ANTIMICROBIAL ACTIVITY AND CYTOTOXIC EFFECTS OF Stachytarpheta
jamaicensis (L.) Vahl CRUDE PLANT EXTRACTS
KU ANIS SHAZURA BT INDERA PUTERA
UNIVERSITI TEKNOLOGI MALAYSIA
ANTIMICROBIAL ACTIVITY AND CYTOTOXIC EFFECTS OF Stachytarpheta
jamaicensis (L.) Vahl CRUDE PLANT EXTRACTS
KU ANIS SHAZURA BT INDERA PUTERA
A dissertation submitted in partial fulfillment of the requirements for the award of the
degree of Master of Science ( Biotechnology)
Faculty of Biosciences and Bioengineering
Universiti Teknologi Malaysia
DECEMBER 2010
iii
Didedikasi khas buat
mereka-mereka yang disayangi,
mereka-mereka yang banyak membantu,
En Indera Putera bin Ku Man,
Pn Sharifah bt Yusuf,
Ku Mohd Ashraff bin Indera Putera
Ku Ashrul Aizat bin Indera Putera
Yang Teristimewa
Terima kasih atas sokongan kalian,
Akhirnya…..sempurna suatu perjalanan.
“ kerana sesungguhnya sesudah kesulitan itu ada kemudahan.
Sesungguhnya kesulitan itu ada kemudahan .” Surah Al-Insyirah, ayat 5-6.
iv
ACKNOWLEDGEMENT
Alhamdulillah, thanks to ALLAH, I am very grateful that I managed to finish
this project on time. In preparing this thesis, I was in contact with many people
including researcher, lecturers and academician. They have contributes towards my
thoughts and my understanding. In particular, I wish to express my thanks towards my
supervisor, Dr Salehhuddin Hamdan for everything that he had done in helping me
finishing my thesis. Moreover, I also would like to express my gratitude towards Miss
Tan Ai Lee and Miss Nurnida from Forest Research Institute Malaysia (FRIM) whom
has helped me directly during plant verification process in my project.
Last but not least, my fellow colleagues whom have support me through the process
especially my friend Nor Asma Husna bt Yusoff. Without cooperation, we can never
get through this. Finally, a million thanks for everyone whom I did not mention in this
limited space but always stays forever in my heart.
v
ABSTRACT
Medicinal plants are known for their ability to treat diseases due to the presence
of bioactive constituents such as alkaloids, phenolic compounds, saponin, tannins and
terpenoids. They can be used as anti-inflammation, antioxidants, antibiotics and anti
infection drugs. Stachytarpheta jamaicensis (L.) Vahl is one of the medicinal plants
found to be useful for treatment of diseases based on its traditional usage. This plant
belongs to the family of Verbenacea and can grow up to 90 to 120 cm tall.
Unfortunately, less research has been done on this plant. In this research, the objectives
are to screen for the presences of phytochemicals in the crude plant extract and its
antimicrobial activity. Antimicrobial activity was performed using disk diffusion
method. The crude extracts were tested for saponin, tannin, flavonoid, phlobatannin,
coumarin, phenolic compound and terpenoids using phytochemicals screening test. It
was found that the extracts contained phytochemicals such as phenolic compound,
tannin, saponin, terpenoids and flavonoid but absences of phlobatannin and coumarin.
From the antimicrobial test, it showed highest inhibition zone on Pseudomonas
aeruginosa with diameter of 10.5 mm by root extracts and slight or less inhibition on
the growth of Streptococcus sp on all three extracts compared to other types of bacteria.
The root extracts are found to inhibit most of the bacteria growth other than leaves and
stem extracts maybe because of the presence of most of the phytochemicals in it. Root
extract can be concluded to be the best parts to be used as antimicrobial drugs. In the
study for cytotoxic effect, leaves extract shows the highest inhibition on the growth of
Hela cancer cells compare to root and stem extract. During incubation of 24 and 72
hours, all of the extracts showed positive results, however, during 48 hours incubation,
the cell activities or growth became unstable. Hence, the extracts can only be exposed to
the cancer cell line at shorter time to avoid the lost of its phytochemicals from time to
time.
vi
ABSTRAK
Pokok ubatan dikenali dengan kebolehan ia untuk mengubati penyakit
disebabkan oleh kehadiran bahan bioaktif seperti alkaloid, fenolic, saponin, tannin dan
terpenoids. Ia boleh digunakan sebagai anti-keradangan, antioksidan, antibiotic dan ubat
antijangkitan. Stachytarpheta jamaicensis (L.) Vahl merupakan salah satu pokok ubatan
yang
diketahui
amat
berguna
untuk
menyembuhkan
penyakit
berdasarkan
penggunaannya secara tradisional. Pokok ini berasal dari keluarga Verbenacea dan
boleh membesar sehingga 90 ke 120 cm tinggi. Malangnya, hanya sedikit kajian
dilakukan berkenaan pokok ini. Objektif kajian ini adalah untuk mengesan kehadiran
jenis fitokimia yang terdapat dalam ekstrak tumbuhan ini serta aktiviti antibakterianya.
Ujian antibakteria dijalankan menggunakan ujian cakera serapan. Ekstrak tumbuhan
tersebut telah diuji untuk kehadiran saponin, terpenoids, tannin, bahan fenol, coumarin,
phlobatannin dan flavonoid, menggunakan ujian fitokimia. Setelah kajian dibuat,
ekstrak tersebut telah didapati mengandungi bahan fenol, tannin, saponin, terpenoid dan
flavonoid tetapi tiada kesan kehadiran coumarin dan phlobatannin. Berdasarkan kepada
ujian antibakteria, ia menunjukkan zon rencatan terbesar pada Pseudomonas aeruginosa
dengan diameter 10.5 mm dan sedikit ataupun tiada zon rencatan pada pertumbuhan
Streptococcus sp.berbanding bakteria lain. Ekstrak akar ditemui dapat menghalang
pertumbuhan kebanyakan bakteria berbanding ekstrak batang dan daun disebabkan oleh
kehadiran lebih banyak fitokimia di dalamnya. Ekstrak akar dapat disimpulkan sebagai
bahagian terbaik dari pokok untuk digunakan sebagai ubat antibakteria. Untuk kajian
kesan ketoksikan, ekstrak tumbuhan menunjukkan rencatan terbaik terhadap
pertumbuhan sel Hela dibandingkan dengan ekstrak akar dan batang. Semasa inkubasi
selama 24 dan 72 jam, kesemua ekstrak member kesan positif kecuali pada 48 jam
inkubasi, aktiviti sel menjadi tidak stabil. Maka, ekstrak tumbuhan ini hanya boleh
didedahkan pada sel untuk jangka masa yang pendek bgi mengelakkan dari kehilangan
fitokimia dari masa ke masa.
vii
TABLE OF CONTENTS
CHAPTER
1
2
TITLE
PAGE
DECLARATION
ii
DEDICATION
iii
ACKNOWLEDGEMENT
iv
ABSTRACT
v
ABSTRAK
vi
TABLE OF CONTENTS
vii
LIST OF TABLES
xi
LIST OF FIGURES
xii
LIST OF ABBREVIATION
xiv
INTRODUCTION
1.1 Background information
1
1.2 Problem statement
2
1.3 Objectives of the research
3
1.4 Scope of the research
3
1.5 Significance of the research
4
LITERATURE REVIEW
2.1 Medicinal plants
5
2.2 Stachytarpheta jamaicensis (L.) Vahl
6
2.2.1 General description
6
2.2.2Studies related to Stachytarpheta species
8
viii
2.3 Bioactive compounds in plants
9
2.3.1 Phenolic compounds
9
2.3.1.1 Flavonoids
9
2.3.1.2 Tannins
11
2.3.1.3 Coumarins
13
2.3.2 Terpenoids
15
2.3.2 Alkaloids
17
2.4 Antibiotics
3
18
2.4.1 Brief history on antibiotic
18
2.4.2 Antibiotic resistance by microorganisms
19
2.5 Antimicrobial properties of medicinal plants
21
2.6 Antimicrobial susceptibility test
22
2.7 Natural compounds and cancer
23
2.8 Cytotoxic assay
24
MATERIALS AND METHODS
3.1 Materials
26
3.1.1 Solutions and reagents
26
3.1.2 Plant Materials
26
3.1.3 Culturing Hela cell
27
3.2 Methods
27
3.2.1 Extraction of Stachytarpheta jamaicensis (L.) Vahl
27
crude extract
3.2.2 Phytochemical tests
28
3.2.2.1 Detection of phenolic compounds
28
3.2.2.2. Test for saponins or Froth test
29
3.2.2.3 Test for coumarins
29
3.2.2.4 Test for terpenoids (Salkowski test)
29
3.2.2.5 Test for flavonoids
29
3.2.2.6 Test for tannin
30
3.2.2.7 Test for phlobatannins
30
ix
3.2.3 Preparation of microbial culture
30
3.2.3.1 Medium preparation
30
3.2.3.2 Microbial culture
31
3.2.3.3 Culturing microorganisms on growth media
31
3.2.3.4 Identification of the bacteria
32
3.2.4 Antimicrobial susceptibility tests
33
3.2.4.1 Preparation of Mueller-Hinton agar
33
3.2.4.2 Preparation of saline solution
33
3.2.4.3 Inoculating microorganisms on Mueller
33
Hinton agar
3.2.4.4 Preparations and application of
34
antimicrobial discs
3.2.5 Recording data and interpreting the results
35
3.2.6 Cytotoxic activity of crude extracts of Stachytarpheta
35
jamaicensis (L.) Vahl on the growth of HeLa cell
3.2.6.1 Seeding Hela cell in 96-well microtiter plate
35
3.2.6.2 Addition of Stachytarpheta jamaicensis (L.)
36
Vahl crude extracts into the Hela cell culture
3.2.6.3 The cytotoxic assay using alamar blue
37
solution
3.2.6.4 Statistical analysis
4
37
RESULTS AND DISCUSSIONS
4.1 Extraction of Stachytarpheta jamaicensis (L.) Vahl crude
39
extracts
4.2 Phytochemicals test
39
4.2.1 Ferric chloride test
41
4.2.2 Gelatin test
41
4.2.3 Alkaline reagent test
42
4.2.4 Test for terpenoids
43
4.2.5 Test for saponin or froth test
44
x
4.2.6 Test for flavonoids
45
4.2.7 Test for tannin
46
4.2.8 Test for coumarin
47
4.2.9 Test for phlobatannins
48
4.3 Identification of bacteria
49
4.4 Antimicrobial susceptibility test
50
4.5 Assessment of cell viability
55
4.6 The cytotoxic activities of Stachytarpheta jamaicensis (L.) 56
Vahl crude extracts on the growth of Hela cells at 24, 48 and 72
hours f exposure times.
4.7 Correlation data between 24, 48 and 72 hours of exposure 61
times of root, stem and leaves extracts.
4.8 Means data of root, leaves and stem extracts for three different 63
exposure times
5
4.8.1 Root extract
63
4.8.2 Leaves extracts
65
4.83 Stem extracts
68
CONCLUSIONS AND RECOMMENDATIONS
5.1 Conclusions
71
5.2 Future studies and recommendations
72
REFERENCES
73
APPENDICES
80
Appendix A
81
Appendix B
82
Appendix C
83
Appendix D
84
xi
LIST OF TABLES
TABLE NUMBERS
2.1
TITLE
The nomenclature classification of Stachytarpheta
PAGE
7
jamaicensis (L). Vahl
2.2
Four main subtypes of coumarin
15
2.3
Discovery of antibiotic and its resistance time
20
4.1
Weight of sample after extraction
39
4.2
Qualitative test for presence of phytochemicals
40
inside stem, root and leaves extracts
4.3
Gram staining bacteria for antimicrobial test
50
4.4
Antimicrobial activity of Stachytarpheta
51
jamaicensis (L.) Vahl by disc diffusion method
4.5
Correlation data obtained from ANOVA test
62
xii
LIST OF FIGURES
FIGURE NUMBER
TITLE
PAGE
2.1
Stachytarpheta jamaicensis (L.) Vahl
7
2.2
The molecular structure of six subclasses of
10
flavonoid
2.3
Molecular structure of tannin
13
2.4
Structure of terpenoids
16
2.5
Structural form of two alkaloids berberin and
18
harmane
2.6
Schematic diagram of disc diffusion method and
23
inhibition zone
3.1
Application of bacterial colonies on Mueller Hinton
34
agar
4.1
Colour changes of extracts during ferric chloride test
41
4.2
Result for gelatin test
42
4.3
Presence of yellow fluorescence for flavonoid test
43
4.4
Formation of reddish brown layer for terpenoids test
44
4.5
Saponin test indicates by froth formation
45
4.6
Extracts with positive test for flavonoids
46
4.7
Brownish green colouration after addition of ferric
47
chloride
4.8
Absence of yellow green colour on filter paper as an
48
indicator for coumarin
4.9
Absence of red deposition after boiling extract with
48
xiii
1% hydrochloric acid
4.10
Inhibition zone for leaves extract
52
4.11
Inhibition zone for stem extract
53
4.12
Inhibition zone for root extract
54
4.13
Reduction of alamar blue indigo blue to pink colour
55
solution after 4 hours incubation
4.14
The effect of Stachytarpheta jamaicensis (L.) Vahl
58
crude extracts on Hela cells after 24 hours exposure
time
4.15
The effect of Stachytarpheta jamaicensis (L.) Vahl
59
crude extracts on Hela cells after 48 hours exposure
time
4.16
The effect of Stachytarpheta jamaicensis (L.) Vahl
60
crude extracts on Hela cells after 72 hours exposure
time
4.17
The mean data from 24 incubation periods
64
4.18
The mean data from 48 incubation periods
64
4.19
The mean data from 72 incubation periods
65
4.20
The mean data for leaves extract after 24 incubation
66
hours
4.21
The mean data for leaves extract after 48 incubation
67
hours
4.22
The mean data for leaves extract after 72 incubation
67
hours
4.23
The mean data for stem extract after 24 incubation
69
hours
4.24
The mean data for stem extract after 48 incubation
69
hours
4.25
The mean data for stem extract after 72 incubation
hours
70
xiv
LIST OF ABBREVIATIONS
AIDS
-
Acquired Immune Deficiency Syndrome
ANOVA
-
Analysis of variance
DNA
-
Deoxyribonucleic acid
FADH
-
Flavin adenine dinucleotide (reduced
form)
FAS
-
Fatty acid synthase
FRIM
-
Forest Research Institute Malaysia
kD
-
Kilo Dalton
LB
-
Luria-Bertani
MH
-
Mueller Hinton
MIC
-
Minimum Inhibitory Concentration
MTT
-
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide)
NA
-
Nutrient Agar
NADH
-
Nicotinamide adenine dinucleotide
(reduced form)
NADPH
-
Nicotinamide adenine dinucleotide
phosphate (reduced form)
PBS
-
Phosphate buffer saline
PDA
-
Potato Dextrose Agar
RNA
-
Ribonucleic acid
USDA
-
United States Department of Agriculture
UV
-
Ultraviolet
WHO
-
World Health Organisation
1
CHAPTER 1
INTRODUCTION
1.1 Background information
Plants are normally being commercialized as a food-based product but they also
have other important roles making them an attractive thing to be explored by the
researcher. They grab the attention of the researchers by showing some medicinal
properties. These medicinal plants have been used for centuries as remedies and the
richest bio-resources of drugs of traditional medicinal systems in pharmaceuticals, folk
medicines, nutraceuticals and synthetics drugs. Medicinal plants or herbal remedies are
valuable in the treatment of various health problems (Das et al., 2010). Previous
research conducted showed that these medicinal plants contain bioactive constituents
with certain physiological that could be used in treatment of diseases (Pieme et al.,
2006). These includes tannins, flavonoids, terpenoids, alkaloids and phenolic
compounds (Hill., 1952). Besides, some of the synthetic drugs used in the medicinal
field also contain the chemicals or active components obtain from these medicinal
plants as an alternative due to the increase of resistance of pathogens towards frequently
used drugs. Despite the modern medicines in the market, most of the plants such as
Murraya koenigii, Curcuma longa, Lawsonia inermis, Ficus deltoidia and Zingiber
officinale are extensively used in the medicinal field especially as a herbal remedies.
Most of these plants extract are used as anti-inflammation, antioxidants, antimicrobial
and in the treatment of cancer.
2
Among them all, flavonoid is the most extensively used constituents because of
its properties to inhibit or kill numerous bacterial strains and also some viral enzymes
(Havsteen B.H., 2002). Flavonoids are active compounds that are usually found in
fruits, vegetables, seeds, stem, flowers, honey and propolis (Cushnie T.P. et al, 2005).
Flavonoid contains substances such as flavonoles, 2-phenyl-3-hydroxy-chromones,
flavones, 2-phenyl-chromones, iso-flavonoles, 3-phenyl-2-hydroxy-chromones and
other compounds which have been characterized in the plants. Unfortunately, not many
research focuses on Stachytarpheta jamaicensis (L.) Vahl. Hence, there are chances that
this plant can be used as antimicrobial agents.
1.2 Problem statement
Nowadays, the production of antimicrobial drugs in pharmaceutical industries
has increased due to the increase in untreatable diseases. This disease cause by
microbes which have become resistant towards commonly used antimicrobial drugs.
Thus, researches try to find other source of compounds which can be turned into
antimicrobial drugs. Recently, attempts made to use herbal plants as one of the
alternative drugs in the treatment of diseases have increased due to the impact it
gives on the human health and disease prevention. Till now, there is less than thirty
researches published on this plant species; Stachytarpheta jamaicensis (L) Vahl and
mostly do not focus on the cytotoxic effect but more to its antimicrobial properties
In this research, the plant extracts of Stachytarpheta jamaicensis (L). Vahl were
determined for its antimicrobial activity and cytotoxic effect. Moreover, the research
on this plant only using the leaves extract, therefore, in this study, the stem and root
extract of this plant was investigated for its antimicrobial activity. Furthermore,
there is less cytotoxic effect regarding this plant species.
3
1.3 Objective of the research
The objectives of this research are:
1. To obtain crude extracts of Stachytarpheta jamaicensis (L.) Vahl.
2. To detect the presence of phytochemical compounds in the crude extracts of
Stachytarpheta jamaicensis (L.) Vahl.
3. To determine the antimicrobial activity of the crude extracts using disk
diffusion technique.
4. To check for cytotoxic activities of the crude extracts of Stachytarpheta
jamaicensis (L.) Vahl on cancer cells
1.4 Scope of the research
The study includes three important steps in which consist of preparation of the
crude
extract
of
Stachytarpheta
jamaicensis
(L).
Vahl,
determinations
of
phytochemicals content in the plant extracts and antimicrobial analysis using disc
diffusion method. Methanol was used as a solvent to extract the bioactive compounds.
The plant was dried and macerated into powder form before mix with methanol to
produce extract. According to previous work, Stachytarpheta jamaicensis (L.) Vahl
contains several phytochemical constituents which are important in the treatment of
diseases examples ulcer, fever and diarrhea. Therefore, determination of the bioactive
compounds using qualitative tests is very important to be carried out. The antimicrobial
test is performed by impregnating the sterile blank disc with diluted plant extract of
various concentrations and measurement of the inhibition zone. Cytotoxic activities of
Stachytarpheta jamaicensis (L.) vahl is done by treating the Hela cancer cells using the
plant extracts and check using alamar blue solution.
1.5
Significance of the research
4
The significance of this research is to identify the possibilities of using
Stachytarpheta jamaicensis (L) Vahl extracts for an antimicrobial drugs based on its
antimicrobial activity. Moreover, the cytotoxic activity of Stachytarpheta jamaicensis
(L.) Vahl on cancer cells also being determine to see whether the extracts can be used in
the treatment of cancer. Hence, it can act as one of the solution to treat cancer due to the
fact that it is the number one cause of death worldwide.
5
CHAPTER 2
LITERATURE REVIEW
2.1 Medicinal plants
Medicinal plants are usually being commercialized into food products because
of their nutritional contents. Besides, medicinal plants are very valuable to human being
as they provide not only food source but also important in the process of making herbal
remedies and medicinal drugs as an alternative in the treatment of diseases such as
cancer and infectious diseases. This is because as time passed, many new and dangerous
diseases evolve apart from the all time popular disease without cure in which is cancer.
The acceptance of the people towards using medicinal plants as a substitute for
chemically synthesize drugs encouraged the scientists to study more about medicinal
plants. Moreover, several researchs have found that these medicinal plants contain
bioactive compounds with useful physiological activity such as alkaloids, tannins,
flavonoids, and phenolics compounds (Edeoga et al, 2005). These bioactive compounds
are important especially as an anti-cancer, anti-inflammation, antimicrobial and
antioxidant. Examples of the medicinal plants that are commonly used as medicine
includes Murraya koenigii (curry leaves), Curcuma longa (curcumin), Lawsonia
inermis (henna leaves), Ficus deltoidia (mistletoe fig) and Zingiber officinale (ginger)
because of the high contents of bioactive compounds in their structure such as roots,
leaves, flowers and fruits.
6
2.2 Stachytarpheta jamaicensis (L.) Vahl
2.2.1 General description
One of the herbal plants that are useful and possess a medicinal value is
Stachytarpheta jamaicensis (L) Vahl. However, few research only have been done on
this plants compared to other plants. This might be because the plants are used mostly
by old folks in a more traditionally way and only being exposed in the medical field
recently. Stachytarpheta jamaicensis (L) Vahl belongs to the family of Verbenaceae and
herbaceous plant that can grow up to 60 to 120 cm tall. This plant also grows in
acclimatized environment such as Malaysia where the native called it by the name of
‘Jolok cacing’ or ‘Selasih dandi’. It habitat are mostly in the tropican America and other
parts of the tropical forest such as in Nigeria, Trinidad and Tobago and acclimatized
tropics ( Sasidharan et al, 2007 and Idu et al., 2006). These plants have woody and
smooth stern especially at the base. The leaves are opposite and whorled, ovate or
oblong elliptic and wide up to 4.5 cm. This Brazilian tea also has short petioles with
widely toothed on the margin smoothed surface on both side of the leaves. It can bears
flowers with mix colors of bluish and white in slender spikes and long rachis. The stems
are dark green and covered in powder which gives its bluish shine (Idu et al., 2006).
7
Table 2.1
The nomenclature classification of Stachytarpheta jamaicensis (L). Vahl
(USDA, 2010)
Kingdom
Plantae
Subkingdom
Tracheobionta
Superdivision
Spermatophyta
Division
Magnoliophyta
Class
Magnoliopsida
Subclass
Asteridae
Order
Lamiales
Family
Verbenaceae
Genus
Stachytarpheta Vahl
Species
Stachytarpheta jamaicensis (L). Vahl
Figure 2.1 Stachytarpheta jamaicensis (L) Vahl
8
2.2.2 Studies related to Stachytarpheta species
Many studies have reported that plants in Stachytarpheta species are normally
used as an anti-inflammation and anti-microbial medicine. Some of its species such as
Stachytarpheta angustifolia, are used locally as anti-infection agent by squeezing the
leaves to form juices and use it on sexually transmitted diseaseas ( Enwuru et al., 2008).
Moreover, these plants also being used in the treatment of diabetic disease ( Ogbonnia
et al., 2009). Almost every parts of this plant have been studied extensively by the
researchers such as stems, barks and leaves.
Stachytarpheta jamaicensis (L.) Vahl itself are also been known for their
antacid, analgesic, anti-helminthic, anti-inflammatory, diuretic, hypotensive , laxative,
lactogogue, purgative, sedative, stomachictonic, spasmogenic, vasilator, vulnerary and
vermifuge (Idu et. al, 2007, Okwu and Ohenhen, 2009). S. jamaicensis (L). Vahl also
have been screen for the antilarval and insectisidal effects of the plant extract towards
mosquito Culex quinquefasciatus and Aedes aegypti (Nazar et al., 2009 and Idu et a.l,
2008). It often used to treat dysentery and intestinal worms by using its leaves (Almeida
et al, 1995). Futhermore, in Malaysia also, ulcer and anti-periodic medicine for malaria
are treated by boiling the leaves with water (Rao et al., 2006, and Sasidharan et al.,
2007).
It also can be used to treat allergies and common respiratory conditions such as
colds, flu, asthma, bronchitis and others. For people with digestive problem, they can
use this plant as remedies because it can treat indigestion, acid reflux, ulcers,
constipation, dyspepsia and slow digestion. For example, stem and barks of this plant
have been used by the Nigerians herbalists to stimulate the gastrointestinal tract for the
treatment of cirrhosis and hepatitis ( Okwu and Ohenhen, 2009). Pregnant women and
patients with low blood pressure usually not recommended using this plant because of
its properties to be abortive and hypotensive (Idu et. al., 2007).
9
2.3 Bioactive compounds in plants
As mentioned earlier, plants are able to synthesize many aromatic secondary
metabolites or commonly known as bioactive compound in examples phenols or its
oxygen-substitute derivatives. Bioactive compounds are extra nutritional compounds
that occur in small quantity in food and are now being studied seriously to check for its
effect on health. Nowadays, many types of bioactive compounds with diverse chemical
structure and function have been discovered and grouped. There are several major
groups of secondary compounds exists in the medicinal plants includes alkaloids,
phenolic compounds and terpenoids. In phenolic compounds, phytochemicals such as
tannins, flavonoids and coumarins amongst the well-known among the researchers.
Occasionally, these substances help the plants and produced as a defense mechanism
against microorganisms, herbivors, and insects. Examples of this defense include the
synthesis of antimicrobial compounds by plants that are infected by bacteria and fungi
known as phytoalexins (Heinrich and Gibbons, 2004). These bioactive compounds are
abundance in parts of plants such as roots, stems, flowers, barks and leaves. Most of
them have been fully studied by many researchers for their ability to treat infectious
diseases.
2.3.1 Phenolic compounds
2.3.1.1 Flavonoids
One of the examples of bioactive compound is phenolic compounds including
their subcategory which is flavonoid. This compound are usually present in all plants
and mostly being discovered in soybeans, nuts, vegetables, fruits, tea and olive oils
(Kris-Etherton et al, 2002). Flavonoid is a plant pigment mostly derived from benzo-γpyrone similar to chromone that gives color to the flower petals. This colored functions
to attract the pollinator animals for pollination purposes (Cushnie et al., 2005 and
Havsteen, 2002). It has a molecular structure consists of two aromatic rings, A and B
10
linked by three carbon bridges. Groups of studies proves that the structures of flavonoid
have certain effects toward antimicrobial, antiviral and antifungal activity
(Cushnie et al, 2005). Flavonoids can be divided into six subclasses comprises of
flavones, flavanones, flavanols, flavonols, isoflavones, and anthocyanidin.
Figure 2.2 The molecular structure of six subclasses of flavonoid (Bruselmans,2005)
Flavonoids have found to inhibit the fatty acid synthase activity. These enzymes
is the key enzyme that catalyzes the synthesis of long chain fatty acid which can be
associated with growth arrest and cell death in cancer patients. Normal tissues have low
fatty acid synthase expressions but in human cancers, it has increase expression mostly
in cancer of the prostate, breast, ovary, endometrium, colon, and lung.
11
Some studies relate the bioactive compound, flavonoids by inhibiting cell
growth, tumor angiogenesis, cell invasion (Brownson et. al, 2002). The increase intake
of the food containing flavonoids frequently associated in reducing the risk of a variety
of cancer such as cancer of the prostate, lung, stomach, and breast. Continuous intake of
tea which contains high amounts of flavanols and flavonols can reduce the risk of
getting breast, prostate, bladder, lung, pancreas, colon, stomach, esophagus, and oral
cavity. One of the studies states that flavanole epigallocatechi-3-gallate produce
cytotoxic effect on prostate cancer cells by looking at its ability to inhibit fatty acid
synthase (FAS). Additionally, researches also have been done on five other types of
flavonoids, luteolin, quercetin, kaempferol, apigenin and taxifolincan also proven to
inhibit cancer cell lipogenesis. Taken into consideration, these findings showed the
possibility of flavonoids to induce apoptosis in cancer cells by looking at its FAS
inhibitory properties ( Brusselmans, 2005).
Flavonoids are commonly synthesis by plant in response to microbial infection.
The antimicrobial activity is probably due to their ability to form a complex with
extracellular and soluble proteins and to complex with bacterial cell wall. Lipophilic
flavonoids may interrupt the microbial membranes (Cowan, 1999). In addition, in
another study, they have found that it can inhibit the DNA synthesis. The B ring of
robinetin, myricetin and (-)-epigallocathecin can intercalate or form hydrogen bond
with the stacking of nucleic acid bases thus explaining the inhibitory action of DNA and
RNA synthesis (Cushnie and Lamb, 2005).
2.3.1.2 Tannins
Tannins are one of the phenolic compounds that are naturally occurring in the
plants (Figure 2.3). It is a polymeric phenolic substance with astringent properties.
These properties give tannins ability to dissolves in water, alcohol and acetone thus
precipitating gelatin from solution (Das et. al., 2010). It combines with protein and
other polymer to form a stable complex (Lim et. al., 2006). Molecular weight of tannins
12
range from 500 to 3000 kD and they are found in most parts of the plants example bark,
wood, leaves fruits and roots. Tannins can be divided into two groups, hydrolyzable and
condensed tannins. Hydrolyzable tannin is basically gallic acid whereas condensed
tannin comes from flavonoid monomers. Formation of tannins might be from the
condensation of flavan derivatives transported to woody tissues of plants (Cowan,
1999).
Tannins have been reported in the treatment of various diseases in humans such
as diarrhea, gastric ulcers, snake bites and wounds (Lim et. al., 2006). Moreover, some
also consider tannin to have antiviral properties. Despite less reports of any involvement
of tannins on antimicrobial activity, Doss et. al, 2009 have showed some positive results
on the antibacterial activity of tannins towards Staphylococcus aureus, Streptococcus
pyrogens, Slamonella typhi, Pseudomonas aeruginosa, Proteus vulgaris and
Escherichia coli.
13
Figure 2.3 The molecular structure of tannins. (Cowan, 1999)
2.3.1.3 Coumarins
Coumarins are also parts of the phenolic groups along with flavonoids and
tannins. They are made of fused benzene and α-pyrone rings and responsible for the
odour release by hay (Cowan, 1999). The name of coumarin originally comes from
‘Coumarou’, a vernacular name of the tonka bean (Dipteryx odorata Willd Fabacea)
where coumarin was first isolated in 1820. As mentioned earlier, coumarin is classified
as a member of benzopyrone family of compounds. It can be divided into two benzo-αpyrones for coumarin and benzo-γ-pyrones where flavonoids one of its member.
Coumarin exists in most of the plant kingdom such as fruits, green tea, and chicory.
They also abundance in higher plants namely Rutacea and Umbelliferae and can be find
at high concentration in fruits followed by roots, stems and leaves (Lacy and
14
O’Kennedy, 2004) and also commonly found in the family of Apiaceae, Asteraceae and
Fabaceae (heinrich and Gibbons, 2004).Table 2.2 shows four main subtypes of
coumarin with features and examples of each subtypes (Lacy and O’ Kennedy, 2004).
Until 1996, there are almost 1300 compound of coumarin have been discovered.
There are also researchers regarding the isolation of coumarin compounds and the
detection of coumarin in plants (Liu et al., 2005, Biavatti et. al, 2004, Celeghini et. al.,
2001). Coumarin are phytoalexins and synthesized de novo by the plant following an
infection by a bacterium or fungus. These phytoalexins are antimicrobial for example
scopoletin which is synthesized by the potato (Solanum tuberosum) followed by
infection of fungal. Hieracium pilosella or mouse ear contain umbilliferone, normally
used to treat brucellosis in veterinary medicine. Its antibacterial drug activity may be
due to the presence of phenol compound (Heinrich and Gibbons, 2004).
Coumarin has been known for its antithrombotic, anti-inflammatory,
vasodilatory, anti-tumour and antimicrobial activities (Lacy and O’ Kennedy, 2004,
Cowan, 1999). There are many studies related to the role of coumarin in the treatment
of diseases. Warfarin is one of the coumarin have been used as an oral anticoagulant
and rodenticide. Moreover, there has been evidence of the antimicrobial properties of
coumarin where it can inhibit Candida albicans in vitro thus can be used as an agent for
treatment of vaginal candidiasis. Derivatives of coumarin like phytoalexins are produce
by carrot in response towards fungal infection (Cowan, 1999). Besides the antimicrobial
activities, the bacteriostatic and anti-tumor of coumarin offers great interest to
researchers to use them as a therapeutic agent. Coumarin and its derivatives 7hydroxycoumarin have anti-tumor activity towards several tumor cell lines. They also
can acts as potential inhibitors of cellular proliferation in diverse carcinoma cell lines.
Furthermore,
4-hydroxycoumarin
and
7-hydroxycoumarin
inhibited
proliferation of gastric carcinoma cell line (Lacy and O’ Kennedy, 2004).
the
cell
15
Table 2.2
Four main subtypes of coumarin with features and examples of each
subtypes (Lacy and O’ Kennedy, 2004).
2.3.2 Terpenoids
Normally we can smell some fragrance release by plants due to the content of
essential oils inside them. These essential oils are secondary metabolites highly
enriched with isoprene based compound. They are called terpenes with general
chemical structure C10H16 and occur as diterpenes, triterpenes, tetraterpenes,
hemiterpenes and sesquiterpenes. If the terpenes contain additional elements such as
oxygen, they are called terpenoids. Terpenoids share the same origin as fatty acids
16
because they are both synthesized from acetate units. The only different between those
two is that they have widespread branching and cyclized.
Examples for terpenoids are methanol and camphor and farnesol and artemisin
(Figure 2.4). Terpenoids have been reported active against bacteria in many researchs
such as by Roberto et. al,.2004, Okwu and Ohenhen, 2009, Edeoga et. al., 2005,
Enwuru et. al., 2008. Essential oils also posses strong antimicrobial properties. 60% of
the essential oil derivatives inhibit the growth of fungi while another 30% goes to
bacteria inhibition. Terpenoids presences in the essential oils of plants are found to be
useful in the control of Listeria monocytogenes.
Figure 2.4 Structure of terpenoids (Cowan M.M.,1999)
17
2.3.3 Alkaloids
Another natural compounds usually found in the plants is alkaloid. It is defined
as heterocyclic nitrogen compounds. However, alkaloid is not uniquely from plants
because they also have been isolated from various animal sources. Morphine is an
alkaloid isolated in 1805 from the opium poppy Papaver somniferum. Plant with
alkaloid such as Ranunculaceae family is found to have antimicrobial properties. As
the time passes, alkaloid draws attention of the researcher because of their physiological
activities in humans and animals. Taxol is the most known form of alkaloid isolated
from Taxus brevivolis useful for anticancer treatment. Many other plants in example
Cephalotaxus harringtona plant containing homoharringtonine can cure leukemias,
while Tapiá can modulate inflammatory disorders (Lopes et. al., 2009). Monsef et. al,
(2004) done a research on the antinociceptive effects of Peganum harmala L. alkaloid
extracts on mouse formalin test.
In this study, they found out that the alkaloid named harmalin to be able to
significantly reduced pain experienced by the animal (Figure 2.5).
Moreover,
antimicrobial activities of alkaloids are explained by its effects on Giardia and
Entamoeba species due to their localization in small intestine. One of the alkaloid,
berberin are found to be effective against trypanosomes and plasmodial. Its reaction is
based on its ability to intercalate with DNA similar to harmane.
18
Figure 2.5
Structural forms of two alkaloids berberine and harmane (Cowan, 2004).
2.4 Antibiotic
2.4.1 Brief history on antibiotic
History of antibiotic first started with the discovery of Penicillin by Alexander
Fleming in 1928 when he first culture Staphylococcus aureus on a plate and found the
growth of a mold formerly known as Penicillum notatum. Around the mold, a clear
zone formed indicating that the growth of Staphylococus aureus has been retarded.
Later, he made some crude extract from the fungus and found that this extract kills
many types of pathogenic bacteria after cultured it on several different types of bacteria.
He also injected the crude into the rabbit and the results showed that the rabbit was not
harmed. The studied further continued by three English scientists in 1938 to produce
large amount of penicillin and they are able to treat the soldiers in World War Two
using the isolated penicillin. The first person successfully treated with Penicillin is a
women name Anne Miller whom developed a streptococcal infection after miscarriage.
They gave her penicillin and she survived after that ( Guilfoile and Calamo, 2006).
Soon after the discovery of penicillin, the next discovery was streptomycin obtain from
microbes in soil by Selman Waksman. This antibiotic was found to cure intestinal
19
diseases. Hence, the development of other antibiotics begun and day by day, more
types of antibiotic were discovered (Stone, 2007).
2.4.2 Antibiotic resistance by microorganisms
The continuously emergence of new antibiotic resistance strains day by day
have become the problems among the people. Microorganisms are always having the
ability to protect themselves against naturally antibiotics by showing resistancy by
exchanging the genetic material with other organisms.
They acquire and adapt
properties of other organisms through this genetic exchange into their own genetic
material to gain new resistancy. Table 2.3 shows the discovery of antibiotics and the
date when the resistance was reported. New disease emerged and affects the human
population such as AIDS, Legionnaire’s and Lyme disease cause by this antibiotic
resistant microorganism. Diseases cause by hantavirus which have not been seen in a
long time also re-emerged in 1993 which affect the Navajo nation (Guilfoile and
Calamo, 2006). As an example, Staphylococcus aureus is a bacteria literally carried by
humans which can cause problems such as mild skin infections, food poisoning, wound
infections, pneumonia and toxic shock syndrome. The World Health Organisation
(WHO) recently reported that more than 95% of Staphylococcus aureus in this world
are resistant to penicillin and 60% are resistant its derivatives called methicillin (Kardar,
2005).
20
Table 2.3 The discovery of antibiotics and the date when the resistance was reported
(Guilfoile and Calamo, 2006).
However, the problems of antibiotic resistance by the microorganisms become
worse as the microorganisms also found to be resistance against the synthetically
synthesize antibiotics.
Thus, alternative methods to fight against this resistance
microorganisms are needed and researchers have struggled to find new antibiotic that
have the capability to inhibit the microorganisms.
21
2.5 Antimicrobial properties of medicinal plants
Even though there are numerous drugs produce in the market, the evolution of
microorganisms has caused the microorganisms to become resistance towards the
antibiotic. Hence, new drugs need to be produced in order to fights against the
microorganisms. For centuries, plants have often being used and act as valuable natural
resources that help to maintain human health. Almost 80% of world populations in
developed countries use traditional medicine derived mostly from medicinal plants.
Many medicinal plants have been discovered everyday around the world. The demands
for medicinal plants are rapidly increasing not only in developed country but also in
developing countries as well. The pressure for the utilization of useful compounds in
medicinal plants as new therapeutic drugs from the also increase due to this matter. The
medicinal plants are very important because of their antimicrobial properties and have
been focused by many researchers because of the bioactive compound present in their
secondary metabolites products ( Nascimento et al., 2000).
Additionally, medicines derived from plants are relatively safer than the
synthetically therapeutic drugs because they offer more affordable treatment.
For
instant, most of the drugs in the market are synthesized from plants. For usage purpose,
the medicinal plants should be collected at the right time, right season and the right
growth stage in order to obtain optimum amount of constituents ( Shahid-Ud-Daula and
Basher, 2009).Nowadays, many studies conducted by the researcher to identify the
antimicrobial characteristics of medicinal plants in their countries in order to find the
cure for certain disease (Lentz et al., 1998; Somchit et al.,2003 and Edeoga et al.,
2005). Variety of species of plans has been tested and they have showed positive effect
on inhibiting the growth of certain microorganisms such as Staphylococcus aureus,
Escherichia coli, Shigella sp, Aspergillus niger, Pseudomonas aeruginosa, Klebsiella sp
and others ( Nascimento et al., 2000).
22
2.6 Antimicrobial susceptibility test
It has been previously mentioned that plants substances are able to act as an
antimicrobial towards pathogen in many research studies. Due to the rapidly emergence
of antibiotic resistance, the spread of resistance gene and negative outcome when using
ineffective antibiotic encouraged many more susceptibility test to be performed. The
sensitivity of the microorganisms towards the antimicrobial agent can be tested using
the antimicrobial susceptibility test. There are two types of antimicrobial susceptibility
test commonly used which are agar dilution method and disc diffusion method. Agar
dilution method is the method where incubation of a standard inoculum of
microorganisms in doubling dilutions of antibiotic in broth and agar which allows the
measurement of minimum inhibitory concentration (MIC) under standard conditions
(Dickert et. al., 1981). The MIC values are used to determine the bacteria susceptibility
to drugs and also to evaluate the activity of new antimicrobial agents. In addition, MIC
values also important because it avoids the excessive use of expensive antibiotic and
minimizes the chances of toxic reactions that larger-than-necessary doses might cause
(Wiegand et al., 2008 and Tortora et. al., 2007)
Second method is the disc diffusion method, a much simpler method compared
to the agar dilution method. The basic principle of disc diffusion test is by placing a disc
impregnated with antibiotic onto the surface of Mueller Hinton (MH) agar swab with
bacterial inoculums. After incubation overnight, the antimicrobial activity was defined
by measuring diameter of inhibition zone around the discs (Isenberg, 1998). The discs
were impregnated with different concentration of chemotherapeutic agents. The drugs
will diffuse from the discs into the agar. The farther the drugs diffuse from the discs, the
lower its concentration. Inhibition zone produces shows the sensitivity of the bacterial
towards the chemotherapeutic agents. Larger inhibition zone, the more sensitive the
bacteria will be (Tortora et al., 2007). Mueller Hinton agar is a protein free medium
developed by Mueller and Hinton in 1941 to culture pathogenic strain of Nisseria. MH
agar contains beef infusion, casein hydrolysate and starch.
The starch act as a
23
protective colloids against the inhibiting effects of certain amino acids ( Odugbemi et
al., 1978).
Figure 2.6
Schematic diagram showing the disc diffusion method for antibiotic
sensitivity test (Parija,2009).
2.7 Natural compounds and cancer
Natural drugs play an important role in the pharmaceutical field. Several plantderived compounds are currently applied on the cancer treatment. Beside antimicrobial
activities, certain plants also have their cytotoxic activity against cancer. There are
many plant-derived cytotoxic compounds studied for further improvement in the cancer
treatment. However, only certain plants have undergoes a number of tests to prove their
cytotoxic activity. Studies conducted mainly in Latin countries resulted in the finding
stated that many plants owned this properties and this is contributed by the presents of
bioactive compound called flavonoids. As mentioned before, flavonoids take part in
inhibiting the enzyme fatty acid synthase which usually expressed in the cancer cells.
Some examples of plants that have cytotoxic activity are Hibiscus tiliaceous,
Hygrophila auriculata, Clerodendron inerme, Blumea lacera and Argemone Mexicana
(Uddin et al, 2009). The action of flavonoids and its actions make it suitable to be used
in the cancer therapies.
24
Till now, there are only nine plant-derived compound approved to be used as an
anticancer drugs which are vinblastine, vincristine, etoposide, teniposide, taxol,
navelbine, taxotere, topotecan and irrinotecan.
The vinblastine and vincristine are
natural compounds isolated from the alkaloids of Catharanchus roseus or Vinca rosea.
These natural compounds are found useful in the treatment of lymphoma and leukemia.
Moreover, the natural compound camptothecin isolated from Camptotheca acuminate
has undergone structural modification in the aim to be used as chemotherapeutic agents.
Normally, the compounds are used in the treatment of gastric, rectal, colon and bladder
cancer (Patel et al., 2010).
Taraphdar and Battacharya (2001) reported several plants useful in the induction
of apoptosis or programme cell death in the cancer cells. One of them are Selanigella
tamariscina (Beauv) or preferably called ‘Keoun Back’, a traditionally medicinal plant
for the therapy of advanced cancer patients in the Orient where they have shown its
ability to modify gene expression and cytokine production. Due to this matter, it is very
important to screen for other bioactive compounds present in the plants so that the fully
functions of the compounds in the plants can be utilized.
2.8 Cytotoxic assay
In order to identify the cytotoxic effects of certain compounds towards the
cancer cells activities, many cytotoxic assays have been produced. Moreover, these
biological assays are also designed to be able to detect the cell viability, cell
proliferation and cell quantification. McMillian et al. (2002) stated that cytotoxicity
assay depends on the metabolic state of the treated cells. The biological assays should
be simple, rapid, highly reproducible, versatile, and capable of handling large numbers
of samples at one time (Voytik-Harbin et al., 1998). There are many kinds of cytotoxic
assay designed and the mostly used are (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide) (MTT) designed by Mossman in 1983. This MTT assay is an
25
assay involves the reduction of tetrazolium salt into colored product formazan
(Mossman, 1983). However, MTT is not effective because it tends to kill the cells
making it imposible to re-culture the cells. As an alternative to MTT assay, alamar blue
is developed. Alamar blue is a water-soluble dye previously used in preliminary studies
for quantifying in vitro viability of various cells (Al-Nasiry et al., 2007). This dye is
very stable, thus, re-culturing cells are possible. It is sensitive oxidation-reduction
indicator that fluorescence and changes colour after reduction by living cells. These
effects are believed comes from the mitochondrial enzymes (Hamid et al., 2004).
Additionally, alamar blue have a few characteristics which are nonradioactive,
nontoxic, water-soluble, and are able to be detected after incubation using either
absorbance or fluorescence spectroscopy (Voytik-Harbin et al., 1998).
26
CHAPTER 3
MATERIALS AND METHODS
3.1 Materials
3.1.1 Solutions and reagents
Chemicals and reagents used in this project were purchased from various
companies. Ethanol used for extraction process was obtained from Fluka Analytical.
The chemicals for phytochemical tests are obtained from Merck KGaA, Germany such
as the ammonia solution while chemicals such as hydrochloric acid, dimethyl sulfoxide,
sulphuric acid, ethyl acetate, sodium chloride and chloroform. For microbial and fungi
culture, nutrient agar (NA), Luria Bertani (LB) agar and Potato Dextrose agar (PDA)
was purchased from Merck KGaA, Germany.
3.1.2 Plant Materials
The plant sample Stachytarpheta jamaicensis (L.) Vahl were collected at
Kampung Behor Mali, Simpang Empat, Perlis and have been identified by Miss Tan Ai
lee from Forest Research Institute Malaysia (FRIM). The herbarium sample for the
plant was prepared with reference number SBID:010/10 and conserved in the Animal
Tissue Culture Laboratory as a specimen sample.
27
3.1.3 Culturing Hela cell
In cell culture, the materials and reagents used were purchased from various
companies and personnel.
The medium for cell growth was obtained from other
postgraduates student in Animal Culture Laboratory, trypsin from Invitrogen, phosphate
buffer saline from Sigma-Aldrich, and alamar blue was from Biosource. Hela cell were
purchased from American Type Culture Collection (ATCC) and were kindly provided
by Animal Tissue Culture Laboratory, Faculty of Biosciences and Bioengineering,
Universiti Teknologi Malaysia, Skudai, Johor. The cells were kept for storage in liquid
nitrogen.
3.2 Methods
3.2.1 Extraction of Stachytarpheta jamaicensis (L.) Vahl crude extract
The plant collected was air-dried for 3 to 4 days and oven-dried in the incubator
oven at temperature 40°C for one week. After the plants were completely dried, the
parts of plants like the roots, stems and leaves were separated. These parts were then
grounded into small pieces using Waring commercial blender. After grounded, the
plants were soaked in the 80% ethanol (v/v) as solvent for seven days. The mixtures
were then filtered using Whatman filter paper 125 mm and the residues were collected
and concentrated using EYELA Rotary Evaporator N-1000. The extraction were
concentrated using rotary evaporator with the temperature around 40°C to 45 °C
because boiling temperature of ethanol is 78.4°C. Therefore, temperature for rotary
evaporator should be half than the actual boiling temperature (Enwuru et al., 2008).
28
3.2.2 Phytochemical tests
3.2.2.1 Detection of phenolic compounds
The detection of phenolic compounds was done using method taken from
Raaman, N. (2006) and were divided into three tests which were ferric chloride test,
alkaline reagent test and gelatin test.
a) Ferric chloride test
50 gm of extract was dissolved in 5 ml of distilled water followed by the
addition of 5% of ferric chloride solutions. The appearance of dark green colour
indicates the presence of phenolic compounds.
b) Gelatin test (Evans,1997)
50 gm of extract was dissolved with 5 ml of distilled water and 2 ml of 1%
solution of gelatin containing 10% sodium chloride was added. The presence of white
precipitate showed presence of phenolic compounds.
c) Alkaline reagent test
Aqueous solutions of the extracts were treated with 10% ammonium
hydroxide solution and yellow fluorescence shows there were flavonoids inside the
extracts.
29
3.2.2.2 Test for saponins or Froth test
1 gm of plant sample was boiled in 10 ml of distilled water for 15 to 20 minutes,
filtered and then shaken. Any persistent froth indicates the presences of saponins.
3.2.2.3 Test for coumarins
Plant sample was mix with distilled water to make it moist and placed in test
tubes. The test tubes were covered with filter paper moistened with 0.1 M sodium
hydroxide solution. The covered test tubes were then placed in a boiling water bath for
15 minutes. After that, the paper were removed and exposed to ultraviolet (UV) light for
one to two minutes and appearance of yellow-green colour within few minutes shows
presence of coumarins.
3.2.2.4 Test for terpenoids (Salkowski test)
5 ml of each extracts were mixed in 2 ml of chloroform and followed by
addition of 3 ml of concentrated sulphuric acid to form a layer. A reddish brown color
was formed on the upper layer indicating the presence of terpenoids and act as a
positive result.
3.2.2.5 Test for flavonoids
Each part of the plant were macerated into small pieces and heated with 10 ml of
ethyl acetate over a steam bath for 3 minutes. Then, the mixture was filtered using
30
Whatman filter paper 125 mm and 4 ml of filtrate shaken with 1ml of 10% diluted
ammonia solution. Yellow colouration after the addition of ammonia solution indicates
the presence of flavonoids.
3.2.2.6 Test for tannin
0.5g of of the dried macerated plant samples were boiled in test tube containing
20 ml of distilled water and further filtered. This was followed by the addition of a few
drops of 0.1% of ferric chloride solutions.
Next, brownish green or blue-black
colouration was observed as an indicator for the presence of tannin.
3.2.2.7 Test for Phlobatannins
An aqeous extracts of the plant sample was boiled in 1% aqeous hydrochloric
acid and the deposition of red precipitate shows positive result for phlobatannins.
3.2.3 Preparation of microbial culture
3.2.3.1 Medium preparation
Nutrient agar was used to culture microbes used for antimicrobial susceptibility
test. Nutrient agar was one of synthetic medium used for culturing non-fastidious
microorganisms. Most bacteria can grow on the surface of the agar to produce small
colonies. In order to make nutrient agar, 20 g of nutrient agar was dissolved in 1 litre of
distilled water. The solution was sterilized using autoclave at 121°C for 10 minutes. The
melted agar was poured into sterile petri dish immediately after it was taken out from
the autoclave to prevent it from hardened. The agar was let cool and hardened in the
petri dish. The petri dish was set upside down to prevent formation of water droplets
31
that will disrupt the growth of microorganism and stored at temperature of 3°C. For the
growth of Escherichia coli, specific medium was used which was Luria-Bertani agar
and in order to prepare 1 liter of agar, 10g of tryptone, 5g of yeast extract, 10g of
sodium chloride and 15g of agar-agar powder were dissolved in 1 liter of distilled
water. Moreover, there were also specific medium to grow fungi which were on Potato
Dextrose agar (PDA) This agar was prepared based on the product manufacturer
instructions by dissolving 39 g in 1 liter of distilled water and sterilized in an autoclaved
at 121°C for 10 minutes before used.
3.2.3.2 Microbial culture
In order to study for the antimicrobial effects of the extracts, there were eight
groups of microorganism select to be tested. These microorganisms were supplied from
Animal Tissue Culture laboratory, Microbiology laboratory and Project laboratory 1 of
Faculty of Biosciences and Bioengineering, Universiti Teknologi Malaysia. These
microorganisms used in this study are Staphylocccus aureus, Escherichia coli,
Pseudomonas aeruginosa, Streptococcus sp., Micrococcus luteus, Bacillus sp., and two
strains of fungi which are Saccharomyces cerevisae and Aspergillus niger. The bacteria
were re-identified using several methods for bacterial identifications.
3.2.3.3 Culturing microorganisms on growth media
After preparing the growth media, all strains of microorganisms were cultured
on to the agar plate and the broth. The cultures were left overnight in an incubator at
37°C for the microorganisms to grow. As for the fungi, they were culture on PDA agar
plate and the plate was left for 5 days for the fungi to grow and form spores.
32
3.2.3.4 Identification of the bacteria
(a) Gram staining method
Gram staining method is a method used to identify the morphology of the
bacteria by using dye to react with the specific cell structures so that these structure may
be visible for example flagella, endospores and cytoplasmic inclusions. The most
widely used stain for bacterial identification is the gram stain. By using gram stain, the
bacteria can be divided into two large groups that are gram positive and gram negative.
The different are based on structure of the cell wall where the gram positive strain will
stain blue-purple and the gram negative will stain pink-red.
The method starts by preparing bacterial smear before applying the dyes. The
solid culture was transferred onto the glass slide and mix with a drop of water to dilute
it. After that, the smear was allowed to air dry and followed by heat fix it using Bunsen
burner several time. However, the glass slide must not be close to fire as it might
become hot and break. After the smear is ready, it was then flooded with crystal violet
and the stain was let stay for 30 seconds. The stain was then wash off using distilled
water. Next, the stain was flooded with iodine solution for 10 seconds and drain off
excess stain using distilled water. Decolorisation of the stain took place by using
alcohol and it was then washed off using distilled water. Finally, the smear was
counterstain using safranin for 30 seconds and wash with water. The smear was dry by
heating. When finished, the glass slide was examined under oil immersion objective.
33
3.2.4 Antimicrobial susceptibility tests
3.2.4.1 Preparation of Mueller-Hinton agar
Mueller Hinton agar is a growth medium used for antimicrobial susceptibility
test by disk diffusion method. The protein free medium have been developed by
Mueller and Hinton in 1941 to isolated pathogenic strains Neisseria The agar are
usually appear as translucent and light amber in colour. Mueller-Hinton agar was
prepared according to the manufacturer suggestion. 34g of the Mueller-Hinton agar
powder was weight and dissolved in one liter of demineralized water. In this case, the
deionized water was used because it was found to be similar to demineralized water.
The solution was then sterilized by autoclaving at 121°C for 18 minutes then pour onto
the petri dish. The agar was let cool and kept at room temperature for one day to seek
for any contamination.
3.2.4.2 Preparation of saline solution
Antimicrobial susceptibility test requires 0.85 % to 0.9 % saline solution for the
dilution of microbial culture before applying onto the plate containing to adjust the
turbidity. Saline solution was prepared by using 4.25 g of sodium chloride and
dissolved in 500 ml of distilled water then autoclaved at 121°C for 18 minutes to
sterilize the solution.
3.2.4.3 Inoculating microorganisms on Mueller Hinton agar
Each bacterial culture was streaked onto nutrient agar to obtain single colonies
and incubate overnight at 37°C. After incubation, one or two single colonies and
inoculate in 0.85% saline solution and adjusted the turbidity to meet the 0.5 McFarland
turbidity standards. The standard is based on the measurement for the absorbance at
wavelength 620 to 625 nm and the turbidity must be around 0.08 to 1 ( Basri and Fan,
34
2005). If the absorbance increase, the addition of more saline solution is required while
addition of more bacterial colonies can increase the absorbance. Next, sterile cotton
swab was used to inoculate the bacterial suspension on Mueller Hinton agar. The
cotton swab must be pressed firmly against the wall of the tube to avoid taking too
much colonies and remove excess fluid.
By using the cotton swab, the bacterial
colonies was streaked onto the surface of the agar three times in the different directions
by rotating the plate each time to ensure that the bacterial distribute evenly on the agar
(Figure 3.1). In addition, around the agar should also be swab with bacterial colonies.
1
3
2
Figure 3.1 Applying the bacterial colonies on Mueller Hinton agar in three directions.
3.2.4.4 Preparations and application of antimicrobial discs
The prepared extracts was diluted to five different concentrations of 5, 10, 15,
20 and 25 µg/µl and sterile filtered using 0.2 µm membrane filter. After preparing the
extracts, it was applied onto 5 mm diameter sterile disc obtain from Whatman filter
paper No. 1. The disc containing the extracts was impregnated on the surface of the
agar within 15 minutes after bacterial inoculum. The discs were placed individually on
the agar using sterile forceps gently. There were six discs on the agar with distances
35
and the plate was duplicated for each bacterial strains. Not more than twelve discs can
be applied onto the agar surface to avoid an overlapping of the inhibition zone by the
extracts.
In this study, there were three control used which were disc containing
solvent, 80% ethanol and disc containing distilled water as the negative control whereas
disc containing commercially prepared antibiotic chloramphenicol 30µg/µl as the
positive control. For antifungal, the spores of the fungi were applied on PDA with the
impregnated discs added onto it. Plate containing extracts impregnated with the discs
were incubate for 24 hours at 37°C for bacteria and 30°C for 7 to 14 days for fungi.
The antibacterial and antifungal activities were measured by the inhibition zone.
3.2.5 Recording data and interpreting the results
The results were collected after 24 hours of incubation period and the inhibition
was measured using ruler in millimeter. This was then compared to the standards in the
literature review. An inhibition zone less than 6 mm was not applicable. Data was then
presented in the form of table. The analysis was perform using SPSS 16.0.
3.2.6 Cytotoxic activity of crude extracts of Stachytarpheta jamaicensis (L.) Vahl
on the growth of HeLa cell
3.2.6.1 Seeding Hela cell in 96-well microtiter plate
The medium used to culture cell was RPMI 1640 supplied by Sigma Aldrich.
The media was supplemented with 10% of fetal bovine serum, 1% of penicillinstreptomycin. The cells were incubated in the CO2 incubator to maintain the pH and
humidity. The incubator contains 95% of carbon dioxide and 5% air. The monolayer
cells were incubated until it achieves 90-95% confluency. Seeding of Hela cells were
performed by taken the cells from 75cm2 tissue culture flask and transfer into 96-well
36
microtiter plates. The old medium was removed by sucking and 10 ml of phosphate
buffer saline (PBS) was added to rinse the flask and clear the debris. Furthermore, 2ml
of trypsin was added to detach the cell from the surface of the flask. The flask then
incubated for 5 minutes in the CO2 incubator. After 5 minutes, the trypsinized cell was
taken out and mixed with 40 ml RPMI 1640 medium in petri dish. Another empty petri
dish was filled with medium only. The medium was then ready to be put into the 96well microtiter plates. There are 12 lanes on the microtiter plates containing 8 well on
each lane. 100µl of medium only was added on wells at Lane 1 and Lane 2 for control
purposes. In addition, another 100µl of medium with cells was added to the rest of the
well. The 96 well microtiter plate containing cells were incubated at temperature 37ºC
in the CO2 incubator until it is suitable for testing purposes.
3.2.6.2
Addition of Stachytarpheta jamaicensis (L.) Vahl crude extracts into the
Hela cell culture
The crude extract of Stachytarpheta jamaicensis (L.) Vahl was prepared at
concentrations of 1000 µg/µL in 10 ml of bijou bottle. The extract was diluted with
80% ethanol to make the stock solution. The extract will be added into the 96-well
microtiter. After the cells achieved confluency around 70%, the old medium was suck
out and washed with 100µl PBS. After that, new medium was added into each well for
cell feeding using multichannel pipette. On lane 3, 50µl of extract was added to the well
and resuspend with the cell. After resuspend, the extract was diluted with two-fold
dilution from lane 3 to lane 10. Thus, there will be 8 different concentrations of extracts
ranges from 1000 µg/µL until 7.8 µg/µL at the end of the dilution process. The cells on
lane 1, 2, 11 and 12 serves as control for the test because they only contain medium and
cells without addition of extracts. The cells containing extracts were then incubated for
24, 48 and 72 hours to observe for the cytotoxic activities in the CO2 incubator at
temperature 37ºC.
37
3.2.6.3 The cytotoxic assay using alamar blue solution
The cytotoxicity assay was performed by preparing the 3 % alamar blue solution
to be added into the cells after incubation. A working stock solution of extract was
prepared and filtered sterilized using nylon membrane filter 0.22 µm. After 24 hours
incubation, the old medium containing extracts were suck out and removed. After
removed the cells, the debris were washed with 100µl of PBS solution. Moreover, 50 µl
of new medium and 50µl of 3% alamar blue solution were added on all wells from lane
1 to lane 11. During the addition of alamar blue solution, the process must be done in
dark condition because alamar blue is sensitive towards light. The final volume of
solution in each well was 100 µl. Then, the 96-well plate was wrapped with aluminium
foil and incubated for 4 hours in CO2 incubator. After 4 hours, the absorbance was read
at wavelength 575 by using BIO-RAD benchmark microplate reader with reference
wavelength of 655 nm. All experiments were performed with four replicates. The
absorbance was used to check for the inhibition of the extracts towards the cell activity.
3.2.6.4 Statistical analysis
All the data collected was presented as means and stardard errors. Statistical
analysis was performed using one-way analysis of variance (ANOVA).
Mutiple
comparisons of the means were done by using Bonferroni test. Significant value of p
<0.05 were taken into consideration. All the statistical analysis were done using SPSS
version 15.0.
38
CHAPTER 4
RESULTS AND DISCUSSIONS
4.1 Extraction of Stachytarpheta jamaicensis (L.) Vahl crude extracts
In solvent extraction process, the solvents used are based on polarity and the
concepts of ‘like dissolves like’ are commonly used. In this study, the polar solvent
used is ethanol because ethanol are said to be one of the most widely used solvent for
the extraction of plant materials containing saturated organic compound (Rosnani
Hasham et.al.,2003, Das et.al.,2010, , Enwuru et.al.,2008). Solvents will diffuse into the
plant tissue and solubilize the compound with similar polarity (Das et. al., 2010).
The extraction methods mostly used are plant homogenization of solvent. It
starts grounded the plant into small finer particle so that the surface area will be
increased thus increasing the time taken for extraction process. Silva et al. (1998)
proposed that the effectiveness of an extraction process depends on the solubility,
stability and functional-group considerations. Next, the steps were proceed by soaking
the grounded parts of Stachytarpheta jamaicensis (L.) Vahl in solvent selected which is
ethanol. The solvent were chosen based on previous research done by Enwuru et al.
(2008) on another family of Verbenacea, Stachytarpheta angustifolia using the similar
solvent, 80% ethanol for extraction. After solvent extraction process, the mixture was
filtered using filter paper to collect only the solution and discard the waste. The filtrate
was concentrated using rotary evaporator and the crude extract obtained was dried in an
39
incubator oven to make sure that the solvent residue was removed from the crude
extract. The temperature during solvent removing process by rotary evaporator must
not exceed 30 to 40°C since thermolabile compound may be degraded by higher
temperature (Silva et al., 1998). The approximate weight of each extracts obtained was
as stated in Table 4.1:
Table 4.1 Quantitative weight of each samples extract taken from Stachytarpheta
jamicensis (L.) Vahl after extraction
Extracts
Weight (g)
Leaves
10.34
Roots
2.25
Stems
13.08
4.2 Phytochemicals test
After performing the phytochemicals tests on the extracts, the following results
were obtained and summarized in the Table 4.2. Negative results shown in Table 4.2
does not mean that the compound did not presence but because the compound may
occur in too low concentration for unambiguous detection.
40
Table 4.2 Qualitative test for the presences of phytochemicals inside stem, leaves and
roots of Stachytarpheta jamaicensis (L.) Vahl extracts
Phytochemical tests
S.jamaicensis
S.jamaicensis (L.)
S.jamaicensis
(L.) Leaves
Roots
(L.) Stems
Phenolic compounds
and flavonoids
i)
Ferric
chloride test
ii)
iii)
Gelatin test
Alkaline
reagent test
Terpenoids (Salkowski
test)
Saponins
Phlobatannin
Tannins
Coumarins
Flavonoid
+
+
+
-
+
-
-
+
+
+
+
+
+
+
+
+
+
+
+
+
Presence of constituents
+ = positive, - = negative
From the Table 4.2, the extracts showed positive results for phenolic compounds
(ferric chloride test), terpenoids, tannins and flavonoid. Crude Leaves extract of
Stachytarpheta jamaicensis (L.) Vahl is positive for ferric chloride, terpenoids, saponins
and flavonoids whereas the roots extract are positive for almost all phytochemicals
except phlobatannin and coumarins. Compare to leaves and roots, stems extract are only
positive for ferric chloride, alkaline test, terpenoids and flavonoids.
The presence of flavonoids in the crude plant extracts in contrast with the
findings from Edeoga et al., (2005) using the plant from the same genus but different
species which is Stachytarpheta cayennensis.
Okokon et. al. (2008) also reported
similar findings regarding the species S. cayennensis where they detected
41
phytochemical such as alkaloids, ipolamide, beta hydroxyipolamide and verbascoside
presence in the plants.
4.2.1 Ferric chloride test
Based on the method proposed by Raaman (2006), the positive result would be
the appearance of green colour indicating the presence of phenolic compound. Thus,
we can see in Figure 4.1 that there are colour changes before and after addition of 5%
ferric chloride solution to the extracts giving it positive results.
(Before)
(After)
Figure 4.1 The colour changes of the extracts to green after the addition of 5% ferric
chloride solution.
4.2.2 Gelatin test
An indicator for positive result for gelatin test is the presence of white
precipitate after the addition of 1% gelatin containing 10% sodium chloride. However,
only roots extract showed positive results for gelatin. The quantity of the white
precipitate also very little and hardly seen through naked eye. This might be due to only
small quantity of gelatin presence in the plants thus explaining the amount of white
precipitates after the test was performed.
42
Figure 4.2 The results for gelatin test. Roots extracts shows white precipitate.
4.2.3 Alkaline reagent test
In alkaline reagent test, 10% ammonium hydroxide solution was added to the
extract and yellow fluorescence shows presence of flavonoid. Based on the Table 4.2, it
showed that only roots and stems showed yellow fluorescence. The yellow fluorescence
will be much more visible if the universal bottle was shaken.
43
(Before)
(After)
Figure 4.3
The presence of yellow fluorescence on roots and stems extracts of
Stachytarpheta jamaicensis (L.) Vahl.
4.2.4 Test for terpenoids
The terpenoids tests showed positive results for all three parts of the plants
indicates that thse whole plants contain high contents of terpenoids. This is due to
formation of reddish-brown color on the upper layer of the extract solutions. Terpenoids
were said to give fragrance characteristic to plants, thus this explains why the plants
release some scent when we smell it.
44
Figure 4.4 Formation of reddish brown layer
4.2.5 Test for saponin or froth test
Saponin is one of natural compounds that derived from parts of plants such as
stems, roots, leaves and flowers.
They were commercially extracted and help in
production of soap. Saponin will produce soap-like foam when they are dissolves and
shaken in water. In this study, saponin test is a test which detects formation of froth
after boiling of extracts in distilled water for 15 to 20 minutes. Table 4.2 shows that
only leaves and roots of Stachytarpheta jamaicensis (L.) Vahl contains traces of
saponin excluding the stems. Idu et al. (2007) have done a research on the leaves of S.
jamaicensis (L.) Vahl where they found traces of saponin inside the leaves after the
addition of 90% ethanol and sulphuric acid. Moreover, these findings also similar to the
results for another species of Stachytarpheta reported by Edeoga et al. (2005) regarding
the presence of saponin inside the plants. The froth formed on the upper layer of the
extracts is persistent for a long time.
45
Formation of froth
Figure 4.5 Saponin test indicates froth formation
4.2.6 Test for flavonoids
Flavonoids test performed in this study was based on the method proposed by
Edeoga et.al (2005). Based on the method, yellow colouration will appear after the
addition of ammonia solution to the filtrate. The yellow colouration appeared as a layer
on the bottom of the test tube. All parts of the plants showed positive results for
flavonoids and leaves gives the highest amount of yellow colouration compare to roots
and stems. Moreover, roots have the least yellow colouration possibly indicate that there
was only a little amount of this bioactive compound inside.
46
Formation of yellow
colouration
on
bottom of test tube
Figure 4.6 Extracts with positive test for flavonoids
4.2.7 Test for tannin
Tannin is one of the bioactive compounds that are soluble in water, alcohol and
acetone and are able to precipitates protein due to its astringent properties (Basri and
Fan, 2005). The result shows that all three parts of the plants contain tannins because of
the brownish green and blue black colouration. This brown colour shows the presence
of gallic and tannin acid inside the plants due to reaction of the extracts with ferric
chloride as reported by Drabble and Nierenstein, 1906. This result also was supported
by research from Idu et al. (2007) giving positive results for tannins in the leaves of
S.jamaicensis (L.) Vahl .
the
47
Figure 4.7 Brownish green colouration after addition of ferric chloride.
4.2.8 Test for coumarin
In Figure 4.8, all three parts of Stachytarpheta jamaicensis (L.) Vahl did not
show any traces of coumarin after test was performed. No yellow green colour appears
after expose under UV light for a few minutes. According to Smith and Gorz, 1965,
coumarin will fluoresce yellow green colour when placed in a strongly alkaline solution
that was exposed to UV light. Coumarin are said to have high concentration in fruits
followed by other parts such as roots, stems and leaves (Lacy and O’Kennedy, 2004).
Unfortunately, this plant do not bears any fruits thus this supports the evidence for the
absence of coumarin inside the plants.
48
Figure 4.8 Absence of yellow green colour on the filter paper shows no presence of
coumarin
4.2.9 Test for phlobatannins
The aqeous extracts of root, leaves and stem of Stachytarpheta jamaicensis was
found not to show any deposition of red precipitate after boiling with 1% aqeous
hydrochloric acid in Figure 4.9. This suggests that the plants did not contain any
phlobatannins compound.
Figure 4.9 Absence of red deposition occur after boiling extract with 1% hydrochloric
acid.
49
4.3 Identification of bacteria
a) Gram staining method
Gram staining method for bacterial identification was developed by Christian Gram
in 1884 to differentiate bacteria based on tissue section. The basic principle of gram
stain is that certain bacteria will retain a blue-black dye complex when staining with
organic solvents such as alcohol or acetone. Formation of the complex occur when
interact between iodine and crystal violet. Tissue of the bacteria did not retain the
complex; however they require the counterstaining with red dye such as safranin. The
bacterial with thin layer of peptidoglycan cannot retain the complex in contrary to the
bacterial with thick layer of peptidoglycan. If the bacterial was found not to retain the
crystal violet-iodine complex when decolorize with alcohol or acetone, this bacterial are
known as gram-negative and the bacterial that are able to retain the dye complex are
called gram-positive. Decolorization with alcohol also leaves small holes in the thin
peptidoglycan layer of the gram-negatives allowing the crystal-violet to diffuse. Differ
from gram-negative, when iodine applied, it forms crystal with crystal violet dye
making it too large to escape through cell wall of gram-positive bacteria. Alcohol will
then dehydrate the peptidoglycan making it much more impossible for the penetration
of crystal violet-iodine complex (Tortora et al., 2007). From the results, there were six
strains of bacterial identified using this method and the results presented in Table 4.3
below. The images of each bacterial staining viewed under 100x magnification of
immersion oil were shown in Appendix A.
50
Table 4.3 Gram staining of the bacterial for antimicrobial test
Microorganims
Staining / Shape
Gram negative
Escherichia coli
Pink, rod
Pseudomonas aeruginosa
Pink, rod
Gram positive
Bacillus sp.
Purple, rod
Micrococcus luteus
Purple, cocci
Streptococcus sp.
Purple, cocci chain
Staphylococcus sp.
Purplee, cocci
4.4 Antimicrobial susceptibility test
The antimicrobial activities of each extracts on the bacterial colonies were
recorded by measuring the diameter of zone inhibition presented by each discs. The
data were recorded as follow in Table 4.4. The data showed that the highest inhibition
zone of the extracts was obtained from the root of S. jamaicensis (L.) Vahl at 5 µg/µl on
Pseudomonas aeruginosa. However, the results are varying based on the
concentrations. All three parts of plant extract showed less inhibition towards
Streptococcus sp. compared to other bacteria. The results were supported by study from
Idu et. al, 2007 on the leaves extract of similar plant on the antimicrobial activities.
Based on the studies, the effects of S. jamaicensis (L.) Vahl varies on different
microorganisms with slightly inhibition on Staphylococcus aureus and Proteus vulgaris.
The lower inhibition zone of the microorganisms may be because of the lower
concentration of extracts used. The extracts were slightly diluted thus; it cannot give a
better inhibition zone when treated on the bacterial inoculum.
51
Table 4.4 Antimicrobial activity of S. jamaicensis ( L.) Vahl extracts determine by the
diameter of inhibition zone (mm).
Microorganisms
Extract
E. coli
Stem
Leaves
5
3.5±3.5
4.5±4.5
Root
7.5±0.5
Stem
7±1.0
Leaves
6.5±0.5
Root
8±0.0
Stem
-
Leaves
Root
Stem
Leaves
3.5±2.7
5
8±0.0
3±3.0
Root
Stem
4±4.0
Leaves
8.5±0.5
Root
4±4.0
Stem
4±4.0
Leaves
3.5±3.5
3.5±3.
5
3.5
Root
10.5±3.
9
8.5±0.
5
Bacillus sp.
S. aureus
Streptococcus sp.
M. luteus
P. aeruginosa
Concentrations (µg/µl)
10
15
20
4±4.0
3.5±3.5
4.5±4. 4±4.0 5±5.0
5
4±4.0 8.5±0. 8±1.0
5
7±1.0 7.5±0. 3.5±3.5
5
6.5±0. 7.5±0. 7±0.0
5
5
3.5±3. 7±1.0 3±3.0
5
3.5±3. 4±4.0
5
3±3.0 -
25
3±3.0
3.5±3.5
8±1.0
5.5±5.5
8.5±1.5
9±1.0
6.5±0.5
-
4 4.0
6.5
4±4.0
3±3.0
7.5±0.5
3.5±3.5
3.5±4.0
-
3.5±3.
5
7.5±0.
5
7±0.0
3±3.0
3±3.0
3±3.0
9±0.0
7.5±0.5
9±.1.0
6.5±0.
5
-
3±3.0
3.5±3.5
4±4.0
-
8±.0
7±0.0
9.5±0.5
7±0.0
7.5±1.0
9±2.95
Analysis of the data for inhibition zone of the bacteria by the extracts using oneway ANOVA test is significant with p < 0.05. This means that the bacterial are related
to the inhibition zone produces. However, there are no inhibition zones recorded by any
52
crude extracts of the plant on the growth of both fungi Aspergillus niger and
Saccharomyces cerevisae.
Diameter of inhibition zone (mm)
10
9
8
7
6
5
4
3
2
1
0
E.coli
S.aureus
Bacillus sp.
M.luteus
Streptococcus P.aeruginosa
sp.
Microorganisms
Figure 4.10 The inhibition zone of S.jamaicensis leaves extract
Based on the Figure 4.10 above, it shows the inhibition zone of S.jamaicensis
leaves extract on the growth of six strains of bacteria. According to the data, highest
inhibition can be seen on the growth of Micrococcus luteus with 8.3 mm. This followed
by inhibition on Pseudomonas aeruginosa and Bacilus sp. However, the extract did not
inhibit much on Staphylococcus aureus, Escherichia coli and Streptococcus sp. Idu et.
al, 2007 also did antimicrobial test for the leaves extract of S.jamaicensis and the result
was similar to the results obtained where the leaves are found to have an antimicrobial
activity towards Pseudomonas aeruginosa, Bacillus
Staphylococcus aureus.
sp., Escherichia coli and
53
Diameter of inhibition zone (mm)
16
14
12
10
8
6
4
2
0
E.coli
S.aureus
Bacillus sp.
M.luteus
Streptococcus P.aeruginosa
sp.
Microorganisms
Figure 4.11 The inhibition zone of S.jamaicensis stems extract.
Figure 4.11 above shows the inhibition zone of stem extract from S.jamaicensis
towards six strains of bacteria. The histogram shows that highest inhibition of stem
extracts was on Bacillus sp. and Pseudomonas aeruginosa with 6.1 mm and 8.5 mm
each. In contrast, the extract did not show any inhibition on Streptococcus sp. at any
concentrations might be because Streptococcus sp. is resistant to the compounds
presence in the extract.
54
Diameter of inhibition zone (mm)
12
10
8
6
4
2
0
E.coli
S.aureus
Bacillus sp.
M.luteus
Streptococcus P.aeruginosa
sp.
Microorganisms
Figure 4.12 The inhibition zone of root extract from S.jamaicensis.
According to Figure 4.12, the root extract gives better inhibition to all six strains
of bacteria except for Streptococcus sp. compare to stem and leaves extracts. The
extract are found to inhibit the growth of Pseudomonas aeruginosa with the highest
inhibition zone produce is 8.5 mm followed by Escherichia coli with 7.2 mm. The
lowest inhibition of the root extract is on Micrococcus luteus with diameter of 3 mm
and no inhibition was found on Streptococcus sp.
55
4.5 Assessment of cell viability
For cytotoxic assay, the Hela cells were seeded in 96-well plates until
confluency to ensure that the cells are in their optimum performances to be used. The
concentrations of extracts used for treatment ranged from 1000 µg/µL to 7.8 µg/µL.
The treatment was conducted at exposure time of 24, 48 and 72 hours. After addition of
extracts and incubate for 24 hours, the cells viability were assess using dye such as
alamar blue (resazurin). The viability of the extracts were determined by the reduction
of the mitochondrial by resazurin into resorufin which can be detected both
colorimetrically and fluorometrically (McMillian et al, 2002). The oxidized form of
alamar blue enters the cytosol and thus converted to its reduced form when it accepts
electrns from NADPH, FADH, FMNH and NADH (Al-Nasiry et al, 2007). Alamar blue
was reduced to purple and pink from blue. The living cells will reduce the indigo blue
dye to fluorescent pink color while dead cells will remain indigo blue colour. The
absorbance of alamar blue was measured at wavelength 575 nm using microplate
reader.
Figure 4.13
The reduction of alamar blue indigo blue colour to pink colour solution
after incubation for 4 hours.
56
The absorbance data collected were used as a means value and graphs was plot
consists of absorbance versus concentrations to observe for cell viability. Based on the
graphs, the cell activities towards extracts of different concentration can be seen and
analysed.
4.6 The cytotoxic activities of Stachytarpheta jamaicensis (L.) Vahl crude extracts
on the growth of Hela cells at 24, 48 and 72 hours f exposure times.
The cytotoxic activities of crude extract of Stachytarpheta jamaicensis (L.) Vahl
on the activities of Hela cancer cells after exposure time of 24, 48 and 72 hours were
shown in Figure 4.14, 4.15 and 4.16 below. The effects of the crude extracts are shown
based on its concentrations ranged from 1000 µg/µl to 7.8 µg/µL. Based on the figure
4.14 shown below, after 24 hours of incubation period, the cell viability was increase
when the concentrations of the extract decrease. The cells started to die when the
concentration of the extracts added was 1000 µg/µL and increased gradually due to low
concentration of extracts. Thus, this can be concluded that the cells activities were
reduced at a higher concentration of extracts. The extracts loss its effects when it is
more diluted. All three types of extracts give effects towards the cell viability.
After 48 hours of incubation, the figure 4.15 shown increased in cell viability for
stem and leaves extracts at the higher concentration compared to root extract. In
contrast, the root extract gives positive results at concentration of 1000, 500 and 250
µg/µL and did not gives any effect for concentrations lower than that. Stem extract also
shows similar results with root extract where the cell viability increase until
concentration of extract is 15.63 µg/µL but decrease with lower concentration in
example 7.8 µg/µL. Leaves extract gives better inhibition compared to another two
because the mean graph increase gradually.
57
In addition, during the incubation for 72 hours in the incubator as shown in
figure 4.16, all three extracts showed positive inhibition towards the activities of Hela
cells. All cell death occur at highest concentration which were 1000 µg/µL and 500
µg/µL, even though at concentration 125 µg/µl, the cell growth increase suddenly but
decrease back after that.
Based on all three figures below (see Figure 4.14, 4.15, 4.16), we can concluded
that the best inhibition occur at 24 and 72 exposure time compared to 48 exposure time.
This is because during 48 hours exposure time, the cytotoxic effect of the extracts
towards the cells activities was unstable.
0.12
0.1
Absorbance(nm)
0.08
0.06
Root(nm)
Leaves(nm)
Stem(nm)
0.04
0.02
0
1000
500
250
125
62.5 31.25 15.63
7.8
Concentrations (µg/µL)
Figure 4.14 The effect of Stachytarpheta jamaicensis (L.) Vahl crude extracts on Hela
cells after 24 hours exposure time
58
0.25
Absorbance (nm)
0.2
0.15
Roots(nm)
Leaves(nm)
0.1
Stem(nm)
0.05
0
1000
500
250
125
62.5
31.25
15.63
7.8
Concentrations (µg/µL)
Figure 4.15 The effect of Stachytarpheta jamaicensis (L.) Vahl crude extracts on Hela
cells after 48 hours exposure time.
59
0.16
0.14
Absorbance(nm)
0.12
0.1
0.08
Root
leaves
0.06
Stem
0.04
0.02
0
1000
500
250
125
62.5
31.25
15.63
7.8
Concentrations (µg/µL)
Figure 4.16 The effect of Stachytarpheta jamaicensis (L.) Vahl crude extracts on Hela
cells after 72 hours exposure time.
60
4.7 Correlation data between 24, 48 and 72 hours of exposure times of root, stem
and leaves extracts.
Using analysis of variance (ANOVA) by SPSS 15.0 software, it shows statistical
data of correlation between 24, 48 and 72 hours of exposure times for each extracts.
Based on Table 4.17, best correlation can be seen on leaves extract compared to the root
and stem extracts. For leaves extract, the correlation between 24, 48 and 72 were very
good with significance level of 0.01. This also goes the same with the stem extracts
because there was correlation between three exposure times. The data were significant
with 0.05 and 0.01. For root extract, correlation would be best between 24 hours and 72
hours only. During 48 hours, there is no correlation might be because there are other
compounds in the extracts which interrupts. Moreover, increase in incubation time
might cause the medium for the cells to deplete and disrupts the growth of the cells thus
suspend the activities of the extracts towards the cells.
61
Table 4.5 The correlation data obtained from ANOVA test from (a) Root(b) Leaves (
c) Stem.
24 hours
24 hours
Pearson Correlation
.658(**)
36
-.152
.377
36
1
.000
36
-.053
.377
36
36
.761
36
.658(**)
-.053
1
.000
.761
36
36
36
24 hours
1
36
.597(**)
48 hours
.597(**)
.000
36
1
72 hours
.673(**)
.000
36
.687(**)
.000
36
.673(**)
.000
36
36
.687(**)
.000
36
N
Pearson Correlation
Sig. (2-tailed)
N
72 hours
Pearson Correlation
Sig. (2-tailed)
72 hours
-.152
Sig. (2-tailed)
48 hours
48 hours
1
N
(a)Root
24 hours
48 hours
72 hours
Pearson Correlation
Sig. (2-tailed)
N
Pearson Correlation
Sig. (2-tailed)
N
Pearson Correlation
Sig. (2-tailed)
N
.000
36
1
36
(b) Leaves
24 hours
Pearson Correlation
Sig. (2-tailed)
48 hours
N
Pearson Correlation
72 hours
Sig. (2-tailed)
N
Pearson Correlation
Sig. (2-tailed)
N
24 hours
1
36
48 hours
.413(*)
.012
36
72 hours
.193
.259
36
1
.684(**)
.000
36
.413(*)
.012
36
36
.193
.259
.684(**)
.000
1
36
36
36
( c ) Stem
62
4.8 Means data of roots, leaves and stem extracts for three different exposure
times
4.8.1 Root extract
Based on figure 4.17, the bar chart showed the significant between the
concentrations of extracts on Hela cells. The blank or medium without cells are
significant with concentrations ranged from 1000 µg/µL to 31.25 µg/µL whereas for
concentrations below 31.25 µg/µL, it was significant with the higher concentrations.
This shows that the amounts of living cells on blank (a) were significant with other
concentrations (b).
According to Figure 4.18, the cells growth increase at concentration of 125
µg/µl and further decrease slowly again. Most of the concentrations were significant
with the blank sample containing only living cells (a) and related. The data were
significant with 0.005.
Furthermore, Figure 4.19 shows the mean data for root extract during 72 hours
incubation periods. Smallest standard error showed that there are less error occurred
during the experiment. The cell viability also showed no difference among them from
higher concentration to the lowest concentration. Based on the figure, it showed that the
cells growth increase and less inhibition occur. This can be concluded by saying that
even after 72 hours incubation time, the root extract did not have any effects towards
Hela cells growth.
63
0.1
c
c
15.63
7.8
a,b,c
0.09
a
0.08
0.07
b
b
b
b
500
250
125
62.5
b
0.06
0.05
0.04
0.03
0.02
0.01
0
0
1000
31.25
Figure 4.17 The mean data for 24 incubation periods showing control (a) is significant
with all samples with different concentrations of root extracts.
0.3
b
0.25
Absrobance (nm)
a,b
a,b
0.2
0.15
a
a
a
a
0
1000
500
250
a,b
a
0.1
0.05
0
125
62.5
31.25
15.63
7.8
Concentration (µg/µl)
Figure 4.18 The mean data for 48 incubation periods showing the significant between
the control (a) and the samples with different concentration of root extracts.
64
0.14
a
a,b,c
Absorbance (nm)
0.12
b
b
b
b
a,b,c
c
c
0.1
0.08
0.06
0.04
0.02
0
0
1000
500
250
125
62.5
31.25
15.63
7.8
Concentration (µg/µl)
Figure 4.19 The mean data for 72 incubation periods showing significant between
control (a) and samples with extracts.
4.8.2 Leaves extracts
Figure 4.20 showed the mean data for leaves extract during 24 incubation hours.
The increase in the absorbance means that the cells growth increases based on the
concentration of extracts. Based on figure 4.20, the absorbance increases when the
concentration decrease showed that at lower concentration, the extracts cannot inhibit
the cell growth. Thus, higher concentrations of leaves extract were needed to suspend
the Hela cells growth.
After 48 hours incubation time, the absorbance increased when the
concentrations decrease (see Figure 4.21). Compare to the blank sample with no extract
added, the absorbance increase when the concentrations of leaves extract decrease.
Therefore, the cell death occurs at higher concentration and more cell alive when at
65
lower concentration. Moreover, the sample without extract added (a) was significant
with all concentration of extracts from 1000 µg/µl until 7.8 µg/µl.
Additionally, after 72 incubation hours of leaves extracts with Hela cells, the
extracts were found to cause slightly small effects on the cell growth because the
absorbance of the cells did showed any clear difference between them. The significant
value between also can be seen in the Figure 4.22 where all the absorbance of the cells
are significant with the control without extracts.
0.12
a
c
0.1
Absorbance (nm)
b
b
b
b
1000
500
250
c
c
0.08
0.06
0.04
0.02
0
0
125
62.5
31.25
15.63
Concentration (µg/µl)
Figure 4.20 The mean data for leaves extract after 24 incubation hours showing the
significant between control (a ) and samples containing extracts (b) and ( c).
66
0.2
0.18
a
a
Absorbance (nm)
0.16
a
0.14
a
a
a
a
a
a
0.12
0.1
0.08
0.06
0.04
0.02
0
0
1000
500
250
125
62.5
31.25
15.63
7.8
Concentration (µg/µl)
Figure 4.21 The mean data for leaves extract after 48 incubation hours where all the
samples and extract was not significant.
0.16
0.14
a
b
0.12
Absorbance (nm)
a,b,c
a,b
a,b,c
500
250
a,b
a,b,c
c
a,b,c
0.1
0.08
0.06
0.04
0.02
0
0
1000
125
62.5
31.25
15.63
7.8
Concentration (µg/µl)
Figure 4.22 The mean data for leaves extract after 72 incubation hours showing
differences in significant level for control (a) and samples with extracts.
67
4.83 Stem extracts
Stem extracts was incubated for 24 hours with Hela cells and identify for its
effect towards the Hela cells activities. Based on Figure 4.23, the effects of stem
extracts on cell growth can be seen where absorbance increase when the concentrations
decrease. This can be concluded that stem extracts do have effects towards cell death,
however, the effects is not so good compared to leaves extract because the difference
gap of absorbance between the concentrations were close. The inhibition best occur at
higher concentrations compare to lower concentrations of extracts.
At low
concentrations, the cells were able to grow and not inhibit by the extracts.
After 48 incubation hours, the effects of stem extracts on the growth or activities
of Hela cells can be seen where there are not so many differences amongst the
concentrations of extracts (see Figure 4.24). However, the data is not significant at all
with the value of 0.582 which is higher than significant value p<0.05. This might be
because of the condition of extracts which was not consists of pure compound. Hence,
the presence of other compound might influence the cytotoxic effect of stem extracts
towards Hela cells.
During 72 hours incubation time, the stem extracts also did not show any
significant effects towards the Hela cells growth. The absorbance was similar between
all concentrations of extracts ranged from 1000 µg/µl to 7.8 µg/µl. The significant
value was 0.040. Besides, when comparing the absorbance of the control and the
absorbance of other s, there were only slightly differences suggesting that stem extracts
after 72 incubation hours were not suitable to cause any cytotoxic effects towards the
Hel cells. This might be because the compounds in the extracts become dysfunctional
after certain duration of time.
68
0.12
Absorbance (nm)
0.1
a
a,c
a,b,c
1000
500
0.08
a,c
c
b
a,b,c
a,b,c
15.63
7.8
a,b,c
0.06
0.04
0.02
0
0
250
125
62.5
31.25
Concentration (µg/µl)
Figure 4.23 The mean data for stem extract after 24 incubation hours showing (a) is
significant with all the concentrations of extracts used in the treatment.
0.2
a
0.18
Absorbance (nm)
0.16
0.14
a
0.12
a
a
a
a
1000
500
250
125
a
a
a
0.1
0.08
0.06
0.04
0.02
0
0
61.25
31.25
15.63
7.8
Concentration (µg/µl)
Figure 4.24 The mean data for stem extract after 48 incubation hours showing (a) is
not significant with the absorbance of cell growth after treatment.
69
0.16
0.14
a
a
0
1000
Absorbance (nm)
0.12
a
a
a
500
250
a
a
31.25
15.63
a
a
0.1
0.08
0.06
0.04
0.02
0
125
62.5
7.8
Concentration (µg/µl)
Figure 4.25 The mean data for stem extract after 72 incubation hours showed no
significant between the control and the samples with extracts added.
70
CHAPTER 5
CONCLUSIONS AND RECOMMENDATIONS
5.1 Conclusions
As a conclusion, extraction of S. jamaicensis (L.) Vahl have been successfully
performed and crude extract was obtained from solvent extraction and rotary
evaporation process. The test on phytochemicals inside the plants shows that there were
presence of many phytochemicals such as flavonoid, saponin, tannin, phenolic
compounds and terpenoids in the three parts of the crude plant extracts. None of any
plants part showed positive results for all the phytochemicals tested. Coumarin and
phlobatannins did not present in any parts of the crude plant extracts. Among three
different parts of S.jamaicensis plants tested, crude root extract showed the presences
for most of phytochemicals tested compared to the other two. The highest inhibition
zone recorded was on the growth of Pseudomonas aeruginosa, Micrococcus luteus and
Escherichia coli. Slight inhibition or less occurs on Streptococcus sp. suggesting that
the plant extracts did not shown any effect on the growth of the microorganism. Based
on the cytotoxic effect of S.jamaicensis (L.) Vahl on Hela cells, the leaves extract were
found to be the best extract amongst all three extracts where it can inhibit more cell
growth compare to the others. This might be because of the presence of compound such
as flavonoids which is known for its function in treatment of cancer.
71
5.2 Future studies and recommendations
In this research, the qualitative test was found to be able to detect for the
presence of the natural compounds in the plant but not being able to quantify the
amount of the compound inside. Thus, quantitative test can be performed to optimize
the amount of compound and tested it on cells. The percentage of inhibition of the
cancer cells, IC50 can also be determined by comparing the inhibition between cancer
cells and normal cell lines in order to check for its ability whether can be used as a drug
in cancer treatment.
72
REFERENCES
Almeida C. E., Karnikowski M.G.O., Foleto R. and Baldisserotto B. (1995). Analysis
of antidiarrhoeic effect of plants used in popular medicine, Rev Saudé Publica,
29(6), 428-433.
Al-Nasiry. S., Geusens. N., Hanssens. M., Luyten. C. and Pijnenborg. R. (2007). The
use of alamar blue assay for quantitative analysis of viability, migration and
invasion of choriocarcinoma cells, Human Reproduction, 22 (5), 1304-1309.
Awah F.M., Uzoegwu P.N., Oyugi J.O.,Rutherford J., Ifeonu.P., Xiao J.Y., Fowke
K.R., and Michael O.E.(2010). Free radical scavenging and immunomodulatory
effect of Stachytarpheta angustifolia leaf extract, Food Chemistry, 119, 14091416.
Basri. D.F. and Fan. S. H.(2005). The potential of aqeous and acetone extracts of galls
of Quercus infectoria as antibacterial agents. Indian Journal of Pharmacology.
37 (1),26-29.
Bent H. Havsteen. (2002). The biochemistry and medical significance of the flavonoids.
Pharmacology and Therapeutics, 96, 67-202.
Bioavatti M.W., Koerich C.A., Henck C.H., Zucatelli E., Martinelli F.H., Bresolin T.B.
and Leiti S.N. (2004). Coumarin Content and Physicochemical Profile of
Mikania laevigata Extracts. Zeitschrift für Naturforschung.C, Journal of
Biosciences, 59c, 197-200.
Brusselmans K., Vrolix R., Verhoeven G. and Swinnen J. V. (2005). Induction of
Cancer Cell Apoptosis by Flavonoids is Asociated with Their Ability to Inhibit
Fatty Acid Synthase Activity. The Journal of Biological Chemistry, 280(7),
5636-5645.
73
Chariandy C.M., Seaforth C.E., Phelps R.H., Pollard G.V. and Khambay B.P.S. (1999).
Screening of medicinal plants from Trinidad and Tobago for antimicrobial
and insecticidal properties. Journal of Ethnopharmacology, 64, 265-270.
Celeghini R.M.S., Vilegas J.H.Y and Lanças F.M. (2001). Extraction and Quantitative
HPLC Analysis of Coumarin in Hydroalcoholic Extracts of Mikania
Glomerata Spreng. (“guaco”) Leaves. Journal of Brazilian Chemical Society,
12(6), 706-709.
Cowan M.M. (1999). Plant Products as Antimicrobial Agents. Clinical Microbiology
Reviews, 12(4), 564-582.
Cushnie. T. P. T. and Lamb. A. J. (2005). Antimicrobial activity of flavonoids.
International Journal of Antimicrobial Agents. 26, 343-356.
Das K., Tiwari R.K.S. and Shrivastava D.K., (2010). Techniques for evaluation of
medicinal plant products as antimicrobial agent: Current methods and future
trends. Journal of Medicinal Plant Research. 4(2), 104-111.
David L. Lentz , Alice M. Clark , Charles D. Hufford , Barbara Meurer-Grimes , Claus
M. Passreiter , Javier Cordero , Omar Ibrahimi and Adewole L. Okunade.
(1998). Antimicrobial properties of Honduran medicinal plants. Journal of
Ethnopharmacology, 63, 253-263.
Dickert. H., Machka. K. and Braveny. I. (1981). The Uses and Limitations of Disc
Testing of Bacteria Diffusion in the Antibiotic Sensitivity. Infection 9, 18-24.
Edeoga H.O.,Okwu D.E. and Mbaebie B.O.(2005). Phytochemicals constituents of
some Nigeria medicinal plants. African Journal of Biotechnology, 4(7), 685-688.
74
Elfrides E.S. Schapoval, Mara R. Winter de Vargas, Ce´lia G. Chaves, Raquel Bridi,
Jose´ A. Zuanazzi and Ame´lia T. Henriques. (1998). Antiinflammatory and
antinociceptive
activities
of
extracts
and
isolated
compounds
from
Stachytarpheta cayennensis. Journal of Ethnopharmacology, 60, 53-59.
Enwuru, N. V., Ogbonnia, S. O, Nkemehule, F, Enwuru, C. A. and Tolani, O., (2008).
Evaluation of antibacterial activity and acute toxicity of the hydroethanolic
extract of Stachytarpheta angustifolia (Mill) Vahl. African Journal of
Biotechnology, 7(11), 1740-1744.
Gislene G. F. Nascimento; Juliana Locatelli; Paulo C. Freitas and Giuliana L. Silva.
(2000). Antibacterial activity of plant extracts and phytochemicals on antibiotic
resistant bacteria. Brazilian Journal of Microbiology, 31, 247-256.
Guilfoile. G. P. and Calamo I. E. (2006). Antibiotic Resistant Bacteria. United States of
America: Infobase Publishing.
Hamid. R., Rotshteyn. Y., rabadi. L., Parikh. R. and Bullock. P. (2004). Comprisons of
alamar blue and MTT assays for high through-put screening. Toxicology in
Vitro, 18, 703-710.
Heinrich. M. and Gibbons. S. (2004). Fundamentals of Pharmacognosy and
Phytotherapy. Spain: Elsevier Health Sciences.
Idu, M., E.K.I. Omogbai, G.E. Aghimien, F. Amaechina, O. Timothy and S.E.
Omonigho. (2007). Preliminary phytochemistry, antimicrobial properties and
acute toxicity of Stachytarpheta jamaicensis (L.) vahl. leaves. Trends in
Medical. Research. 2(4), 193-198.
75
Isenberg. H. (1998). Essential Procedures for Clinical Microbiology. Washinton DC:
ASM Press.
Ivan Kosalec,Stjepan Pepeljnjak,Marina Bakmaz and Sanda Vladimir-Knežević.,
(2005). Flavonoid analysis and antimicrobial activity of commercially available
propolis products, Acta Pharm, 55, 423–430.
Lacy. A. and O’ Kennedy. R.(2004). Studies on Coumarins and Coumarin-Related
Compounds to Determine their Therapeutic Role in the Treatment of Cancer.
Current Pharmaceutical Design, 10, 3797-3811.
Lim S.H., Darah I. and Jain K. (2006). Antimicrobial Activities of Tannin Extracted
From Rhizophora apiculata Barks. Journal of Tropical Forest Sciences, 18(1),
59-65.
Liu R., Sun Q., Shi Y. and Kong L. (2005). Isolation and Purification of Coumarin
Compounds From The Root of Peucedanum decursivum (Miq.) Maxim by HighSpeed Counter-Current Chromatography. Journal of Chromatography A., 1076,
127-132.
Lopes F. CM., Rocha .A., Pirraco. A., O Regasini. L., Silva D. HS., Bolzani. V. S.,
Azevedo., Carlos I. Z. and Soares. R. (2009). Anti-angiogenic effects of
pterogynidine
alkaloid
isolated
from Alchornea
glandulosa.
BMC
Complementary and Alternative Medicine. 9(15).1-11.
McMillian M.K., Li. L, Parker J.B, Patel., Zhong. Z., Gunnet. J.W., Powers W.J. and
Johnson. M. D. (2002). An improved resazurin-based cytotoxicity assay for
hepatic cells. Cell Biology and Toxicology. 18,157-173.
76
Melanchauski. L.S.,Broto. A.P.G.S., Moraes. T.G., Nasser. A.L.M., Said. A., Hawas.
U.W., Rashed. K., Vilegas. W. and Hiruma-Lima. C.A. (2010). Gastroprotective
and Antisecretory Effects of Ailanthus excelsa (Roxb). Journal of Natural
Medicines.64, 109-113.
Monsef H. R., Ghobadi. A. and Iranshahi. M. (2004). Antinociceptive effects of
Peganum harmal L. alkaloid extract on mouse formalin test. Journal of
Pharmacy and Pharmaceutical Sciencies. 7(1), 65-69.
Mossman T. (1983). Rapid colorimetric assay for cell growth and survival:Application
to proliferation and cytotoxicity assays. Journal of Immunological Methods, 65,
55-63.
Odugbemi. T., McEntegart. M. and Hafiz. S. (1978). Effects of Various divalent cations
on the survival of Neisseria gonorrhoeae in liquid media. British Journal of
Venereal Disease. 54. 239-242.
Ogbonnia. S.O., Nkemehule. F.E. and Anyika. E.N.,(2009). Evaluation of acute and
subchronic toxicity of Stachytarpheta angustifolia (Mill) Vahl (Fam.
Verbenaceae) extract in animals. African journal of Biotechnology. 8(9), 17931799.
Okwu D. E. and Ohenhen. O.N. (2009). Isolation, Characterization and Antibacterial
Activity of Lanostane Triterpenoid from the Leaves of Stachytarpheta
jamaicensis Linn Vahl. Der Pharma Chemica. 1(2), 32-39.
Parija. S. C. (2009). Textbook of Microbiology and Immunology. Haryana,India:
Elsevier.
77
Patel. B., Sattwik. D., Prakash. R. and Yasir. M. (2010). Natural Bioactive Compound
with Anticancer Potential. International Journal of Advances in Pharmaceutical
Sciences. 1, 32-41.
Rosnani H., Norsaupiah S., Mohamad Roji. S., Ramlan. A. A., Nasrul Fikri. C. P.
(2003). Extraction of Oleoresin from Zingiber zerumbet Rhizobium:
Comparative Study on Yield, Zerumbone and Curcumin Content. International
Conference of Chemical and Bioprocess Engineering. 2003. Universiti Malaysia
Sabah.
Shahid. Ud-Daula A. F. M. and Basher. M. A. (2009). Phytochemical screening, plant
growth inhibition, and antimicrobial activity studies of Xylocarpus granatum.
Malaysian Journal Pharmaceuticals Sciences. 7(1), 9-21.
Shaikh J. Uddin, I. Darren Grice and Evelin Tiralongo. (2009); Cytotoxic Effects of
Bangladeshi Medicinal Plant Extracts. eCAM Advanced Access, p 1-6.
Silva. G.L., Lee. I.K. and Kinghorn. A. D. (1998). Special Problems with the Extraction
of Plants. in Cannell. R.J.P. (Ed.), Natural Products Isolation. (pp. 343-363).
Totowa NJ: Humana Press Inc.
Smith. W. K. and Gorz. H. J. (1965). Sweetclover Improvement.In Norman. A. G. (Ed.).
Advances in Agronomy (pp 164-223). London: Academic Press Inc.
S. Nazar ,S. Ravikumar, G. Prakash Williams, M. Syed Ali and P. Suganthi, (2009).
Screening of Indian coastal plant extracts for larvicidal activity of Culex
quinquefasciatus. Indian Journal of Science and Technology, 2(3), 24-27.
Somchit. M. N., I. Reezal, I. Elysha Nur and A.R. Mutalib. (2003). In vitro
antimicrobial activity of ethanol and water extracts of Cassia alata. Journal of
Ethnopharmacology, 84, 1-4
78
Stone J. (2007,26 April) The history of antibiotic.Articlesbase.
S. Sasidharan, L. Yoga Latha, Z. Zuraini, S. Suryani, S. Sangeetha, L. Shirley. (2007).
Antidiarrheal and antimicrobial activities of Stachytarpheta jamaicensis (L).
Vahl leaves. Indian Journal of Pharmacology, 39(5), 245-248.
Taraphdar. A. K., Roy. M. and Bhattacharya. R. K. (2001). Natural products as inducers
of apoptosis:Implication for cancer therapy and prevention. CURRENT
SCIENCE. 80(11), 1387-1396.
Tortora. G. J., Funke. B. R., Case. C. L. (2007). Microbiology An Introduction. 9th ed.
San Francisco: Pearson Benjamin Cummings.
Uddin S.J., Grice D. and Tiralongo. E. (2009). Cytotoxic Effects of Bangladeshi
Medicinal Plant Extracts. eCam Advanced Access, 1-6.
Voytik-Harbin S.L., O Brightman. A., Waisner B., Lamar C.H. and Badylak S. F.
(1998). Application and the evaluation of alamar blue assay for cell growth and
fibroblasts. In Vitro Cell Development Biology- Animal, 34, 239-246.
Wiegand I., Hilpert K. and Hancock. R.E.W. (2008). Agar and broth dilution methods
to determine the minimal inhibitory concentration (MIC) of antimicrobial
substances. Nature Protocols,3, 163-175.
79
APPENDIX A
(a)
( c)
(b)
(d)
(e)
(f)
Gram stain of six strains of bacteria.(a) P.aeruginosa (b) S.aureus. (c ) E.coli (d)
Bacillus sp.(e) Streptococcus sp.(f) M. luteus. The staining were viewed under 10x
magnification by oil immersion.
80
APPENDIX B
(a)
(b)
( c)
The crude extracts obtained from Stachytarpheta jamaicensis (L.)Vahl. (a) Root (b)
Stem (c ) Leaves.
81
APPENDIX C
82
The SPSS data analysis for inhibition zone of Stachytarpheta jamaicensis
83
APPENDIX D
(a)
(b)
(c )
(d)
Example of the inhibition zone produces by the extracts on the growth of bacteria after
24 hours incubation time.