... 17
3.4.6 Test for Cardiac glycosides (Keller-Kiliani test) .......................................................... 17
3.4.7 Test for Terpenoids (Salkowski test) ............................................................................ 17
3.5 Determination of the antibacterial potential of the aqueous crude extract ...................... 17
3.5.1 Micro-organisms tested ................................................................................................ 17
3.5.2 Preparation of standard bacterial suspensions .............................................................. 17
3.5.3 Preparation of inoculum ............................................................................................... 18
3.6 Data analysis ....................................................................................................................... 18
CHAPTER FOUR: RESULTS AND DISCUSSION ................................................................... 19
4.1 Results ................................................................................................................................. 19
4.1.1 Extraction yield of Portulaca Oleracea leaves ............................................................ 19
4.2 Discussion of the Results .................................................................................................... 21
CHAPTER FIVE: CONCLUSIONS AND RECOMMENDATIONS ......................................... 22
5.1 Conclusions ......................................................................................................................... 22
5.2 Recommendations ............................................................................................................... 22
REFERENCES ............................................................................................................................. 23
APPENDICES .............................................................................................................................. 25
Appendix I: Research Activities ............................................................................................... 25

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 LIST OF TABLES
Table 1. Percentage yield of the DCM/methanolic extract of Portulaca oleracea ...................... 19

Table 2. Secondary metabolites identified in extract .................................................................... 19

Table 3. Zone of inhibition (mm) of extract on E. coli ................................................................. 19

Table 4. Zone of inhibition (mm) of extract of portulaca oleracea on S. aureus .......................... 20

viii
 LIST OF FIGURES
Figure 1: Portulaca oleracea plant leaves and flowers .................................................................... 6

Figure 2: Basic structure of some important plant derived phenolics ............................................ 9

Figure 3: Basic structures of some important plant derived Flavonoids ...................................... 10

Figure 4: Basic structures of some important plant derived Alkaloids ......................................... 10

Figure 5: Basic structures of some important plant derived Terpenoids ...................................... 11

Figure 6: Basic structures of some important plant derived tannins ............................................. 12

Figure 7: Mean ZOI of extracts on E. coli ................................................................................... 20

Figure 8: Mean ZOI of extracts on S. aureus................................................................................ 20

ix
 LIST OF ABBREVIATIONS AND ACRONYMS
E. coli Escherichia coli
S. aureus Staphylococcus aureus
P. oleracea Portulaca oleracea
ZOI Zone of inhibition
℃ Degree centigrade
μL Microliter
L Litre
mL Mililitre
mm Millimeter
Psi Pascals square inch

DCM Dichloromethane

DMSO Dimethyl Sulfoxide

x
 DEFINITIONS OF OPERATIONAL TERMS
Zone of inhibition: circular area around the spot of the antibiotic in which the bacteria colonies
do not grow, used to measure the susceptibility of the bacteria towards the antibiotic.

Phytochemicals: Bioactive chemical compounds found in fruits, vegetables, grains, and other
plant foods that may provide desirable health benefits beyond basic nutrition to reduce the risk of
chronic diseases.

Antibiotics: Medicines that fight bacterial infections in people and animals; they work by killing
bacteria or by making it hard for bacteria to grow and multiply.

Antibacterial: Any substance that destroys or otherwise inhibits the growth of bacteria.

Antibacterial resistance: Ability of a bacteria to multiply under conditions that would otherwise
inhibit other members of the strain.

xi
 ABSTRACT
The aim of the study was to assess the phytochemical compounds and antibacterial potential of
Portulaca oleracea plant leaves on selected bacteria. Shade dried samples were ground to
powder and extracted using maceration method with methanol and dichloromethane as solvents
in the ratio of 1:1. Standard phytochemical screening procedures were followed to test for the
presence of phytochemicals. The antibacterial activity was achieved using agar well diffusion
method. Phytochemical screening revealed the presence of terpenoids, steroids, alkaloids,
saponins and cardiac glycosides in the crude extract whereas flavonoids and tannins were absent.
The results obtained from the antibacterial activity revealed that the extract considered exhibited
significant antibacterial activity against certain bacteria. The leaves extracts showed higher
antibacterial activity on E. coli and S. aureus with mean zone of inhibition of 27.3±0.1 mm and
10.7±0.1 mm respectively. The leaf extract had higher antibacterial effects on E. coli compared
to S. aureus. The difference in the activity was due to the difference in the concentration of anti-
bacterial agents. Thus, E.coli was regarded as susceptible to the leaf extract as the mean zone of
inhibition was within the break for standard such as ampicillin ≥ 21 mm, amoxicillin ≥ 21 mm
and ceftriaxone ≥21 mm. Characterization of these plant extracts for pure compounds, toxicity as
well as their synergistic effects is recommended.

xii
 CHAPTER ONE: INTRODUCTION
1.1 Background of study
Many efforts have been made to discover new antimicrobial compounds from various kinds of
sources such as micro-organisms, animals, and plants. One of such resources is folk medicines.
Systematic screening of them may result in the discovery of novel effective compounds. The
increasing prevalence of multi drug resistant strains of bacteria and the recent appearance of
strains with reduced susceptibility to antibiotics raises the specter of untreatable bacterial
infections and adds urgency to the search for new infection-fighting strategies. For these reasons,
medicinal plants are important substances for the study of their traditional uses through the
verification of pharmacological effects and can be natural composite sources that act as new anti-
infectious agents (Weese, 2011).

Herbal medicine is still the mainstay of about 75-80% of the whole population, mainly in
developing countries, for primary health care because of better cultural acceptability, better
compatibility with the human body and fewer side effects. However, the last few years have seen
a major increase in their use in the developed world (Tong, 2015).

Nowadays multiple drug resistance has developed due to the indiscriminate use of commercial
antimicrobial drugs commonly used in the treatment of infectious disease. In addition to this
problem, antibiotics are sometimes associated with adverse effects on the host including
hypersensitivity, immune-suppression and allergic reactions. This situation forced scientists to
search for new antimicrobial substances. Given the alarming incidence of antibiotic resistance in
bacteria of medical importance, there is a constant need for new and effective therapeutic agents
(Tong, 2015).

Because of the side effects and the resistance that pathogenic microorganisms build against
antibiotics, recently much attention has been paid to extracts and biologically active compounds
isolated from plant species seed in herbal medicine. Plant-based antimicrobials represent a vast
untapped source of medicines and further exploration of plant antimicrobials needs to occur.
Antimicrobials of plant origin have enormous therapeutic potential. They are effective in the
treatment of infectious diseases while simultaneously mitigating many of the side effects that are
often associated with synthetic antimicrobials (Narasinga, 2003).

1
All medicinal, plant contains certain active constituent, it responsible to some pharmacological
activity. The medicinal actions of plants are unique to a particular plant species or group,
consistent with the concept that the combination of secondary products in a particular plant is
taxonomically distinct. Therefore, there is a need to develop alternative antimicrobial drugs for
the treatment of infectious diseases from medicinal plants.

1.2 Problem Statement

Infectious diseases have become a very big problem to the community today as a result of their
easy wide spread across a very large population in a short time. People have tried to make use of
the available antibiotics despite the many side effects caused by these drugs. Nevertheless, these
diseases have become resistant due to their mother causing germs being no longer affected by the
intake of the available antibiotics. This has left a very big challenge on how the community can
overcome these disease-causing germs easily without getting side effects as well as eliminating
the resistant germ strains. One of the remedies could be herbal medicine. The use of Portulaca
oleracea plant leaves in treating infectious diseases has not been exploited and yet it could have
a potential to kill these germs. Thus, the assessment of the antibacterial potential of Portulaca
oleracea plant leaves has been explored in this study.

1.3 General objective

The general objective of this research was to assess the phytochemical compounds and
antibacterial potential of Portulaca oleracea plant leaves on selected bacteria.

1.3.1 Specific objectives

The above general objective was based on the following specific objectives:

2. To determine the phytochemical compounds of Portulaca oleracea plant leaves.

3. To determine the antibacterial potential of Portulaca oleracea plant leaves on the
selected bacteria.

1.4 Scope of the study

The study involved the phytochemical analysis and determination of the antibacterial potential of
Portulaca oleracea plant leaves on two selected bacteria (Gram positive; Staphylococcus aureus
and Escherichia coli). The plant material was collected from Ngogwe village in Wakiso district
and transferred to the Department of chemistry at Kyambogo University for analysis.the research
activity took a duration of two months, from September to October 2021.

2
1.5 Justification
The study was conducted in order to document the phytochemical components and antibacterial
activity of Portulaca oleracea ethanolic crude extract. This comes as a result of the community
dependency on this plant to treat their infections without any scientific knowledge about it.

3
 CHAPTER TWO: LITERATURE REVIEW
2.1 Purslane (Portulaca oleracea)
This is one of the members from Family portulacaceae and the genus Portulaca. The ‘weed’
purslane (Portulaca oleracea ) is a weed of open agricultural habitats, especially in turf grass and
field crops areas. It revealed tremendous nutritional potential and has indicated the potential use
of this herb for the future (Alam et al., 2014).

The plants are characterized by its taller upright growth habit and larger leaves and seeds.
Purslane is a nutritious vegetable which can be consumed by human being and it was mentioned
in Egyptian texts from the time of the Pharaohs. Portulaca oleracea can be eaten raw as a salad,
eaten cooked as a sauce in soups or eat as green vegetables. Portulaca oleracea provides a rich
plant source of nutritional benefits. According to Simopoulos and Salem. (2016), P. oleracea is
one of the green plants which is rich in omega fatty acids and a linolenic acid (Chowdhary et al.,
2013).

2.1.1 Origin
It was first identified in the United States in 1672 in Massachusetts. The name Portulaca is
thought to be derived from the Latin „Porto‟ meaning „to carry‟ and „lac‟ meaning milk, since
the plant contains a milky juice; oleracea from Latin, meaning „pertaining to kitchen gardens‟,
referring to its use as a vegetable. The use of this plant as a vegetable, spice and medicine has
been known since the times of the ancient Egyptians and was popular in England during the
middle Ages (Chowdhary et al., 2013).

2.1.2 Classification
Kingdom - plantae
Subkingdom - tracheobionta
Super division - spermatophyte
Division - magnoliophyte
Class - magnoliopsida
Subclass - caryophyllidae
Order - caryophyllales
Family - portulacaceae
Genus - portulacae L.

4
Species - Portulacae oleracea L.

2.1.3 Propagation and distribution

Purslane is distributed all over the world; Portulaca oleracea is a herbaceous annual, native of
many parts of Europe, found in the East and West Indies, China, Japan and Ascension Island,
and though found also in the British Isles is not indigenous there. It is a weedy summer annual
species that is abundant throughout the world, invading vegetable gardens, bare areas, low-
maintenance lawns, ornamental plantings, and agricultural areas (Bae, 2004).

It is particularly well adapted to the warm, moist conditions found in California’s irrigated
agricultural and ornamental sites. It has been cultivated in India and the Middle East and has
been popular in Europe since the Middle Ages. Common purslane germinates in California from
February to March in the southern desert areas to late spring in cooler areas when soil
temperature reaches about 60°F. For an early crop, the seed is best sown under protection in
early spring and can then be planted out in late spring. Outdoor sowings in situ take place from
late spring to late summer, successional sowings being made every two to three weeks if a
constant supply of the leaves is required (Harborne & Baxter, 2009).

It germinates very near to or at the soil surface in large numbers after an irrigation or rain. Most
of the tiny seedlings die, but the survivors grow rapidly and can produce flowers in a few weeks.
The fleshy stems of common purslane can remain moist and viable for several days after
cultivation and hoeing, and reroot to form “new” plants when gardens or fields are irrigated.
Because of its ability to produce large numbers of seeds, common purslane can rapidly colonize
any warm, moist site. It requires a moist light rich well-drained soil in a sunny position. Plants
will not produce good quality leaves when growing in dry conditions (Karimi, G. et al., 2010).

The plants take about six to eight weeks to produce a crop from seed and can then be harvested
on a cut and come again principle, providing edible leaves for most of the summer. Common
purslane is low in stature and forms dense mats. These vegetative mats utilize available moisture
and nutrients and screen out light to the soil surface, preventing emergence of other seedlings.
Common purslane is unsightly, reducing purslane can limit summer vegetable production and
reduce the efficiency of harvesting nut crops, such as almonds and walnuts, from the orchard
floor (Mohamed & Hussein, 2016).

5
2.1.4 Botanical Description of Portulaca oleracea
Portulaca oleracea is a succulent annual herb. Stems of Portulaca oleracea are sometimes
flushed red or purple, not articulated, prostrate or decumbent, less often erect, diffuse, much
branched; leaf axils with a few inconspicuous stiff bristles (Figure 1).

Figure 1: Portulaca oleracea plant leaves and flowers

The leaves are alternate or occasionally sub-opposite, with short petiole and flat leaf blade,
obovate, 10-30 × 5-15 mm, base cuneate, and apex obtuse, rounded and truncated. The flowers
of Portulaca oleracea are in clusters of three to five, 0.4-0.5 cm in diameter, surrounded by
involucre of two to six bracts. Sepals are green, helmeted, ca. 4 mm, apex acute and keeled.
Petals 5, yellow, obovate, 3-5 mm, slightly connate at base, apex retuse (Narasinga, 2003).
Stamens 7-12, circa 12mm; anthers are yellow. The ovary is glabrous with four to six lobed
stigmas. Capsule ovoid, ca. 5 mm. Seeds are glossy black when mature, never iridescent,
obliquely globose-reniform, 0.6-1.2 mm; testa cells stellate, usually with central peg like
tubercle, sometimes without and then surface granular (Hassawi & Kharma, 2006).

Seed production of these plants is very high (one plant can introduce up to 10,000 seeds to the
environment. It has a slightly sour and salty taste. The stems, leaves and flower buds are all
edible.

2.1.5 Benefits of Portulaca oleracea

The young leaves are a very acceptable addition to salads, their mucilaginous quality also
making them a good substitute for okra as a thickener in soups. Older leaves are used as a

6
potherb. The seed can be ground into a powder and mixed with cereals for use in gruels, bread,
pancakes. The plant is antibacterial, antiscorbutic, depurative, diuretic and febrifuge. The fresh
juice is used in the treatment of strangury, coughs and sore

The leaves are poulticed and applied to burns, both the leaves and the plant juice are particularly
effective in the treatment of skin diseases and insect stings. A tea made from the leaves is used in
the treatment of stomach aches and headaches. The leaf juice is applied to earaches, it is also said
to alleviate caterpillar stings (Tong, 2015). The leaves can be harvested at any time before the
plant flowers; they are used fresh or dried. This remedy is not given to pregnant women or to
patients with digestive problems. The seeds are tonic and vermifuge. They are prescribed for
dyspepsia and opacities of the cornea.

To complete the range of its applications, one could mention its use as an insecticide, in which
case its juice is poured on to anthills, and also its ornamental use in Roman and medieval
gardens. Another authority declared that the distilled water took away pains in the teeth, the
seeds, bruised and boiled in wine, were given to children as a vermifuge

In Africa, the whole plant is considered antiphlogistic (takes the heat out) and bactericide in
bacillary dysentery, diarrhoea, haemorrhoids, enterorrhaghia. It has been used in prescriptions as
an antidiabetic. Externally it is used as a cataplasm of fresh leaves for maturing of abscesses. The
seeds are also calmative and will help slake a thirst. An infusion is used as anthelmintic for
children to expel roundworms, in high doses as an emetic and also as a cooling drink, with a mild
diuretic effect

In Nigeria the plant is used as a diuretic. The bruised leaves are used in external application for
erysipelas, treatment of burns and are applied topically to swellings. In Benin area, the plant
along with other ingredients is taken as an aid to the development of the foetus.

2.2 Photochemistry
Phytochemicals are biologically active and naturally occurring chemical compounds found in
plants. It provides health benefits for humans further than those attributed to macronutrients and
micronutrients. Recently, phytochemical was reported to have roles in protection of human
health when their dietary consumption is significant.

7
Dietary phytochemicals can be found in fruits, vegetables, legumes, whole grains, nuts, herbs
and also spices. Costa et al had reported that the phytochemical will accumulate in different parts
of the plants including stems, roots, leaves, flowers, fruits or even seeds. These compounds are
known as secondary plant metabolites which have biological properties such as antioxidant
activity, antimicrobial effect, modulation of detoxification enzymes, stimulation of immune
system, decrease of platelet aggregation and modulation of hormone metabolism and anticancer
property.

Recently, researches had reported that many phytochemicals can used to protect human against
diseases. Phytochemicals can be classified as primary or secondary constituents based on their
role in plant metabolism. The examples of primary constituents are common sugars, amino acids,
proteins, purines and pyrimidines of nuclei acids, chlorophyll and others. Secondary constituents
are the remaining plant chemicals including alkaloids, terpenes, flavonoids, lignans, plant
steroids, curcumines, saponins, phenolic and glucosides (Ghufran, 2017). Many constituents of
Portulaca oleracea have been isolated, including flavonoids, alkaloids, fatty acids, terpenoids,
polysaccharides, vitamins, sterols, proteins, and minerals.

2.2.1 Phenolic Compound

Phenolic phytochemicals which are widely distributed in plant kingdom are the largest category
of phytochemicals. Phenolic are hydroxyl group (-OH) containing class of chemical compounds
where the (-OH) bonded directly to an aromatic hydrocarbon group. Phenol (C6H5OH) is
considered the simplest class of this group of natural compounds.

Phenolic compounds are a large and complex group of chemical constituents found in plants.
They are plant secondary metabolites which play an important role as defence compounds.
Phenolics exhibit several properties that bring benefits to human beings. Its antioxidant
properties are important as protecting agents against free radicalmediated disease processes.
Phenolic acids form a diverse group that includes the widely distributed hydroxybenzoic and
hydroxycinnamic acids (Zhou et al., 2015). Based on the previous study by Uddin et al., (2012),
total phenolic content (TPC) of the edible aerial parts of P. oleracea at different growth stage
was reported. The shoots were collected from 15, 30, 45 and 60 days old plants. The result shows
that the TPC value for 15 days old plant was significantly lower compared to 30, 45 and 60 days
old plants. The TPC of 60 days mature plant was slightly lower than those plants under

8
developing stage. It can be concluded that TPC content was higher when the plant was under
developing stage.

H CO
3

H OH
HO O
OH
O O
O O
HO
HO OH
OH H OH

Figure 2: Basic structure of some important plant derived phenolics

2.2.2 Flavonoid Compound

Flavonoids are polyphenolic compounds that are ubiquitous in nature. More than 4,000
flavonoids have been found in many vegetables, fruits and beverages like tea, coffee and fruit
drinks. The flavonoids have played a major role in successful medical treatments of ancient
times until today. Flavonoids are ubiquitous among vascular plants and occur as aglycones,
glucosides and methylated derivatives. According to Harborne and Baxter (2009), more than
4000 flavonoids have been found within the parts of plants normally eaten by humans and
approximately 650 flavones and 1030 flavanols are known. Small amount of aglycones are
frequently present and occasionally represent a considerably important proportion of the total
flavonoid compounds in the plant.

In the Portulaca oleracea plant, the flavonoids levels vary according to the part of the plant; the
highest levels are present in the root followed by stem and the leaf; and seven different
flavonoids are present in this plant, including kaempferol, myricetin, luteolin, apigenin, q
uercetin, genistein, and genistin (Figure 3). However, only kaempferol and apigenin have been
found in ethanolic extracts of leaves and stems, with the levels in the former being higher.
Portulacanones B–D, three homoisoflavonoids compounds, display selectively cytotoxic
activities against three human cancer cell lines.

9
 OH

OH OH OH

HO O HO O HO O

OH
OH O OH O OH O

Kaempfero Apigenin
l

Figure 3: Basic structures of some important plant derived Flavonoids

2.2.3 Alkaloids
In addition to flavonoids, another important chemical found in this plant is alkaloids including
dopa, dopamine, and noradrenalin. The content of dopamine and noradrenalin is higher in leaves
compared to stem and seeds. The amount of dopamine and noradrenalin obtained from leaves
varies according to the solvents used in the extraction process, suggesting that the levels of these
compounds are dependent on the solvents used during the extraction process. Oleraceins A,
Oleraceins B, Oleraceins C, Oleraceins D, Oleraceins E (figure 4) are cyclodopa alkaloids
isolated from this plant (Petropoulos et al.,, 2019).

HO H
OH HO H
O COOH OH
O N O COOH
O N
OH
O C OH
OH O C
OH
OH OH

Oleraceins A OCH
Oleraceins B

Figure 4: Basic structures of some important plant derived Alkaloids

2.2.4 Terpenoids
Terpenes are among the most widespread and chemically diverse groups of natural products.
They are flammable unsaturated hydrocarbons, existing in liquid form commonly found in
essential oils, resins or oleoresins. Terpenoids includes hydrocarbons of plant origin of general
formula (C5H8)n (Figure 5) are classified as mono-, di-, tri- and sesquiterpenoids depending on

10
the number of carbon atoms. Diterpenes (C20) are classically considered to be resins and taxol,
the anticancer agent, is the common example.

Some of the terpenoid compounds found in Portulaca oleracea include; Portuloside A,

Portuloside B, Portulene, Lupeol (Figure ) (Parham et al., 2020).

Portuloside B Portuloside A

Figure 5: Basic structures of some important plant derived Terpenoids

2.2.5 Tannins
These are widely distributed in plant flora. They are phenolic compounds of high molecular
weight. Tannins are soluble in water and alcohol and are found in the root, bark, stem and outer
layers of plant tissue. They are acidic in reaction and the acidic reaction is attributed to the
presence of phenolics or carboxylic group. They form complexes with proteins, carbohydrates,
gelatin and alkaloids. Tannins are divided into hydrolysable tannins and condensed tannins.

Tannins are used as antiseptic and this activity is due to presence of the phenolic group.
Common examples of hydrolysable tannins include theaflavins (from tea), daidezein, genistein
and glycitein (Figure 6) (Dabbou et al., 2019).

11
 Figure 6: Basic structures of some important plant derived tannins

2.2.6 Other chemical constituents

Portulaca oleracea is also an excellent source of omega3 fatty acids, which is usually present in
oil and fat of fishes but not normally found in plants. Omega-3 fatty acids play an important role
in the enhancement of immune function and prevention and treatment of hypertension, coronary
artery disease, cancer, and other inflammatory and autoimmune disorders. It includes 𝛼-linolenic
acid and linoleic acid, which are essential for normal growth, health promotion, and disease
prevention in humans (Zhou et al., 2015).

Polysaccharides found in Portulaca oleracea are potential therapeutic agents for the treatment of
diabetes mellitus owing to their modulation of blood lipids, metabolism, and decrease of blood
glucose. In addition, vitamins have also been isolated from the leaves of this plant. It contains the
highest content of vitamin A which is a natural antioxidant playing an important role in vision,
maintaining healthy mucus membranes and protecting against lung and oral cavity cancers
among green leafy vegetables (Petropoulos et al., 2019).

This plant also contains ascorbic acid, 𝛼-tocopherol, and B-complex vitamins, for example,
niacin, pyridoxine, and riboflavin. Furthermore it is rich in minerals like phosphorus, manganese,
icon, calcium selenium, and the amino acids isoleucine, proline, leucine, lysine, phenylalanine,
methionine, cystine, valine, threonine, and tyrosine (Sicari et al., 2018).

12
2.3 Antimicrobial Agents
An agent which can kill microorganisms or stop their growth is known as an antimicrobial agent
or antimicrobial medicine. Antimicrobial medicines are categorized based on the primary
microorganisms they act against such as bacteria and viruses (Alam et al., 2014). Antimicrobial
agents are divided into two groups based on their different chemical substances.

The first group is synthetic antimicrobial agents (chemical antimicrobial agents) including
antibiotic drugs and metal and metal oxide nanoparticles including silver, silver oxide, and so on.
Antibiotics and other chemical antimicrobial agents play a big role as antimicrobial agents, but
they lead to various side effects. One of the main side effects is the generation of free oxygen
radicals (ROS). ROS are very toxic and have been thought to play a main role in producing
cancer.

The second group is related to herbal antimicrobial agents, such as clove, portulaca, tribulus,
eryngium, cinnamon, turmeric, ginger, thyme, pennyroyal, mint, fennel, chamomile, burdock,
eucalyptus, primrose, lemon balm, mallows, and garlic. These biomaterials can act as free radical
scavengers and can therefore block the production of ROS (Chowdhary et al., 2013).

2.3.1 Anti-microbial Activity of Portulaca oleracea

Ramesh and Hanumantappa, (2011) reported the phytochemical and anti-microbial activity in the
aerial parts of chloroform and ethanolic extracts of Portulaca oleracea by agar diffusion method.
The antimicrobial activity in Portulaca oleracea are used in against five bacteria for example
Bacillus cereus and Klebisilla pneumonia and three fungi such as Aspergillus fumigates and
Nerospora crassa (Asia et al., 2014).

Ethanolic crude extract of P. oleracea showed maximum effect on organism such as

Staphylococcus aureus, Klebisilla pneumonia and Nerospora crassa. Whereas chloroform extract
of Portulaca oleracea showed moderate effect on Klebisilla pneumonia, Aspergillus niger and
Nerospora crassa. The result of this research supported the folklore usage of the studied plant and
shows that the extract of this studied plant contains compounds which have anti-microbial agent
in the form of drugs for the therapy of infectious diseases caused by pathogens (Karimi et al.,
2010).

13
Based on the previous study of Bae, (2004), antimicrobial effect of P. oleracea extracts on food
borne pathogens was assessed. His study had found that ethyl acetate extract was having highest
antimicrobial activity against Staphylococcus aureus and Shigella dysentrica compared to
petroleum ether, chloroform and methanol extracts. Strong antimicrobial activity was found from
the ethyl acetate extract of P. oleracea to against Staphylococcus aureus at 4000 ppm
concentration (Asia et al., 2014).

2.3.2 Pharmacological activities of Portulaca oleracea

2.3.2.1 Anti-arthritic Activity
Jagan et al. (2012) reported the anti-arthritic activity of Petroleum-ether extract of Portulaca
oleracea Linn by Fruends Adjuvant arthristis model in male wistar rats. The extracts of
Portulaca oleracea were at the dose of 100, 200 and 300 mg/kg/p.o and standard as
Indomethacin at a dose of 100 mg/kg. Maximum of inhibition which is about 77.82% was
observed on 21st day. This study revealed the anti-arthritic activity of aqueous extract of
Portulaca oleracea (Sicari et al., 2018).

2.3.2.2 Hepatoprotective Activity

Based on the previous study of Prabhakaran et al. (2010), the suspensions of methanol and
petroleum ether extracts of whole plant parts of P. oleracea in carboxy methyl cellulose were
evaluated for hepatoprotective activity in Wister albino rats by inducing hepatic injury with D-
galactosamine (400 mg/kg). At the dose levels of 200 and 400 mg/kg, altered biochemical
parameters were significantly restored when compared to d-galactosamine and Silymarin treated
groups.

Karimi et al. (2010) reported the aqueous and ethanolic extract of P. oleracea against cisplatin
induced acute renal toxicity in rats. Treatment with aqueous and ethanolic extracts in the highest
dose (0.8 and 2 g/ kg), 6 and 12 hour before cisplatin injection reduced blood urea nitrogen and
serum creatinine.

2.3.2.3 Neuronal Activity

The neuronal activities of aqueous extract of stem and leaves of Portulaca oleracea with a dose
of 1.5ml/kg in adult rats for 12 days were reported by Abdel et al., (2012). This study showed
significant decrease in the Ca2+ level in cerebral cortex by about -25.2% at p<0.05. There was

14
significantly decrease in dopamine content in spinal cord but significantly increase in dopamine
content in cerebellum, cerebral cortex, thalamus and hypothalamus of rats. This study concluded
that Portulaca oleracea has the potential as neurotransmitters, which plays an integral part of
many neurodegenerative disorders.

2.3.2.4 Anti-inflammatory Activity

According to Chan et al. (2000), P. oleracea sub sp. sativa was evaluated for further work
because of its abundant availability from reliable sources. The 10% ethanolic extract of the aerial
parts showed significant anti-inflammatory activity in the carrageenan-induced hind paw oedema
and the cotton pellet-induced granuloma models in rats. Besides that, significant analgesic
activity were also found in the hot-plate and tail flick models (in mice and rats, respectively)
after intraperitoneal administration (Azuka & Abu, 2014).

2.3.2.5 Skeletal Muscle Relaxant Activity

Parry et al. (1993) reported the skeletal muscle relaxant activity of aqueous extract of the stems
and leaves of P. oleracea. It was found that the extract of P. oleracea able to stop the twitch
contraction of the directly stimulated rat hemidiaphragm preparation. The effect of the extract
mimic qualitatively the action of potassium oxalate which is one of the known constituent of
Portulaca oleracea on the diaphragm was also reported (Petropoulos et al., 2019).

Removal of potassium ions from the methanol extract by passing it through a cation exchange
resin reduced the inhibitory effect of the extract. A positive relationship between the
concentration of potassium ions in the extract and the effects of potassium chloride of similar
molarity were reported. Hence, it can be concluded the relaxant effect observed on the isolated
rat diaphragm is because of the potassium ion content of Portulaca oleracea.

15
 CHAPTER THREE: MATERIALS AND METHODS
3.1 Materials
Test tubes, conical flasks, glass beakers, filter papers, measuring cylinder, analytical balance,
distilled water, grinder, spatula, SPSS software package, glass containers, Portulaca oleracea
plant leaves and petri dishes.

3.2 Sample collection and preparation

The Fresh plants/plant parts were collected from Ngogwe village wakiso district. Fresh plant
materials were washed under running tap water, air dried, and then homogenized to fine powder
and stored in airtight bottles.

3.3 Preparation of the aqueous crude extract

The leaves were finally milled to a coarse powder using mechanical grinding machine, so as to
enhance effective contact of solvent with sites on the plant materials. Powdered material (200 g)
was placed in a 2.5 L Winchester bottle and methanol (2 L) measured using a measuring cylinder
(1 L) added and plugged with cotton. The powder material was extracted with methanol for 72
hours at room temperature with continuous stirring. After 72 hours the supernatant was collected
by filtration using a cotton and a filter funnel. The solvent was then evaporated on a rotary
evaporator model strike 300 to make the crude extract. The residues obtained was stored in
airtight bottles in a refrigerator for analysis.

3.4 Phytochemical analysis of aqueous crude extract

3.4.1 Test for Saponins
Aqueous Crude extract (1 g) was weighed using the analytical balance and then mixed with
distilled water (5 mL) in a test tube and shaken vigorously. Five drops of olive oil was then
added using a dropper. The formation of stable foam will be taken as an indication for the
presence of saponins.

3.4.2 Test for Alkaloids

The aqueous crude extract (2 mL) was measured using a measuring cylinder (10 mL) and mixed
with Wagner’s reagent (2 mL). Reddish brown colored precipitate indicated the presence of
alkaloids.

16
3.4.3 Test for anthraquinones
To Aqueous crude extract (1 mL) in a test tube was added benzene (2 mL). This was filtered then
followed by ammonia solution (3 mL, 10%). A green solution which turns brown with formation
of a ring indicated presence of anthraquinones.

3.4.4 Test for Tannins

Sample (1 mL) was measured using a measuring cylinder (10 m)L into a test tube and then
Potassium ferricyanide (0.008 M, 1mL) was added. Ferric chloride1 (0.02 M, 1mL) containing 1
mL of 0.1 M dilute hydrochloric acid was added and observed for blue-black coloration.

3.4.5 Test for Quinones

Dilute sodium hydroxide solution (4 mL) was added to crude extract (1 mL). Blue green or red
coloration indicated the presence of quinones.

3.4.6 Test for Cardiac glycosides (Keller-Kiliani test)

Aqueous crude extract (1 mL) was treated with glacial acetic acid (2 mL) containing one drop of
ferric chloride solution. This was underlayed with concentrated sulphuric acid (1 mL). A brown
ring of the interface indicated a deoxy sugar characteristic of cardenolides. A violet ring
appeared below the brown ring, while in the acetic acid layer, a greenish ring formed just
gradually throughout thin layer.

3.4.7 Test for Terpenoids (Salkowski test)

Aqueous crude extract (5 mL) was measured using measuring cylinder (10 mL) and mixed with
chloroform (2 mL) and concentrated sulphuric acid (3 mL) carefully added to form a layer. A
reddish brown colouration of the inter face formed showed positive results for the presence of
terpenoids.

3.5 Determination of the antibacterial potential of the aqueous crude extract

3.5.1 Micro-organisms tested
The following strains of bacteria were used: Escherichia coli and Staphylococcus aureus. The
organisms were maintained in stock culture.

3.5.2 Preparation of standard bacterial suspensions

The average number of viable Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus
organisms per ml of the stock suspensions was determined by means of the surface viable
counting Technique as by Miles and Misra, (1938). About (108-109) colony-forming units per

17
ml was used. Each time, a fresh stock suspension was to be prepared; the experimental
conditions was maintained constant so that suspensions with very close viable counts would be
obtained.

3.5.3 Preparation of inoculum

Nutrient agar plates was swabbed (sterile cotton swabs) with 24 hour old - broth culture of
bacteria wells (10 mm diameter and about 2 cm a part) was made in each of these plates using
sterile cork borer. Stock solution of aqueous crude extract was prepared at a concentration of 1
mg/mL. Aqueous crude extract (100 µL) was added using a sterile syringe into the well and
allowed to diffuse at room temperature for 2 hrs. Control experiments comprising inoculums
without plant extract was set up.

The plates were incubated at 37°C for 24 h for bacterial pathogens and the diameter of the
inhibition zone (mm) was measured. Triplicates were maintained and the experiment was
repeated thrice, for each replicate the readings was taken in three different fixed directions and
the average values was recorded.

3.6 Data analysis

The zones of inhibition and the minimum inhibitory concentration of the extracts were measured
and recorded. The mean zones of inhibition of the extracts were calculated to assess and compare
the susceptibility of the bacteria. The results were displayed on bar graphs. Graphs of mean
zones of inhibition in millimeters against the extracts were plotted in each case using Microsoft
excel 2013.

18
 CHAPTER FOUR: RESULTS AND DISCUSSION
4.1 Results
4.1.1 Extraction yield of Portulaca Oleracea leaves
In natural products research, extraction yield measures a solvent’s efficiency to extract
compounds of interest from matrices. The yields obtained in this study were expressed as
percentages of the initial mass of the macerated dry leaf and fruit powders (Table 1).

Table 1. Percentage yield of the DCM/methanolic extract of Portulaca oleracea

Initial weight of the extract(g) Weight of extract(g) Percentage yield (%)
200 3.072 1.536

Table 2. Secondary metabolites identified in extract

Phytochemical Inference

Alkaloids +

Cardiac glycosides +++

Flavonoids ⁻

Phenols ++

Quinones +

Saponins ++

Steroids +++

Tannins ⁻

Terpenoids +++

Note: +++ represents very high, ++ indicates moderate, + indicates little/traces, and − indicates
absent.

Table 3. Zone of inhibition (mm) of extract on E. coli

Extract 1 2 3 Mean±SD

Leaves extract 36.0 35.0 11.0 27.3±0.1

Positive control 35.0 10.0 34.0 26.3±0.1
Negative control 0.0 0.0 0.0 0.0 ±0.0

19
Table 4. Zone of inhibition (mm) of extract of portulaca oleracea on S. aureus

Extract 1 2 3 Mean ±SD

Leaves extract
11.0 11.0 10.0 10.7±0.1

Positive control
22.0 21.5 21.0 21.5±0.1

Negative control
0.0 0.0 0.0 0.0 ±0.0

Figure 7: Mean ZOI of extracts on E. coli

Figure 8: Mean ZOI of extracts on S. aureus

20
From the graphs, the leaves extracts showed higher antibacterial activity on E. coli than S. aureus
with mean zone of inhibition of 27.3±0.1 mm and 10.7±0.1 mm respectively. The zone diameter
of complete inhibition is a function of the quantity of antibacterial agents and the susceptibility
of the bacterium.

4.2 Discussion of the Results

The results obtained from this investigation revealed that the extract considered exhibited
significant antibacterial activity against certain bacteria. The leaves extracts showed higher
antibacterial activity on E. coli and S. aureus with mean zone of inhibition of 27.3±0.1 mm and
10.7±0.1 mm respectively. The leaf extract had higher antibacterial effects on E. coli compared
to S. aureus. The difference in the activity was due to the difference in the concentration of anti-
bacterial agents. Thus, E. coli was regarded as susceptible to the leaf extract as the mean zone of
inhibition was within the break for standard such as ampicillin ≥ 21 mm, amoxicillin ≥ 21 mm
and ceftriaxone ≥21 mm
The secondary metabolites as well as the reported phytochemical compounds in
Methanol/Dichloromethane of Portulaca oleracea leaves could be responsible for the observed
antimicrobial activities.
Phytochemical screening of leaves showed the presence of several phytochemicals such as
alkaloids, phenols, saponins, quinones, Terpenoids, steroids, cardiac glycosides as shown in
Table 1.

The antibacterial activity shown by this plant materials may be linked to the presence of steroids,
cardiac glycosides, alkaloids and saponins which were reported to possess antimicrobial
activity(Bourgaud et al., 1994).
For example, saponins and alkaloids have reported antimicrobial activities which are attributed
to both their direct action against microorganisms or suppression of microbial virulence factors
(Daglia, 2012). Saponins inhibit microbial growth through precipitation of microbial proteins,
rendering such nutritional proteins unavailable to the microorganisms(Pradhan et al., 2009)..
Alkaloids exert their bactericidal effect through penetrating cells, intercalating microbial DNA
and targeting several nucleic acid enzymes which inflict irreversible damages to microbial cells
(Yi et al., 2007).

21
 CHAPTER FIVE: CONCLUSIONS AND RECOMMENDATIONS
5.1 Conclusions
High yield of extract was obtained from the leaves. Portulaca oleracea has the potentials for
production of antibacterial agents. The leaves contain alkaloids, phenols, saponins, quinones,
steroids, and cardiac glycosides. Staphylococcus aureus and Escherichia coli were both observed
susceptible to the extracts at 1 g/ml.
From the results obtained, Portulaca oleracea leaves showed superior antibacterial activity
against S. aureus and E. coli. Portulaca oleracea leaves can be used for making alternative
antibiotics for treating infections caused by E. coli and S. aureus.

5.2 Recommendations
Based on the findings from this study, it is recommended that further research should be carried
out to test Portulaca oleracea leaves extracts on other pathogenic microorganisms.
Further evaluation should be performed with the isolated pure compounds to give a definite
conclusion of the bioactive compounds contributing to the antibacterial activity of the Portulaca
oleracea leaves extract.
With definite findings of those responsible compounds, extracts of portulaca Oleracea can then
be used as antibacterial agents in foods, drug formulations, cosmetics products and other
ingredients.

22
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 APPENDICES
Appendix I: Research Activities

Portulaca oleracea Leaves Shade drying the leaves Sample Extraction

Leaves extract Rotary evaporation Determination of antibacterial

activity

25