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NI
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ISOLATION OF CHEMICAL COMPOUNDS

FROM METHANOL EXTRACT OF
Peperomia pellucida
NIROSA A/P RAMAN
FAKULTI SAINS DAN TEKNOLOGI

20
12
UNIVERSITI MALAYSIA TERENGGANU

2012
ISOLATION OF CHEMICAL COMPOUNDS FROM

METHANOL EXTRACT OF Peperomia pellucida
By
NIROSA A/P RAMAN
A PITA report submitted in partial fulfillment of

the requirements for the award of the degree of
Bachelor of Science (Chemical Sciences)
DEPARTMENT OF CHEMICAL SCIENCES

FACULTY OF SCIENCE AND TECHNOLOGY
2012
JABATAN SAINS KIMIA

FAKULTI SAINS DAN TEKNOLOGI
KIM 4998/ 4999

PENGAKUAN DAN PENGESAHAN LAPORAN
Adalah ini diakui dan disahkan bahawa laporan penyelidikan bertajuk: ISOLATION
COMPOUNDS FROM METHANOL EXTRACT OF
OF CHEMICAL
Peperomia pellucida
diperiksa dan semua
dikemukakan kepada
oleh NIROSA A/P RAMAN, No. matrik: UK20028 telah

pembetulan yang disarankan telah dilakukan. Laporan ini
Jabatan Sains Kimia sebagai memenuhi sebahagian daripada
keperluan memperolehi Ijazah SARJANA MUDA SAINS (SAINS KIMIA), Fakulti

Sains dan Teknologi, Universiti Malaysia Terengganu.
Disahkan oleh:
..
Penyelia Utama
Nama:
Cop Rasmi:
Tarikh: ..........................
..
Ketua Jabatan Sains Kimia
Nama:
Cop Rasmi:
Tarikh: ............................
ii
DECLARATION
I hereby declare that this PITA research report entitled Isolation of Chemical
Compounds from Methanol Extract of Peperomia pellucida is the result of my own
research except as cited in the references.
Signature
Name
Matric No
Date
:.............................
: Nirosa a/p Raman
: UK20028
:............................
ACKNOWLEDGEMENTS
I would like to thank all those people made this thesis possible and enjoyable
experience for me. First of all, I wish to express my sincere gratitude to the
administration of the Department Of Chemistry, University Malaysia Terengganu for
giving me an opportunity to take up final year project during my final year of studies.
I am grateful to my supervisor, Dr Habsah bt Mohamad for her kind guidance and
sincere reprimands throughout my project commenced. Her kind consideration has
given me an opportunity to experience the real research world that I am about to
endure in near future. She has shown and taught me on how a life as a natural chemist
and a researcher in general is and the steps that an excellent personality would take to
make it a successful one. I would like also to thank my friends especially my research
group mate, R. Hemala, Nor Nadia, Nur Dalila, Nor Nabila, Tee Tek Jun, Nurul
Hidayah, Wan Siti Azierah and Yong Hwee Chin for their constant encouragement
and guidance.
I also would like to express my appreciation to all the chemists and lab assistants that
have helped and guided me throughout my project completion. Special thanks to Miss
Kamariah, Mr Tan Hock Seng, Mrs Desy Fitra and Miss Siti Aishah for helping with
my project in the lab. They have placed a lot of belief in me that it has eventually
helped me to become independent and confident with my skills and abilities.
Finally, my loving gratitude would go to my parents, Mr and Mrs. Raman Muthusamy
for their support, help and encouragement throughout completion of my final year
project. They have always been my inspiration in walking through the thick and thin
of a working life. Their sacrifices and hard work have all paid off well. Thank you all
once again who has directly or indirectly made my final year project a success.
ISOLATION OF CHEMICAL COMPOUND FROM METHANOL EXTRACT

OF PEPEROMIA PELLUCIDA
ABSTRACT
In this study, Peperomia pellucida was subjected to isolation of chemical compounds
and biological activity determination. Five compounds were successfully isolated
where two of them (PPM-1 and PPC-5) were partially characterized. The isolation of
methanol extract was performed by using various chromatographic techniques such as
dry column vacuum chromatography, gravity column chromatography and preparative
thin layer chromatography technique. The structures of the compounds were
determined using IR, UV-Vis, and NMR spectroscopy. Air-dried Peperomia pellucida
plant sample was used. The sequential maceration utilized three different organic
solvents with increasing polarity, namely dichloromethane, ethyl acetate and methanol.
The phytochemical screening showed that the crude extract is rich in alkaloid and
saponin. Methanol crude extract also exhibited the presence of flavonoid during
phytochemical screening. The crude extract only displayed in vitro antimicrobial
activity on 3 bacteria which are Bacillus cereus, Micrococcus sp., and Streptococcus
uberis. However, no activity was reported for the other five bacteria (Klebsiella
pneumonia, Escherishia coli, Vibrio alginolyticus, Vibrio parahaemlyticus and Vibrio
mimicus). Antioxidant activity of methanol crude extract was determined using DPPH
(2, 2-diphenyl picrylhydrazyl) radical scavenging assay. The extract showed
antioxidant activity with IC50 value 6.41 1.34 mg/ml. The result also showed that the
free radical scavenging activity is proportional to the concentration of the crude
extract. Total phenolic content of methanol crude extract was determined
spectrophotometrically using Folin-Ciocalteu method where gallic acid was used as
standard. Total phenolic content in methanol crude extract of Peperomia pellucida
was represented by GAE value and the GAE value for methanol crude extract is 3.5
mg/g sample. Pharmacognostical identification was also done on air dried Peperomia
pellucida plant sample and different colour was produced when the sample was mixed
with different chemical reagents.
PEMENCILAN SEBATIAN KIMIA DARIPADA EKTRAK METANOL

PEPEROMIA PELLUCIDA
ABSTRAK
Dalam kajian ini, Peperomia pellucida telah digunakan bagi pemencilan sebatian
kimia dan penentuan aktiviti biologi. Lima sebatian kimia telah berjaya dipencilkan
dan dua daripadanya telah ditentukan struktur kimia. Dua sebatian kimia yang telah
berjaya dalam penentuan struktur kimia ialah PPM-1 dan PPC-5. Pemencilan ekstrak
metanol telah dilaksanakan menggunakan pelbagai teknik kromatografi seperti
kromatografi vakum turus kering, kromatografi turus graviti dan kromatografi lapisan
nipis preparatif. Penentuan struktur telah ditentukan dengan menggunakan teknik
spektroskopi infra-merah, ultralembayung dan resonan magnetik nuklear. Peperomia
pellucida yang dikering udara di bawah teduhan cahaya matahari telah digunakan.
Pengekstrakan berperingkat dilakukan menggunakan tiga pelarut organik mengikut
peningkatan polariti iaitu diklorometana, etil asetat dan metanol. Penyaringan fiokimia
menunjukkan bahawa ekstrak metanol mempunyai kandungan alkaloid dan saponin
yang tinggi. Ekstrak metanol juga menunjukkan kehadiran flavonoid semasa
penyaringan fitokimia. Selain itu, ekstrak metanol sampel hanya menunjukkan aktiviti
anti-mikrobial pada tiga bakteria iaitu Bacillus cereus, Micrococcus sp., dan
Streptococcus uberis tetapi ia tidak mempunyai sebarang aktiviti terhadap lima
bacteria yang lain (Klebsiella pneumonia, Escherishia coli, Vibrio alginolyticus,
Vibrio parahaemlyticus dan Vibrio mimicus). Penentuan aktiviti anti-oksidan oleh
ekstrak metanol ditentukan menggunakan assai radikal DPPH (2,2-dipenil
pikrihidrazil). Ektrak metanol menunjukkan aktiviti anti-oksidan dengan nilai IC50
6.41 1.34 mg/ml. Keputusan tersebut juga menunjukkan bahawa aktiviti radikal
bebas sejajar dengan kepekatan ekstrak. Tambahan pula, kandungan fenol bagi ekstrak
metanol juga ditentukan secara spektrofotometrik menggunakkan kaedah FolinCiocalteu. Kandungan fenol di dalam ektrak metanol Peperomia pellucida telah
diwakilkan dengan nilai GAE. Nilai GAE bagi ektrak metanol ialah 3.5 mg/g sampel.
Pengenalpastian farmakognostik telah dilakukan pada sampel kering Peperomia
pellucida. Pelbagai warna telah dihasilkan semasa sampel tersebut dicampurkan
dengan pelbagai bahan kimia.
TABLE OF CONTENTS
TITLE PAGE
BORANG PENGESAHAN DAN KELULUSAN LAPORAN KIM4999
DECLARATION
ACKNOWLEDGEMENT
ABSTRACT
ABSTRAK
TABLE OF CONTENTS
LIST OF FIGURES
LIST OF PLATES
LIST OF SCHEMES
LIST OF TABLES
LIST OF ABBREVIATIONS
CHAPTER 1.0
CHAPTER 2.0
INTRODUCTION
1.1 Introduction in Natural Products
1.2 Peperomia pellucida
1.3 Significance of the study
1.4 Objectives of the study
LITERATURE REVIEW
2.1 Traditional Uses of Peperomia pellucida
2.2 Biological Activity studies on Peperomia pellucida
2.3 Previous Chemical Compounds Isolated from
Peperomia pellucida
2.4 Previous Phytochemical Screenings on
Peperomia pellucida
2.5 Chemical Variation in Genus Piperceae
2.5.1 Peperomia glabella
2.5.2 Peperomia blanda
vii
PAGE
i
ii
iii
iv
v
vi
vii
x
xi
xii
xiii
xiv
1
3
6
6
7
7
8
10
15
16
16
CHAPTER 3.0
METHODOLOGY
3.1 Collection and Preparation of Plant Sample
19
3.2 Solvent Extraction
19
3.2.1 Chemical Reagents used for cold extraction
21
3.2.2 Apparatus which used for cold extraction
21
3.3 Phytochemical Screenings
21
3.3.1 Alkaloid Test
21
3.3.2 Triterpenoid/ Steroid Test
21
3.3.3 Saponin Test
23
3.3.4 Flavonoid Test
24
3.3.5 Chemical Reagents used in the phytochemical
screenings
25
3.3.6 Apparatus which used in the phytochemical
screenings
25
3.3.7 Phytochemical Screening Reagent
3.3.7.1 Dragendroffs Reagent
26
3.4 Chemical Compound Isolation Techniques
26
3.4.1 Analytical Thin Layer Chromatography (TLC) 26
3.4.2 Dry Column Vacuum Chromatography (DCVC)
3.4.2.1 Column Packing
28
3.4.2.2 Sample Crude Extract Application
28
3.4.2.3 Sample Crude Extract Elution
28
3.4.3 Gravity Column Chromatography (CC)
3.4.3.1 Column Preparation
29
3.4.3.2 Fraction Elution and Fraction Collection 29
3.4.4 Preparative TLC (PTLC)
29
30
3.4.4.2 Sample Fraction Application
30
3.4.4.3 Sample Fraction Detection and Elution 30
3.4.5 Chemical Compounds Isolation Schemes
31
3.5 Characterization of Chemical Compounds by
Spectrophotometer Techniques
3.5.1 Fourier Tranform Infra Red Spectrometer (FT-IR)32
3.5.2 Ultra Violet Spectrometer (UV)
33
3.6 Characterization of Chemical Compounds
Using Spectroscopy Technique
3.6.1 Nuclear Magnetic Resonance Spectroscopy
(NMR)
33
3.7 Antibacterial Assay
3.7.1 Chemicals used
34
3.7.2 Microorganisms used
34
3.7.3 Antibacterial Activity Assay
34
3.8 Free Radical Scavenging AssayDetermination of DPPH Scavenging Activity
35
3.9 Total Phenolic Content
37
3.10 Determination of Powdered Sample Colour
with Different Chemical Reagents
38
CHAPTER 4.0
CHAPTER 5.0
RESULTS AND DISCUSSIONS

4.1 Peperomia pellucida Plant Sample Extract
4.2 TLC Profiling of MeOH Crude Extract
4.3 Phytochemical Screenings
4.3.1 Alkaloid Test
4.3.2 Triterpenoid/Steroid Test
4.3.3 Saponin Test
4.4 Isolation of Chemical Compounds of
Peperomia pellucida
4.4.1 Isolation of Chemical Constituents
of Fraction M-2
of Fraction M-3
of Fraction M-4
4.5 Characterization of Chemical Compounds
4.5.1 PPM-1
4.5.2 PPC-5
4.6 Antimicrobial Test
4.7 Free Radical Scavenging Assay-Antioxidant Activity
4.8 Total Phenolic Content
4.9 Determination of Powdered Sample Colour
with Different Chemical Reagents
CONCLUSION
5.1 Conclusion
5.2 Future Recommendation
REFERENCES
APPENDIX
CURRICULUM VITAE
39
39
40
41
41
42
43
43
44
45
47
49
49
49
60
71
73
74
75
76
77
78
84
86
LIST OF FIGURES
No
3.1
3.2
4.1
4.2
4.3
4.4
4.5
4.6
4.7
4.8
4.9
4.10
4.11
4.12
4.13
4.14
4.15
4.16
4.17
4.18
4.19
Figures
TLC Sheet
Mechanism of DPPH acceptor
Infrared Spectrum of compound PPM-1
UV Spectrum for Compound PPM-1 in CHCl3
1
H NMR Spectrum for Compound PPM-1 in
3
Deuterated-chloroform (CDCl )
13
C NMR Spectrum for Compound PPM-1 in
3
13
APT, DEPT 135 and C NMR Spectra for Compound PPM-1
3
in Deuterated-chloroform (CDCl )
HMBC Spectrum for Compound PPM-1 in
3
HMQC Spectrum for Compound PPM-1 in
3
COSY Spectrum for Compound PPM-1 in
3
Suggested fragments from PPC-5
Infrared Spectrum of compound PPC-5
UV Spectrum for Compound PPC-5 in CHCl3
1
H NMR Spectrum for Compound PPC-5 in
3
1
H NMR Spectrum for Compound PPC-5 in
3
Deuterated-chloroform (CDCl ) (Expansion 1)
13
C NMR Spectrum for Compound PPC-5 in
Page
28
37
53
54
68
HMBC Spectrum for Compound PPC-5 in
3
HMQC Spectrum for Compound PPM-1 in
3
COSY Spectrum for Compound PPM-1 in
3
Free Radical Scavenging Activity by MeOH Extract of
Peperomia pellucida
Gallic Acid Standard Curve Graph
1
0
55
56
57
58
59
60
63
64
65
66
67
69
70
71
74
75
LIST OF PLATES
No.
1.1
4.1
4.2
4.3
4.4
4.5
4.6
Plates
Picture of Peperomia pellucida with descriptions
TLC Profiling of MeOH Crude Extract
TLC Profiling of M-1 until M-6
TLC Profiling of M-2-1 until M-2-11
Isolation and TLC Profiling of Fractions towards Isolation of PPC-5
and PPM-4
Isolation and TLC Profiling of Fractions towards Isolation of PPM-1,
PPM-2 and PPM-3
Page
5
42
46
47
47
49
49
LIST OF SCHEMES
No.
3.1
3.2
3.3
3.4
3.5
3.6
3.7
Schemes
Schematic Diagram of Overall Extraction Procedure
Schematic Diagram of Alkaloid Test
Schematic Diagram of Triterpenoid/Steroid and Saponin Test
Schematic Diagram of Flavonoid Test
Schematic Diagram of Overall Chemical Compound Isolation
from Peperomia pellucida MeOH crude extract
Chemical Compounds isolation scheme of fraction M-2
Chemical Compounds isolation scheme of fraction M-3
xii
Page
21
23
25
26
32
32
33
LIST OF TABLES
No.
3.1
3.2
3.3
3.4
3.5
4.1
4.2
4.3
4.4
4.5
4.6
4.7
4.8
4.9
4.10
Table
Page
Stages of Turbidity to Categorize Alkaloid
23
Qualitative Scale for Triterpenoid/Steroid Test
24
Scale for Classification of Saponin in Emulsion Test
24
Classification of Flavonoid
26
Powdered Peperomia pellucida Sample with Different Reagents
39
Weight and Percentage Yield of Plant Sample and Crude Extract
40
Phytochemical Test Result on MeOH Crude Extract of
Peperomia pellucida
45
The Fraction M-1 until M-6 weight
45
Infra-red Characteristics Bands for Compound PPM-1
51
UV Absorbance Data Analysis of PPM-1
51
Infra-red Characteristics Bands for Compound PPC-5
61
UV Absorbance Data Analysis of PPC-5
62
1
13
1D-NMR ( H and C NMR) and 2D-NMR assignments for PPC-5
63
Antimicrobial Result for MeOH Crude Extract of Peperomia pellucida 72
Colour Characteristics of Powdered Peperomia pellucida with Different
Chemical Reagents
75
13
LIST OF ABBREVATIONS
ABBREVIATIONS
AS
CC
CH3COOH
CHCl3
CH3COCH3
COSY
d
DCM
DCVC
DEE
DMSO
DPPH
EtOAc
EtOH
FeCl3
FT-IR
Hep G2
HCl
HMBC
HMQC
HNO3
H2SO4
IC50
KOH
L6
m
MCF 7
Me
MeOH
min
MS
NMR
NOESY
Anisaldehyde
Column Chromatography
Acetic Acid
Chloroform
Acetone
Correlated Spectroscopy
duplet
Dichloromethane
Dry Column Vacuum Chromatography
Diethyl ether
Dimethyl sulfoxide
1,1-diphenyl-2-picrylhydrazyl
Ethyl Acetate
Ethanol
Ferric Chloride
Fourier Transform Infrared Spectrometer
Human Leukemia Cancer Cells
Hydrochloric Acid
Heteronuclear Multiple Bond Correlation
Heteronuclear Multiple Quantum Correlation
Nitric Acid
Sulphuric Acid
The Concentration of Compound that Inhibits Oxidation
of Radical by 50% Relative to The Control
Potassium Hydroxide
Normal Cell
multiplet
Breast Cell
Methyl
Methanol
Minute
Mass Spectroscopy
Nuclear Magentic Resonance Spectroscopy
Nuclear Overhauser Effect Spectroscopy
14
PE
PTLC
q
Rf
s
sp.
t
TLC
UV
1D
2D
Petroleum Ether
Preparative Thin Layer Chromatography
quartet
Retention Factor
singlet
Species
triplet
Thin Layer Chromatography
Ultraviolet Spectrometer
one dimensional
two dimensional
15
CHAPTER 1
INTRODUCTION
1.1
Introduction to Natural Products
Plant extracts which exist in the form of pure compounds or standardized extracts are
extracted from natural products (Sayeed, 2007) and it provides millions chances for
new drug discoveries. Based on the survey done by World Health Organisation
(WHO) a long time ago, it shows that more than 80% of world population is
depending on traditional medicine for their primary healthcare needs (Tshikalange et
al, 2004; Gordon & David, 2007; Himal et al., 2008; Suhanya et al., 2009; Egwuche et
al., 2011).
Natural products are assumed as pure and it possesses good properties. It is assumed
like that due to adjective natural incorporated when modes of treatment or
pharmaceutical product sources are made of it. The sources of natural products come
from various sources such as plants, animals, prebiotic origin or microbes (Satyajit,
2006; Cera et al., 2011). Natural products mainly consist of few classes of bioactive
compounds such as terpenoids, polyketides, amino acids, peptides, proteins,
carbohydrates,
lipids,
nucleic
deoxyribonucleic acid (DNA).
acid
bases,
ribonucleic
acid
(RNA),
and
This leads the interest of many researchers to explore the diversity of local medicinal
plants for its valuable medicinal properties. The literature review shows that there are
many plants that have medicinal traits contain phytochemicals such as alkaloids,
essential oils, aldehydes, phenols, and ethanol or water soluble compounds. All these
phytochemical compounds are essential in therapeutic application to fight against
microorganisms such as bacteria, viruses, fungi and pathogens (Suhanya et al., 2009).
The bioactive compounds in the natural products contribute to the advancement of
natural products (Sayeed, 2007; Cera et al., 2011). In recent years, many new drugs
are developed to fight against these dangerous microorganisms due to the new
discoveries on natural products medicinal properties.
Natural products have made significant improvements in the new drug discovery
where vincristine, taxol and etoposide are the drugs of choice in treatment of cancer
chemotherapy (Lixin, 2005; Gordon & David, 2007; Donald, 2008). Determination of
pharmacology, toxicology and therapeutics of natural product plants are very essential
in the production of natural product based drugs or treatments. Understanding the
history, species origin, availability, chemical and physical properties of natural
products contributes to the studies done on drug discovery or product development
using natural products (Cera et al., 2011).
Even though this world is moving further towards modern medicine but the long
known traditional medicine based on plants or natural products are not forgotten either.
Traditional medicine already placed a secured position until now because of its
remarkable properties (Pulak, 2011). At this new era, the general public somehow has
developed insecurity feelings towards synthetic medicine and having an opinion that
naturally developed drugs are much safer. Many of them prefer to treat themselves
with traditional treatment because of the cost and safety suspicion on synthetically
derived drugs (Mutee et al., 2010). It is important to be able to measure the amount of
the active ingredient present in certain natural products to develop a large number of
useful drugs. Furthermore, it is more economical to extract them out from plant
material rather than synthesizing it.
1.2
Peperomia pellucida
Peperomia pellucida is an herb species of the Piperaceae family and 1000 over
species of Peperomia pellucida distributed mainly in Central and South America,
Africa, Australia, and South-East Asian countries (Leosvaldo et al., 2006; Paola et al,
2007; Akinnibosun et al, 2008; Pulak & Arun, 2011). Fifty to ninety species dispersed
in South-East Asia (Kiew, 1999; Khan et al., 2008; Mutee et al., 2010; Nguyen et al,
2010; Cera et al., 2011; Ganiyat et al., 2011). There are few other common name for
Peperomia pellucida such as shiny bush (in English), ketumpangan air (in Malay),
ulasiman-bato (in Tagalog) and phak krasang (in central Thailand) (Kiew, 1999).
Taxonomy of the plant is as follows (Mosango, 2000; Pulak & Arun, 2011; Cera et al.,
2011).
Kingdom: Plantae
Subkingdom: Tracheobiota
Superdivision: Spermatophyta
Division: Magnoliophyta
Class: Magnoliopsida
Subclass: Magnoliidae
Order: Piperales
Family: Piperaceae
Genus: Peperomia
Species: Peperomia pellucida (L.) Kunth
The plant is a small fleshy herb where it can grow up to 45 cm tall (Ferdinand, 2009).
The tiny dot-like seeds are attached to several fruiting spikes and when it is crushed, it
has mustard like odour (Kiew, 1999; Pulak, 2011). Peperomia pellucida grows in
clumps, thriving in loose, humid soils and a tropical to subtropical climate with
translucent green heart-shaped fleshy and shiny waxy succulent alternate and ovate
leaves, with terminal and axillary efflorescence, at the opposite side from leaves,
develops well in loose and humid soil by the tree shadows (Arrigoni-Blank et al.,
2004; Ritson & Duvita, 2007; Akinnibosun et al., 2008; Ferdinand, 2009; Nguyen et
al, 2010; Egwuche et al., 2011; Pulak, 2011, Cera et al., 2011).
It is also has a thread like but angular trailing stem and the flower are very tiny,
bisexual growing in the form of cord like spikes arising from leaf axils, 1 to several,
terminal and axillary or leaf-opposed, well spaced, and unremarkable (Arrigoni-Blank
et al., 2004; Ritson & Duvita, 2007; Akinnibosun et al., 2008; Egwuche et al., 2011;
Pulak, 2011, Cera et al., 2011).
Leaf
Translucent green, heart

shaped, fleshy, shiny waxy
Succulent alternate and ovate
leaves
Whitish green underneath
Fruit/ Seed
Dot like seeds attached to

several fruiting spikes
Flowers are very tiny and
Ste
m
Thread like angular trailing
stem
Fleshy, translucent pale
green
Succulent
Root
Plate 1.1
Shallow root
Picture of Peperomia pellucida with descriptions
1.3
Significance of the study
The general aim of this study is to utilize Peperomia pellucida as it is an invasive

weed with little or no economic value at present. The extraction of active compounds
from this plant goes beyond its known ethno medicine uses such as furuncles (boils),
eye inflammation and skin sores. In particular, it is aimed to determine if there is a
potential bioactive compound from the plant by screening the antioxidant
antimicrobial activity that might have some applications in the treatment of those
infections that have increasingly developed resistance to conventional antibiotics.
Currently, there is no detailed scientific paper on its bioactive properties. In Malaysia

particularly, there is no publication on compound isolation of Peperomia pellucida
plant although this plant can be easily collected though out the year. This study
enables a huge contribution to further studies especially on pharmaceutical
development and focus on the other bioactive possibility in different area in Malaysia
even to other countries.
1.4
Objectives of the study
The objectives of the studies are:

1. To extract MeOH crude extract from Peperomia pellucida plant sample.
2. To isolate, purify and characterize the chemical compounds from MeOH
crude extract of Peperomia pellucida.
3. To conduct phytochemical screenings verifying the presence of
phytochemicals such as alkaloid, steroid, saponin and flavonoid in the
crude extract and evaluate the biological activities of MeOH crude extract
of Peperomia pellucida.
CHAPTER 2
LITERATURE REVIEW
Peperomia pellucida is a common thriving terrestrial plant that grows on humid soil.
Peperomia pellucida is from genus Piperaceae and it has many valuable properties
such
as
medicinal
properties,
antioxidant,
antimicrobial
and
many more.
Phytochemical profile from genus Piperaceae is classified by occurrence of alkaloids,

phenylpropanoids, benzopyrans,
chromenes, several nor/seco-compounds
and
prenylated hydroquinones (Leosvaldo et al., 2006). Species of Peperomia are well

known ornamental plants and have found application in folk medicine for the
treatment of inflammation, asthma, and gastric ulsers and as analgesic and
antibacterial agents (Felippe et al., 2008; Ganiyat et al., 2011; Cera et al., 2011).
2.1
Traditional Uses of Peperomia pellucida
Peperomia pellucida has been known for its medicinal properties (Bayma et al.,
2000). The infusion or decoction of leaves and stems are used to treat gout and
arthritis where it is included in daily meal as salad (Susie et al., 2000; Khan &
Omoloso, 2002; Mutee et al., 2010; Nguyen et al, 2010; Pulak, 2011; Pulak & Arun,
2011). The plant is used in traditional medicine in treatment of measles, small pox,
male impotence, mental disorders, and breast cancer (Aziba et al., 2000; Khan &
Omoloso, 2002; Ganiyat et al., 2011).
Egwuche et al., (2011) stated that Peperomia pellucida is used as emollient and
diuretic. It is also stated that cardiac arrhythmia and cough can be controlled and
cured with Peperomia pellucida. Peperomia pellucida leaves are used in the treatment
of headache, fever, eczema, abdominal pains and convulsions (Arrigoni-Blank et al.,
2004; Alam et al., 2008; Ferdinand, 2009; Alam et al., 2010; Ganiyat et al., 2011; Lee
et al., 2011). In folk medicine, this species is employed on abscesses, furuncles, and
skin sores, as well as eye inflammation (conjunctivitis) (Arrigoni-Blank et al., 2002;
Su et al., 2005; Pulak, M., 2011, Cera et al., 2011).
It is traditionally used to treat boils, skin wounds, trauma, and bleeding (Su et al.,
2005; Khan & Omoloso, 2002; Ritson & Duvita, 2007; Mutee et al., 2010). Arthritic
pains can be relieved by consuming Peperomia pellucida but according to Manila
Medical Society, patient can be attacked by CNS depression which is caused by
Peperomia pellucida (Khan et al., 2008; Alam et al., 2010). In South America,
Peperomia pellucida are used medicinally in a way where the fresh stem and leaves
juice is extracted and used to treat eye inflammation (Pulak, 2011).
2.2
Biological Activity Studies On Peperomia pellucida
Based on Lee et al., (2011) research revealed that Peperomia pellucida leaf extract
contains anticancer properties. It also has antibacterial properties. The antibacterial
properties were shown clearly when Peperomia pellucida extract inhibited the growth
of eight bacteria which were Edwardsiella tarda, Escherichia coli, Flavobacterium sp.,
Pseudomonas aeruginosa, Vibrio cholera, Klebsiella sp., Aeromonas hydrophilla and
Vibrio alginolyticus while it also limits the growth of Salmonella sp. and Vibrio
parahaemolyticus. Moreover, antioxidant properties exhibited by Peperomia pellucida
leaf extract by inhibiting 30% of 1,1-diphenyl-2-picrylhydrazyl (DPPH), free radical.
Significant antibacterial activities against ten bacteria were also shown by the plant
extract. The bacteria used for the assay were Escherichia coli, Staphylcoccus aereus,
Bacillus subtilis, Pseudomonas aeruginosa, Klebsiellae pneumonae, Salmonellae
typhi, Candida albicanas, Rhizopus stolon, Aspergillus niger and Penicullum notatum.
Other than that, Peperomia pellucida showed antioxidant properties as well. It was
determined by comparing it with the standards which are butylated hydroxyl anisole
(BHA), ascorbic acid and -tocopherol (Ganiyat et al., 2011).
Evalution of anti-inflammatory effect was done using the carrageenan-induced rat

hind paw edema method in Mutee et al., (2010) research. Chloroform (CHCl3) and
methanol (MeOH) extract did not show any significant activity while carrageenan-1
induced rat hind paw edema was significantly reduced (p< 0.05) by 1000 mg kg of
Peperomia pellucida extract compared to the control (p<0.01) (Mutee et al., (2010).
Determination of in vitro antibacterial, antifungal and cytotoxic activities was done on

patuloside A which is a xanthone glycoside (Alam et al., 2010). Weak antifungal
activities against Aspergillus flavus and Candida albicans were shown by the
compound. Besides that, a significant antibacterial activity against four Gram-positive
bacteria (Bacillus subtilis, Bacillus megaterium, Staphylcoccus aureus, Streptococcus
-haemolyticus) and six Gram-negative (Escherichia coli, Shigella dysenteriae,
Shigella sonnei, Shigella flexneri, Pseudomonus aeruginosa, Salmonella typhi) was
shown by patuloside A. LC50 of the compound against brine shrimp nauplii was 18.24
/mL when determination of cytotoxic of the compound evaluated.
Maria et al., (2002) stated that phenophases I and II of aqueous Peperomia pellucida
extract shows a considerable anti-inflammatory activity. The assay was performed on
wistar rats using rat paw edema test induced by carrageenan. Other than that, toxicity
of crude extracts from Peperomia pellucida also was determined where methanol,
hexane and ethyl acetate fractions were toxic while buthanol and water fractions were
non toxic. It was determined through brine shrimp lethality tests (Ganiyat et al., 2011).
Khan and Omoloso (2002) also proved that methanol extract of Peperomia pellucida
has good antimicrobial activity. The crude fractions also exhibited antibacterial
properties where the fractions were more active compared to the crude extract.
Microorganisms used in the assay were bacteria, fungi and protozoan obtained from
stock cultures.
Short report by Aziba et al., (2001) showed a significant analgesic activity on acetic
acid-induced writhing in mice when methanol extract of Peperomia pellucida aerial
parts given orally at doses ranging from 70 21 mg/kg.
2.3
Previous Chemical Compounds Isolated from Peperomia pellucida
Alam et al., (2010) isolated a xanthone glycoside, patuloside A (3--Dglucopyranosyloxy-1,5,6-trihydroxy-9H-xanthene-9-one) (1). The isolated compound
1
characterized using liqud chromatography/electrospray-mass spectroscopy, NMR ( H,

13
C, JMOD, COSY, NOESY, HMBC, and HMQC).

O
HO
OH
OH
CH2OH
O
OH
HO
OH
Govindachari et al., (1998), Su et al., (2005), and Chen and Dong (2006) isolated and
characterized few secolignans. The compounds were 2-methylene-3-[(3,4,5trimethoxyphenyl)(5-methoxy-3,4-methylenedioxyphenyl)methyl]butylrolactone (2),
2, 3 trans 2 methyl 3 - [(3 hydroxyl 4, 5 - dimethoxyphenyl) (5 methoxy
3,4- methylenedioxyphenyl)methyl]butylrolactone (3) and peperomins A (4), B (5), C
(6) (Chen & Dong, 2006) and E (7) (Govindachari et al., 1998).
R3
R1
R4
R2
R5
R6
R8
R7
R1
2
R2
CH2
R3
R4
R5
OCH3 OCH3 OCH3
CH3 OCH3 OCH3 OH
OCH2O
R6
R7
R8
OCH2O
OCH3
OCH2O
OCH3
OCH3 OCH3
OCH2O
OCH3 OCH3 OCH3 OCH3
OCH2O
OCH3 OCH3 OCH3 OCH3 OCH3 OCH3
CH2
OCH2O
OCH3 OCH3 OCH3 OCH2O
Su et al., (2005) research, thirteen compounds were isolated where five of it were new
compounds. The isolated compounds were characterized using IR, UV, NMR and MS
spectroscopy techniques. Thirteen isolated compounds were 7,8-trans-8,8-trans-7,8cis 7, 7 bis (5 methoxy 3, 4 - methylenedioxyphenyl) 8 acetoxymethyl 8-hydroxymethyltetrahydrofuran, 7,8trans8,8trans-7,8cis7-(5methoxy3,4methylenedioxyphenyl) - 7 - (4 hydroxyl - 3, 5 - dimethoxyphenyl) - 8, 8 diacetoxymethyltetrahydrofuran, peperomins A, B, C, E, sesamin (8), isoswertisin
(Webby
&
Markham,
1994),
7,8-trans-8,8-trans-7,8-cis-7-(5methoxy3,4-
methylenedioxyphenyl)-7-(4hydroxyl3,5-dimethoxyphenyl)8acetoxymethyl8hydroxymethyltetrahydrofuran (9),
7,8trans8,8trans-7,8cis7,7-(4-hydroxyl-3,5-dimethoxyphenyl)-8,8diacetoxymethyltetrahydrofuran (10) and 5,6,8trimethoxy 4 - (2, 4, 5 trimethoxyphenyl) 3, 4 dihydro 1(2H) - naphthalenone also known as
dihydronaphthalenone (11).
O
O
O
O
O
O
R1
OCH3
HO
R2
H3CO
OCH3
OAc
R 3O
R1
R2
OCH2O
10
OCH3 OH
R3
H
Ac
OCH3 O
H3CO
OCH3
H3CO
OCH3
OCH3
11
Other than that, a novel dimeric ArC2 compound (pellucidin A) (12), apiol (13), and
dill-apiol (14) was isolated by Bayma et al., (2000). Structural confirmation was done
with IR, MS, 1D and 2D NMR spectroscopy.
H
H
H
H
OMe
MeO
OMe
OMe
OMe
OMe
12
R1
O
O
R3
R2
R1
13
OCH3
14
R2
R3
OCH3
OCH3
H
OCH3
Manalo et al., (1983) isolated few compounds from Peperomia pellucida plant and the
compounds are 2,4,5-trimethoxy styrene (15), campesterol (16), stigmasterol (17), and
-sitosterol (18).
OCH3
H3CO
OCH3
15
H
H
HO
16
H
H
H
HO
17
H
H
HO
18
Aqil et al., (1994) isolated few flavonoid compounds such as pellucidation (19),
pellucidatin-8-neoliesperid (20), apigenin (21), acacetin (22) and isovitexin (23).
R4
R1
H3CO
R2
R3
OH
2.4
R1
R2
R3
R4
19
OH
OCH3
20
O-glc-rha
OCH3
21
22
OCH3
23
glc
OH
OCH3
OCH3
OCH3
OCH3
OH
Previous Phytochemical Screening on Peperomia pellucida
Analysis of methanol extract from Peperomia pellucida leaves by using GC/MS

revealed that it contains phytol (37.88%), 2-naphthalenol, decahydro- (26.20%),
hexadecanoic acid, methyl ester (18.31%) and 9,12-octadecadienoic acid (Z,Z)-,
methyl ester (17.61%) (Lee et al, 2011).
Other than that, phytochemicals such as alkaloids, flavonoids, carbohydrates,

triterpenoids, tannins and steroids were present in Peperomia pellucida stem extract
(Pulak, 2011). The article also states that proteins and saponins were absent in the
stem crude extract. Egwuche et al., (2011) research revealed that tannins, saponins,
alkaloids and cardenolides present in Peperomia pellucida methanol crude extract.
The screening done on powdered dried sample and also revealed that anthraquinones
were absent in the crude extract. Ganiyat et al., (2011) identified that phytochemicals
such as alkaloids, carbohydrate, tannins, resins, steroids and phenol were present in
Peperomia pellucida leaf extract.
Ritson and Duvita (2007) proven that alkaloids, saponins, steroids, triterpenoids,
flavonoids and phenol were present in the Peperomia pellucida plant extract.
Alkaloids were present in ethanol crude extract, hexane fraction, chloroform fraction
and aqueous fraction. Saponins are only present in ethanol crude extract and aqueous
fraction. Besides that, steroids were absent in ethanol crude extract and flavonoids
were present in ethanol crude extract and aqueous fraction. Other than that, phenol
were absent in hexane fraction and chloroform fraction.
2.5
Chemical Variation in Genus Piperceae
2.5.1 Peperomia glabella

This plant is an epiphyte used in Venezuelan folk medicine as an antiasthmatic.
Peperomia glabella has shown to contain 1 secolignan of butenolide skeleton. In the
previous study, 2-hydroxy-4,6-dimethoxyacetophenone (24) extracted from leaves of
Peperomia glabella (Marisi et al., 2006). Acetophenone derivatives have shown many
interesting biological properties such as anti-inflammatory, cytotoxic, and choleretic
activities.
OH
O
CH3
H3C
O
CH3
24
2.5.2 Peperomia blanda

Peperomia blanda H.B.K. is a perennial herb that typically grows in wet rock crevies.
Lidiane et al (2007) isolated 5 tetrahydrofuran lignans and 2 flavones from aerial parts
of Peperomia blanda. The characterized lignin compounds are 4-hydroxy-4,5methylenedioxy-3,5,3-trimethoxy-7,7-epoxylignan
(25),
4,5-methylenedioxy-
3,4,5,3-tetramethoxy-7,7-epoxylignan (26), 4-hydroxy-3,4,5,3,5-pentamethoxy-
7,7-epoxylignan (27), 4,5,4,5-dimethylenedioxy-3,3-dimethoxy-7,7-epoxylignan

(28), 9-hydroxy-4,5-methylenedioxy-3,4,5,3-tetramethoxy-7,7-epoxylignan (29).
MeO
RO
MeO
OMe
R
25
26
Me
MeO
MeO
OMe
OH
MeO
OMe
27
O
O
MeO
OMe
28
OH
MeO
MeO
O
O
MeO
OMe
29
The antitripanosomal properties of these novel lignans have been determined as well
in the studies. The structures of the isolated compounds were elucidated by
interpretation of their spectroscopic data including by HMQC, HMBC and NOESY.
Four of the lignans were diastereomeric while one was of mixed biosynthetic origin.
All but one of the lignans exhibited high in vitro trypanocidal activity when assayed
against epimastigotes of Trypanosoma cruzi strain Y.
CHAPTER 3
METHODOLOGY
3.1
Plant Collection and Preparation of Plant Samples
Approximately 5 kg of whole fresh Peperomia pellucida plants were collected at a

palm oil estate in Linggi, Negeri Sembilan on July 2011. The fresh plants were
washed with clean running water to remove all the soils and dirt on the plants
(Akinnibosun et al., 2008). During the cleaning process, the fresh plants that has cut or
infections were discarded to make sure the accuracy of the research. The mass of fresh
plants weighed before it is air dried. The weight of air dried plant samples was
measured after the sample was dried fully. Dried plant samples then were grinded into
powder form using laboratory mill and stored in the air tight container for laboratory
analysis (Mohamad & Wong, 1999; Ebrahimzadeh et al., 2008; Alam et al., 2010; Aja
et al, 2010).
3.2
Solvent Extraction
The crude extraction of the powdered plant samples was based on solvent polarity.
The powdered plant samples were extracted with less polar solvent first before it was
extracted with increasing solvent polarity. Firstly, the grounded plant samples soaked
in dichloromethane (DCM) at room temperature for two days before it was filtered to
collect the extract filtrate. The process then repeated until almost clear filtrate was
obtained. The soaked plant samples filtered using Buchner vacuum filter set with
Whatmann filter paper no 2.
The solvent in the extract was evaporated using rotary evaporator at 40 C under
reduced pressure (Alam et al., 2010). The left over solvent then removed by leaving
the crude extract in the laminar fume hood for nearly a week. Secondly, the DCM
residue soaked in ethyl acetate (EtOAc) for six times with the same procedure again.
Finally, the EtOAc residue then extracted with methanol (MeOH) for six times again
by the same procedure as DCM extraction.
The crude extract obtained then used for pure chemical compound isolation,
phytochemical screenings and biological activity assay. The summary of the
extraction procedure and chemical compound isolation scheme is shown in the
schematic diagram 3.1.
5kg of whole fresh Peperomia pellucida handpicked
Plant samples were air dried
The air dried samples were grounded into powder form
Samples were soaked in DCM
DCM extract
DCM Residue
EtOAc Residue
EtOAc Extract
Soaked in MeOH
MeOH Crude Extract
TLC Profiling
Chemical Compound Isolation
Phytochemical Screening
Biological Activity Assay
Scheme 3.1
Schematic Diagram of Overall Extraction Procedure
20
3.2.1 Chemical reagents used for crude cold extraction

Technical grade DCM, technical grade EtOAc, technical grade MeOH.
3.2.2 Apparatus used for crude cold extraction
Conical flask 2 L, filter funnel, Buchner filter vacuum set, Whatmann filter paper
No 2, rotary evaporator, round bottom flask 500 ml, spatula, dropper, vial bottles.
3.3
Phytochemical screenings
Phytochemical screenings is a qualitative method to verify the presence of

phytochemicals such as alkaloid, steroid, saponin and flavonoid in the crude extract.
There are four main phytochemical test conducted to methanol crude extract that were
alkaloid test, triterpenoid/steroid test, saponin test and flavonoid test. The screening
methods were based on standard procedures outlined in Houghton and Amala (1998).
3.3.1 Alkaloid test
300 mg of methanol crude extract dissolved in 15 ml of ammoniacal chloroform (10%
ammonia). Then, the solution was filtered. The filtrate then was added with 5 to 10
drops of sulphuric acid (H2SO4) 2 M (Ayoola et al., 2008; Hatil & Mohammed, 2010).
The mixture was shaken vigorously and allowed to settle to form two distinctive
layers that are organic and acidic layer (Hatil & Mohammed, 2010). The acidic layer
was pipette out using pipette into test tube. Two drops of Dragendroffs reagent were
added into the test tube containing acidic layer (Houghton and Amala, 1998; Surya &
John, 2001; Himal et al., 2008; Aja, 2010; Abulude et al., 2010; Hatil & Mohammed,
2010). Finally, the changes were observed and compared with the Table 3.1 that
shows the stages of turbidity used to categorize alkaloid. Scheme 3.2 shows the
schematic diagram of alkaloid test (Houghton and Amala, 1998).
300 mg MeOH crude extract was dissolved in 15 ml ammoniacal chloroform

The solution was filtered
The filtrate were added with 5-10 drops of 2 M H2SO4
The mixture was then shaken well and allowed to settle to form two layers
The acidic layer pipette out into test tube
Two drops of Dragendroffs reagents was added into test tube containing acidic layer
The changes to the solution was observed and compared with Table 3.1
Scheme 3.2
Table 3.1
Schematic Diagram of Alkaloid Test

Stages of turbidity to categorize alkaloid
Observation
Scale
Result
No changes
Negative
Transparent solution with faint trace of precipitate
+1
Positive
Mostly transparent solution with some precipitate
+2
Positive
Translucent solution with definite precipitate
+3
Positive
Immediate precipitate formation
+4
Positive
Large amount of precipitate formed immediately
+5
Positive
Source: Houghton & Amala (1998)

200 mg of methanol crude extract was diluted in 20 ml ethanol and boiled in water
bath until the mixture boils. The mixture was then filtered and the filtrate was
concentrated until it dries in the water bath. The residue after that was diluted with 10
ml of diethyl ether.
The solution again was filtered and the filtrate was dried at room temperature. The
dried sample was then added with one or two drops of H2SO4 and 3-5 drops of acetic
anhydride (Houghton and Amala, 1998; Himal et al., 2008). Finally, the colour
changes of the solution was observed and classified according to Table 3.2 (Houghton
and Amala, 1998).
Table 3.2
Qualitative Scale for Triterpenoid/Steroid Test

Observation
Triterpenoid
Steroid
No Changes
Negative
Negative
Red Colouration
Positive
Negative
Green/ Blue Colouration
Negative
Positive

3.3.3 Saponin Test
The residue that did not dissolve in diethylether was used in saponin test. The residue
was dissolved in distilled water (Hatil & Mohammed, 2010). The solution was then
transferred into test tube and distilled water was added until of the test tube (Hatil &
Mohammed, 2010). The test tube was shaken vigorously and the time taken to form
emulsion was recorded and tabled according to Table 3.3 (Houghton and Amala,
1998). Scheme 3.3 shows overall triterpenoid, steroid and saponin test schematic
diagram (Houghton and Amala, 1998).
Table 3.3
Scale for Classification of Saponin in Emulsion Test

Observation
Scale
Result
Emulsion remains less than 30 minutes
Negative
Emulsion remains between 30 minutes and 1 hour
+1
Positive
Emulsion remains 1 hour
+2
Positive
Emulsion remains 2 hours
+3
Positive
Emulsion remains 3 hours
+4
Positive
200 mg MeOH crude extract + 20 ml EtOH

Shaken vigorously
Filtered
Filtrate
Heated until dryness
Added with 10ml DEE
Filtered
Residue
Filtrate
Added with 3-5 drops
of acetic anhydride
Added with 1/2 drops
of H2SO4
Residue
observed
for bubble
(Saponin Test)
Observed for colour changes in the solution

Red Colouration
(Positive Triterpenoid)
Scheme 3.3
Green/Blue Colouration
(Positive Steroid)
Schematic Diagram of Triterpenoid, Steroid and Saponin Test

200 mg of methanol crude extract dissolved in ethanol. The solution pipette into two
different test tubes which was labelled as test tube A and test tube B. Test tube A was
set as control (blank). Test tube B was added with two small pieces of Magnesium
(Mg) tape and 0.5 ml of concentrated hydrochloric acid (HCl) (Houghton and Amala,
1998; Himal et al., 2008; Hatil & Mohammed, 2010). Finally, the colour change was
observed by comparing it with test tube which set as control and classified according
to Table 3.4 (Houghton and Amala, 1998). Scheme 3.4 shows the schematic diagram
of flavonoid test (Houghton and Amala, 1998).
Table 3.4
Classification of Flavonoid
Colouration
Component
No changes
Negative
Orange to Red
Flavones
Red to Cream
Flavanol
Cream to Magenta
Flavanon
Green to Blue
Flavanon
200 mg MeOH crude extract + EtOH
Solution filtered
Test Tube A
(control)
Test Tube B
Added with Mg tape
Added with 0.5ml of concentrated HCl
Colour changes observed
Scheme 3.4
Schematic Diagram of Flavonoid Test
3.3.5 Chemical reagents used in the phytochemical screenings

Analytical grade acetic acid (Merck, Germany), acetic anhydride (Merck, Germany),
ammonia (Merck, Germany), bismuth subnitrate, chloroform (Merck, Germany),
diethyl ether (Merck, Germany), distilled water, ethanol (Merck, Germany),
hydrochloric acid (Merck, Germany), iodine (Merck, Germany), magnesium tape,
mercuric chloride, potassium iodide, and sulphuric acid (Merck, Germany).
3.3.6 Apparatus used in the phytochemical screening
Beaker 50 ml, pipette, dropper, test tubes, vials, glass rod, filter funnel, Whatmann
filter paper No 1.
3.3.7 Phytochemical Screening Reagent

3.3.7.1 Dragendroffs Reagent
Based on Houghton and Amala (1998), stock solution and working solution were
prepared. For stock solution, bismuth subnitrate (oxynitrate; 1.7 g) was mixed with 80
ml of distilled water and 20 ml of glacial acetic acid. Then, potassium iodide solution
(50% w/v, 100 ml) was added. After that, the stock solution was shaken until
dissolved and kept in dark bottle.
For working solution, 100 ml stock solution was mixed with 200 ml of glacial acetic
acid and distilled water was added until the final volume of the solution is 1 L. Finally,
the Dragendroffs reagent was stored in dark glass bottle after it is prepared.
3.4
Chemical Compound Isolation Techniques
Chemical compound isolation was done to separate impurities and chemical

compounds to collect pure compounds from methanol crude extract. Chromatographic
techniques involved the distribution of compounds in methanol crude extract between
two phases in the chromatographic techniques which are mobile phase and stationary
phase. Separation of compounds depends on the affinity towards the phases and based
on polarity of the compounds.
3.4.1 Analytical Thin Layer Chromatography (TLC)
The TLC sheet used was plastic TLC sheet pre-coated with Kieselgel F254 and
purchased from Merck, Germany. Firstly, the TLC sheet was measured and labelled
according to the Figure 3.1. Then, TLC sheet was spotted small single spot with
chosen dry sample fraction which was diluted using suitable solvent. After that, the
TLC sheet was placed in the developing chamber which filled with the suitable
solvent system (Houghton & Amala, 1998; De et al, 2010).
The amount of the solvent system should not be more than the spot on the sheet. The
TLC sheet placement allows the suitable solvent system to move up the sheet. The
solvent system moved up based on capillary action and the compounds in the sample
fraction were dissolved in the solvent and moved together with the solvent. The
movement of the compounds based on solubility and diffraction differences
(Houghton & Amala, 1998).
TLC was taken out after the solvent system reached the solvent front. It was air dried
to allow the solvent to dry out and then the sheet was observed under UV short wave
(254 nm) and UV long wave (336 nm). UV short wave was used to identify
compounds containing conjugated double bonds and some fluorescent compounds that
usually have extended electron system. UV long wave was used to identify some
fluorescent compounds. After that, the sheet was placed in closed iodine chamber to
leave the sheet get contact with iodine vapour and was removed after a while. This
was done to identify many types of compounds particularly if there is double bonds
present in the compounds (Houghton & Amala, 1998).
Finally, the TLC sheet was sprayed with anisaldehyde (AS) reagent. AS reagent was
sprayed to observe terpenoid compounds that will give purple, blue or red spot. It was
also sprayed to observe some other compounds like lignans, sugars and flavonoids.
AS reagent were prepared by mixing 0.5 % of AS in sulphuric acid, glacial acetic acid
and methanol in the ratio 5: 10: 85 accordingly (Houghton & Amala, 1998).
Solvent Front
5 cm
Single small spot

1
cm
Figure 3.1: TLC sheet
3.4.2 Dry Column Vacuum Chromatography (DCVC)

3.4.2.1 Column Packing
The column packing was started with packing 6-7 cm loose silica gel Kieselgel PF254
on sintered glass filter which has porosity 3 and 10 cm in diameter. The glass funnel
was then fixed on Buchner flask. The loose silica gel was levelled and pack compactly
by fixing vacuum. A filter paper was placed on top of the packed silica gel to prevent
it from outside surrounding. Voids and channels in the packed silica were checked by
eluting hexane (Houghton & Amala, 1998).
3.4.2.2 Sample Crude Extract Application
First of all, the crude extract was mixed with silica gel Kieselgel 60 (0.063- 0.200
mm) (Merck, Germany). Crude was dissolved in suitable solvent, added with silica gel
and run under reduced pressure using rotary evaporated to remove out all the solvent
and to obtained dry n powdery crude sample. The dried crude sample was then
transferred into mortar to make the sample into fine powder and finally added on top
of packed column. After that, the sample was covered with filter paper and pressed
using flat apparatus while making it compact with vacuum (Houghton & Amala,
1998).
3.4.2.3 Sample Crude Extract Elution
The prepared dry column which was fixed with vacuum poured with suitable solvent
system by gradient elution. The solvent system was poured based on increasing
solvent polarity. Solvent fractions were collected after a particular solvent system
eluted out the column. The elution continued to take place until all the solvent fraction
was poured and eluted out. The collected fractions then transferred into rotary
evaporator to remove all the solvent. The fractions obtained are slurry and it was
transferred into vial bottles and placed in laminar flow hood for few days for the
remaining solvents to dry out. The dried fractions were then used for TLC analysis to
combine all the similar fractions (Houghton & Amala, 1998).
3.4.3 Gravity Column Chromatography (CC)

CC was done to separate compounds in the fractions. The separation took place in the
column packed with silica gel Kieselgel 60 (0.040-0.063 mm) (Merck, Germany). The
silica gel functioned as stationary phase. Firstly, cotton was placed at the bottom of
the column. Then, the column was packed with silica gel which diluted with suitable
solvent. The suitable solvent acted as mobile phase. The column was tapped slowly to
make the silica gel to be compact. Initial mobile phase were poured into the column to
check for voids and channels (Houghton & Amala, 1998).
3.4.3.2 Fraction Elution and Fraction Collection
Mobile phase were poured into the column based on increasing solvent polarity and
suitable solvent system was determined via best separation shown by TLC analysis
done on the chosen sample fraction. Fractions collected in vial bottles and left to dry
in laminar flow hood for few days and TLC analysis done on the dried fractions. The
compounds that have similar Rf value combined and a particular fraction was chosen
for further compound isolation. Rf was calculated based on the formula below
(Houghton & Amala, 1998).
Rf =
Distance solute moved

Distance solvent front moved
3.4.4 Preparative Thin Layer Chromatography (PTLC)

It was the easiest method in isolating pure chemical compounds. The restriction for
this method was that the maximum sample load that can be loaded was only less than
40 mg (Houghton & Amala, 1998). So the weight of the obtained pure compounds
was very small.

Firstly, the 60 g of silica gel Kieselgel 60 PF254 for preparative layer chromatography
containing gypsum was dissolved in 120 ml of cold distilled water and it is was
shaken hard for approximately 10 minutes. The suspension was then poured on top of
the plate with the measurement of 0.1 cm x 20 cm x 20 cm. After that, the plate was
0
left to dry in the air and in the oven at 105 C for a day (Houghton & Amala, 1998; De
et al, 2010).
3.4.4.2 Sample Fraction Application
The sample fraction was dissolved in suitable solvent and the sample loaded on the
baseline of the dried preparative plate for 13 cm long starting 2 cm from bottom and
3.5 cm from the left edge (Houghton & Amala, 1998). Then, the plate was developed
in closed tank containing chloroform acetone 9.5:0.5 solvent system until 16 cm from
the baseline.
3.4.4.3 Sample Fraction Detection and Elution
After the plate was fully developed, it was left to dry and after that, it was observed
under UV light (long wave 365 nm and shortwave 254 nm) to detect and mark the
eluted band of the loaded sample. The marked band was then scraped off carefully
from the plate onto Whatmann filter paper No 1. The scraped silica sample was
filtered with acetone into pre weighed vial. The filtrate was then allowed evaporating,
the weight of the vial was measured after that and finally the weight of pure
compound was calculated (Houghton & Amala, 1998). The summary of the chemical
compound isolation scheme is shown in the schematic diagram 3.5, 3.6 and 3.7.
PPM (33.75 g of MeOH crude extract)

DCVC silica gel Kieselgel 60 PF254 for
preparative layer chromatography
Chloroform, Acetone, Methanol gradient
M-1
0.22 g
M-2
1.84 g
PPC-5
10.1 mg
Scheme 3.5
PPM-4
4.5 mg
M-3
2.12 g
PPM-1
3.4 mg
M-4
1.25 g
M-5
7.85 g
PPM-2
2.29 mg
PPM-3
4.8 mg
M-6
13.72 g
Schematic Diagram of Overall Chemical Compounds Isolation from

Peperomia pellucida MeOH crude extract
PPM
M-2 (1.84 g)
CC, silica gel Kieselgel 60 (0.0400.063 mm) 25 cm x 1 cm CHCl3,
CH3COCH3, MeOH gradient
M-2-1 until M-2-3
M-2-4 (23.4 mg) M-2-6 (10 mg) M-2-5, M-2-8 until M-2-11
PTLC, silica gel Kieselgel 60 PF254

for preparative layer chromatography
0.1 cm x 20 cm x 20 cm
CHCl3: CH3COCH3 8:2

0.1 cm x 20 cm x 20 cm
CHCl3: CH3COCH3 8:2
PPC-5
10.1 mg
PPM-4
4.5 mg
Scheme 3.6 Chemical Compounds isolation scheme of fraction M-2
PPM
M-3 (2.12 g)
CC, silica gel Kieselgel 60
(0.040-0.063 mm) 3 cm x 20 cm
PE, CH3COCH3, MeOH gradient
M-3-1 until M-3-4
M-3-5 (8 mg) M-3-7 (10 mg)

0.1 cm x 20 cm x 20 cm
PE: CH3COCH3 8:2
M-3-6, M-3-8 until M-3-15

0.1 cm x 20 cm x 20 cm
CHCl3: CH3COCH3 9.5:0.5
PPM-1
10.1 mg
PPM-2
2.29 mg
PPM-3
4.8 mg
Scheme 3.7 Chemical compounds isolation scheme of fraction M-3

3.5
Characterization of Chemical Compounds by Spectrometer Techniques
3.5.1 Fourier Transform Infrared Spectrometer (FT-IR)

Infrared spectra of isolated compounds were obtained from Perkin Elmer 100 Fourier
Transform Infrared Spectroscopy (Raphael, 2006). The isolated compounds were in
the solid form, so it was mixed with KBr powder in the ratio of 1: 7 to form KBr
pallet. The pallet was then air dried and used for analysis. The analysis was about
measurement of absorption band through the vibration of the compound under
-1
-1
infrared. The wavelength range is from 4000 cm to 400 cm .
3.5.2 Ultra Violet Spectrometer (UV)

It was used to record maximum and minimum absorption and also to measure
intensity in terms of log using the equation = A/ c x l where A is absorbance, c is
molar concentration and l is sample cell length. It is also used to identify the
conjugated system of the compound. Chloroform was used to dissolve the compound
and 100 ml of chloroform added to specific concentration. The solution was then
poured into 1 cm sample cell and analysis was done on it to get the UV spectrum.
3.6
Characterization of Chemical Compounds using Spectroscopy Techniques
3.6.1 Nuclear Magnetic Resonance Spectroscopy (NMR)

The pure compounds were subjected to NMR analysis using NMR spectroscopy
which runs at 400 MHz with deuterated solvent and tetramethysilane (TMS) signal as
internal standard. The NMR spectrometer model that was used is Bruker Ultrashield
400. The spectra that were obtained showed chemical shift in ppm and peak
multiplicities as singlet (s), duplet, (d), triplet, (t) and multiplet (m). NMR analyses
were done to determine the structures of the compounds are 1D and 2D NMR (Karge,
2004).
Types of 1D NMR are H (proton) NMR and
13
C (carbon) NMR. Types of 2D NMR
are COSY (correlated spectroscopy), HMQC (heteronuclear multiple quantum

correlation), HMBC (heteronuclear multiple bond correlation) (Karge, 2004).
3.7
Antibacterial Assay
3.7.1 Chemicals used

Standard of Pennicilin G 10 units (Oxoid, UK), Chloramphenicol 50 g (Oxoid, UK),
and Gentamicin 10 g (Oxoid, UK), paper disc 6 mm, nutrient agar, Mueller Hinton
agar (Difco, USA).
3.7.2 Microorganisms used
Eight microorganisms were used for the purpose of antibacterial evaluation. The
microorganisms were Escherichia coli (E.coli) (gram negative), Klebsiella
pneumonia (gram negative), Vibrio alginolyticus, Vibrio mimicus, Vibrio
parahaemolyticus, Bacillus cereus (gram positive), Micrococcus sp. (gram
positive), and Streptococcus uberis (gram positive) were freshly cultured in the
0
appropriate broths overnight before it were used and incubated at 37 C (Tshikalange

et al., 2005; Krittika et al., 2007; Tan & Charles, 2011; Govindappa et al, 2011).
The bacteria were maintained on nutrient agar (NA) and stored at 4 C. The bacteria
cultures were then dissolved in 9 ml autoclave distilled water using sterile saline. The
concentrations of cultures were adjusted with 0.5 McFarland standards to adjust the
turbidity of bacterial suspensions (Igbinosa et al., 2009). All the bacteria were
obtained from Institute of Marine Biotechnology, Universiti Malaysia Terengganu.
3.7.3 Antibacterial Activity Assay
Antibacterial activity assay was determined using disc diffusion method (Ates &
Erdogrul, 2003; Leland et al., 2006; Krittika et al., 2007; Chen et al., 2008; Joy et al.,
2008; Vallinayagam et al., 2009; Suhanya et al., 2009; Alam et al., 2010; Sangeetha,
2010; Govindappa et al., 2011; Tan et al., 2011).
Samples of methanol crude extract were loaded onto each Whatmann No 1 filter paper
discs (, 6 mm) in series of concentration, i.e. 2000 g/disc, 1000 g/disc,
500
g/disc, 250 g/disc and 125 g/disc (Gislene et al., 2000). The paper discs
were placed in the laminar flow hood to remove stock solution solvent (Mohamad &
Wong,
1999). The 200 L suspensions of the bacteria was swabbed using cotton swap on
Muller Hinton agar media.
The discs were located on the surface of the previous inoculated agar. The plates were
0
inverted and incubated for 24 hours at 37 C (Gislene et al., 2000; Tshikalange et al,
2004; Akinnibosun et al, 2008; Shahid-ud-daula & Mohamad, 2009). Clear inhibition
zones around the disc were measured after the incubation period edge of paper disc
(Ates & Erdogrul, 2003). The positive and negative controls were used for each
bacterium.
3.8
Free Radical Scavenging Assay- Determination of DPPH Scavenging

Activity
It is was measured in terms of radical scavenging ability or H- donating by using the

stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) acceptor with the standard (quercetin)
(Kumaran & Joel, 2006; Kai et al., 2007; Ayoola et al., 2008; Chan et al., 2008; Chen
et al., 2008; Nabavi et al, 2008; Ye et al., 2008; Ebrahimzadeh et al., 2008; Nan et al.,
2009; Govindappa et al., 2011; Tan et al., 2011; Mukram et al., 2012). DPPH acceptor
mechanism is shown in Figure 3.2. Using 96 well plates, 20 l of methanol crude
extract and quercetin (in DMSO) was added. 200 l of DPPH (2.37 g in 100 ml) was
added to the mixture and it was left for 30 minutes for incubation at room temperature.
The absorbance was measured at 517 nm using Elisa UV/Visible light readers
(Multiskan ascent, thermo electron corporation) against as DMSO as blank (control)
where the control contained 20 l DMSO and 200 l DPPH (Kumaran & Joel,
2006; Kai et al., 2007; Ayoola et al., 2008; Chan et al., 2008; Chen et al., 2008;
Nabavi et al,
2008; Ye et al., 2008; Ebrahimzadeh et al., 2008; Suhanya et al., 2009; Mutee et al.,
2010; Lee et al., 2011; Govindappa et al., 2011; Tan et al., 2011; Mukram et al.,
2012).
Free radical scavenging activity determined according to the equation below.
Free Radical Scavenging Activity (%) = (Ac As)/ Ac x 100%
where Ac is control absorbance and As is sample absorbance.
N
O 2N
NO2
NO2
Figure 3.2
RH
O2 N
NH
NO2
NO2
Mechanism of DPPH Acceptor
3.9
Total Phenolic Content
With slight modification on Folin-Ciocalteu method and the method was used for the
determination of total phenolic content in methanol extract of Peperomia pellucida
(Chan et al., 2008; Chen et al., 2008; Nabavi et al, 2008; Ebrahimzadeh et al., 2008;
Suhanya et al., 2009; Mutee et al., 2010; Govindappa et al., 2011; Tan et al., 2011).
Firstly, 20 l of MeOH extract was mixed with 100 l of 2 N Folin-Ciocalteu
reagent. The mixture was then left for incubation at room temperature for 8 minutes.
After the incubation, 300 l of Na2CO3 (20 % w/v) was pipette into the previous
mixture.
1.58 mL of distilled water was also poured into the mixture after that. Then, the
mixture was left for incubation for 2 hours at room temperature. Absorbance at 760
nm was measured for the mixture after that. Besides that, gallic acid at concentration
-1
ranging from 0.0625 0.5 mg/ml was prepared to obtain a calibration curve of gallic
acid (Mutee et al., 2010). Finally, total phenolic content was expressed in terms of
Gallic acid Equivalents (GAE). GAE was calculated using the following formula:
C= (c x V) / m
Where C is total phenolic content
c is the concentration of gallic acid from the calibration curve (mg/mL)
V is volume (1 mL)
-3
m is 10 x 10 kg (weight of dried crude extract)
3.10
Determination of Powder Sample Colour with Different Chemical

Reagents
Air dried Peperomia pellucida plant sample in the powdered form was used in this
determination to study on pharmacognostical of Peperomia pellucida. This
observation contributed to the identification of pharmacognostical and stardardization
of the drug in the crude form. The powdered sample was mixed with different
reagents based on the Table 3.5 to observe the colour changes when it is seen in the
naked eye (Pulak, 2011).
Table 3.5
Powdered Peperomia pellucida Sample with Different Reagents

Sample + Chemical Reagent
Powdered Sample
Powdered Sample + 50% HNO3
Powdered Sample + 1N HCl
Powdered Sample + 50% H2SO4
Powdered Sample + Glacial Acetic Acid
Powdered Sample + Iodine Powdered
Sample + 5% KOH
Powdered Sample + MeOH
Source: Pulak (2011)
CHAPTER 4
RESULTS AND DISCUSSION
The aim of the study was to isolate and characterize pure chemical compounds from
methanol extract of Peperomia pellucida. The methodology used to achieve the
objectives were crude extraction, chemical compounds isolation by various
chromatography techniques, and chemical compound structure identification by
various spectroscopy techniques. The structural identification was done based on the
comparison with previous studies.
4.1
Peperomia pellucida Plant Sample Extraction
Collected Peperomia pellucida plant samples were air dried, grinded into powder and
macerated subsequently with dichloromethane (DCM), ethyl acetate (EtOAc) and
methanol (MeOH) solvent. MeOH crude was used for chemical compound isolation
and it is labelled as PPM. Table 4.1 shows the weight and percentage yield of
Peperomia pellucida plant sample and MeOH crude extract.
Table 4.1
Weight and Percentage Yield of Plant Sample and Crude Extract

Plant Sample
Fresh Sample
Air Dried Sample
Sample Soaked in Solvent
Dried Percentage
Crude Extract
MeOH crude
Weight
33.75 g
Weight
9.119 kg
344.7 g
321.9 g
3.78 %
Percentage Yield
10.48%
Maceration of plant sample was done using three different solvent which were DCM,
EtOAc and MeOH. All these three solvents have different solvent polarities where it
extracted compounds with different polarity from the plant. Since DCM has semi
polar solvent polarity therefore it extracted out semi polar lipid-like compounds and
EtOAc extracted out polar chemical compounds since EtOAc is a polar solvent.
Finally, MeOH is also a polar solvent so it extracted out polar compounds which are
more phenolic.
4.2
TLC Profiling of MeOH Crude Extract
TLC profiling was done on methanol crude extract to identify the presence of
chemical compounds. Chemical compounds have different retention factor, Rf values
when it is was separated in different solvent system based on their polarity.
Appropriate solvent system was selected to separate the pure chemical compounds
using column chromatography. Selection of suitable solvent system was done by
analyzing the Rf values of chemical compounds in different solvent systems.
TLC profiling was done on crude extract with solvent system of chloroform/acetone
3:7 and visualised by normal visualisation, UV short wave (254 nm), UV long wave
(365 nm), iodine vapors and anisaldehyde (AS) reagent. Plate 4.1 shows TLC
profiling of MeOH crude extract with TLC.
40
Plate 4.1
4.3
TLC profiling of MeOH crude extract
Phytochemical Screening
Alkaloid, triterpenoid, steroid, flavonoid and saponin test were done on methanol
crude extract. The screening methods were based on standard procedures outlined in
Houghton and Amala (1998).
4.3.1 Alkaloid test
Dragendroffs reagent was used to test the availability of alkaloid in methanol crude
and it showed positive result since precipitate formed immediately. Table 4.2 shows
the alkaloid test result for the crude extract.
The presence of alkaloid was determined based on colour changes observed when
became turbid and
acidic solution was added with Dragendroffs reagent. Acidic
yellow precipitate formed when few drops of reagent was added into the acidic
solution.
According to research done on Peperomia pellucida stem extract by Pulak, (2011),

methanol stem extract was found to have alkaloid. Research done by Ganiyat et al.,
(2011) on Peperomia pellucida leaf extract showed that methanol leaf extract contains
alkaloid. In both of the previous studies, the level of turbidity was not stated.
Currently, there is no research done regarding alkaloid testing on methanol crude
extract with same extraction method.
Methanol crude extract was subjected to triterpenoid/ steroid test and the results are
tabulated in Table 4.2. The observation was done after addition of few drops of acetic
anhydride then followed by addition of few drops of concentrated sulphuric acid. The
observation obtained was no colour change of light brown solution.
This makes the result to show that there is no terpenoid or steroid in methanol crude
extract. This might be due to high quantity of pigment present in the methanol crude
extract which makes it is hard to determine the colour change of the solution.

methanol stem extract was found to have triterpenoids and steroids. Research done by
Ganiyat et al., (2011) on Peperomia pellucida leaf extract showed that methanol leaf
extract contains steroids. Currently, there is no research done regarding triterpenoid
and steroid testing on methanol crude extract with same extraction method.
4.3.3 Saponin Test

Saponin test on methanol crude extract showed positive result of saponin compound.
This conclusion was made after the emulsion remained more than 3 hours. Table 4.2
shows test result of saponin for methanol crude extract. According to research done on
Peperomia pellucida stem extract by Pulak, (2011), methanol stem extract was found
do not contain saponins. Based on the research done by Ganiyat et al., (2011) on
Peperomia pellucida leaf extract, it was found that methanol leaf extract do not
contain saponins. In both of the previous studies, the emulsion level was not stated.
Currently, there is no research done regarding saponin testing on methanol crude
extract with same extraction method.
After subjection of methanol crude extract to flavonoid test, it can be concluded that
methanol crude extract contains flavones compound. This conclusion was done when
change of colour was observed (the solution changed from orange to red) when
magnesium tape was added into concentrated hydrochloric acid. Table 4.2 shows
flavonoid test result of methanol crude extract.

methanol stem extract was found to contain flavonoids. Based on research done by
Ganiyat et al., (2011) on Peperomia pellucida leaf extract, it was found that methanol
leaf extract to contain flavonoids. In both of the previous studies, the component type
of flavonoids was not stated. Currently, there is no research done regarding flavonoids
testing on methanol crude extract with same extraction method.
Table 4.2
Phytochemical
Phytochemical Test Result on Methanol Crude Extract

of Peperomia pellucida
Observation
Conclusion
Screening Test
Alkaloid
Positive alkaloid
+4
(Immediate prepicipitate formation
Triterpenoid/Steroid
Negative triterpenoid
No changes
Negative steroid
(Remains light brown)

Saponin
Positive saponin
+4
(Emulsion remains 3 hours)
Flavonoid
Orange to red
Positive flavonoid
(Flavones)
4.4
Isolation of Chemical Compounds of Peperomia pellucida
Dry column vacuum chromatography (DCVC) packed with preparative TLC silica
(Merck, Kieselgel 60 PF264) was used for first chemical compound isolation and 33.75
g of methanol crude extract was used. The height of the column was 4.5 cm and the
diameter of the column was 10 cm. The solvent system used was chloroform : acetone:
methanol by increasing gradient. This allowed good elution of crude and a total of six
fractions obtained. The recovery percentage was only 80% due to high pigment
contain in the crude extract. Table 4.3 shows the weight of fractions collected prior to
DCVC and Plate 4.2 shows the TLC profiling of the fractions.
Table 4.3
The fraction M-1 u ntil M-6 weight
Fractions Fractions combined

M-1
1
M-2
2
M-3
3-6
M-4
7-9
M-5
10-12
M-6
13-15
Total
Weight (g)
0.22
1.84
2.12
1.25
7.85
13.72
27.00
Plate 4.2
TLC Profiling of M-1 until M-6
4.4.1 Isolation of Chemical Constituent of Fraction M-2

1.84 g of fraction M-2 was subjected to gravity column chromatography packed with
silica gel (Merck, Kieselgel 60 PF254) for further chemical compound separation. The
diameter of the column was 1 cm and the height of the column was 25 cm. The
column was eluted with chloroform: acetone: methanol with increasing gradient. The
the fractions were
number of total collected fractions was 57 fractions and all of
analysed using TLC. 11 combined fractions were obtained after combining the similar
fractions. The combined fractions were labelled as M-2-1 until M-2-11.
The combined fractions were analysed with TLC by visualization with normal, UV
light short wave (264 nm), UV light long wave (365 nm), iodine vapours and
(AS). M-2-4 was chosen to purify with PTLC with solvent
anisaldehyde reagent
system chloroform: acetone 8:2. Yellow liquid PPC-5 was successfully isolated as
pure chemical compound with a weight of 10.1 mg. M-2-6 was chosen to be purified
with PTLC with solvent system chloroform: acetone 8:2. Light yellow PPM-4 (4.5
mg) was successfully isolated. Plate 4.3 shows TLC profiling of M-2-1 until M-2-11
and Plate 4.4 shows isolation and TLC profiling of fractions towards isolation of PPC5 and PPM-4.
Plate 4.3
Plate 4.4
Isolation and TLC profiling of fractions towards isolation

of PPC-5 and PPM-4

Fraction M-3 which weighed 2.12 g was subjected to further isolation with gravity
column chromatography. A column chromatography was packed with silica gel
(Merck, Kieselgel 60 PF254) where the diameter was 3 cm and the height of the
column was 20 cm. Solvent system of petroleum ether gradient with acetone and
methanol was used to elude the compounds. Total of 250 fractions were collected and
further analyzed with TLC. Similar pattern fractions were combined and gave 15
combined fractions. The combined fractions were labelled as M-3-1 until M-3-15.
Fraction M-3-5 was chosen to be purified with PTLC with solvent system petroleum
ether: acetone 8:2. A dark blue band found at PTLC under the examination of UV
light long wave (365 nm). The band was scraped and the silica was filtered. A pure
compound PPM-1 (3.4 mg) was obtained. Fraction M-3-7 was chosen to be purified
with PTLC with solvent system chloroform: acetone 9.5:0.5. A dark blue band was
observed at PTLC under examination of long wave UV light (365 nm). The band then
was scraped off and subjected to filtration to obtain a pure compound, PPM-2 (2.29
mg). A dark green band was observed at PTLC under the examination of short wave
UV light (254 nm). The band was scraped and the silica was filtered. A pure
compound PPM-3 (4.8 mg) was obtained.
Fraction M-3-10 was chosen for further purification with gravity column
chromatography and total 135 fractions were collected. Similar pattern fractions were
combined into 23 fractions labelled as M-3-10-1 until M-3-10-23. The separation was
not efficient and further purification was not successful. Plate 4.5 shows TLC
profiling of M-3-1 until M-3-15 and Plate 4.6 shows isolation and TLC profiling of
fractions towards PPM-1, PPM-2 and PPM-3.
Plate 4.5
Plate 4.6
TLC profiling of M-3-1 until M-3-15
Isolation and TLC profiling of fractions towards

PPM-1, PPM-2 and PPM-3

Fraction M-4 (1.25 g) was subjected to gravity column chromatography isolation
where the diameter was 2.5 cm and the height was 22 cm. The column was packed
with chloroform with acetone and followed by methanol with increasing gradient. 57
fractions were collected and combined into 11 fractions (M-4-1 until M-4-11).
Fraction M-4-6 was subjected to further purification with PTLC (chloroform acetone
8:2) and purification was not successful.
4.5
Characterization of Chemical Compounds
The compounds were characterized with various instruments to look for the actual
structure. The instruments used were IR, UV and NMR spectroscopy.
4.5.1 PPM-1
PPM-1 (3.4 mg) which is odourless was isolated as a colourless solid. Its molecular
structure was characterized using spectral data and comparison with literature values.
The IR spectrum (Figure 4.1) showed a broad band at 3367.07 cm
-1
indicating the
presence of OH (hydroxyl) group in this compound. C-H sp in the structure is

3
-1
clearly shown by the presence of strong C-H stretching (sp ) at 2923.81 cm and
-1
2853.64 cm . A small band at 1746.85 cm
-1
and 1716.00 cm
-1
indicated the
probability of presence of C=O (carbonyl) group. Umbrella bending of C-H showed at

-1
-1
band 1455.75 cm . Furthermore, the absorption band at 1020.16 cm was assigned to

-1
C-O stretching. Finally, a band at 798.52 cm might be attributed by the presence of

C-H out of plane ring bending. The IR characteristics bands are shown in Table 4.4.
Table 4.4
Infra-red characteristics bands for compound PPM-1

-1
Wavenumber (cm )
Assignments
3367.07
O-H stretching
2923.81 and 2853.64
-C-H sp stretching
1746.85 and 1716.00
C=O stretching
1455.75
C-H umbrella bending
1020.16
C-O stretching
798.52
C-H out of plane ring bending
PPM-1 was analyzed by UV-Vis spectroscopy (Figure 4.2) in the wavelength ranging
from 200 nm to 500 nm. Four absorbance peaks were obtained from the spectrum,
where the maximum wavelengths (max) were 329.50 nm, 308.50 nm, 242.50 nm and
221.00 nm (Table 4.5). The solvent cut off point of CHCl3 solvent was max 242.50
nm. max 329.50 nm and 308.50 nm indicated the to * transitions in this compound.
Table 4.5 UV-Vis absorbance data analysis of PPM-1
Number of peak
Wavelength (nm)
Absorbance
329.50
0.1449
308.50
0.1838
The H NMR (Figure 4.3) data of PPM-1 disclosed a triplet (J = 6.8 MHz) at 0.907.
It is also afforded a downfield multiplet at 1.280, a proton singlet at 1.581.
13
The C NMR (Figure 4.4) spectrum of PPM-1 exhibited signals 5 carbon atoms. One
of which was assigned to a methyl group (14.21 ppm), two signals to methylene group
(22.70 ppm and 29.37 ppm), one signal to a long chain methylene group (29.71ppm)
and another one signal to quaternary carbon group (31.94 ppm). At 77.011 ppm,
there is a triplet peak observed which is CHCl3 solvent.
According to DEPT (Figure 4.5) spectra, all this carbon is group into 3 different
proton substituted carbon, the spectra showed one methyl (CH3) group, three
methylene (CH2) groups and one quaternary carbon in this compound.
The triplet signal at 0.907 in the H NMR spectrum which corresponded to the
13
NMR signal at 14.12 ppm showed HMBC (Figure 4.6) correlation to methylene group
(22.70 ppm), a methylene group (29.37 ppm) and quaternary carbon (31.94 ppm).
Further, there were HMBC correlations between 1.29 to methyl group (14.12 ppm),
methylene group (22.70 ppm) and long chain methylene group (29.71 ppm). These
correlations further supported in HMQC. There is no correlations can be seen in
COSY.
All the data were combined to discover the structure of the compound but due to
insufficient data, structure of the isolated compound (PPM-1) was unable to be
determined. The obtained data was insufficient because the quantity of PPM-1 was
very little.
100.0
95
45 2.34
90
46 8.49
85
80
75
39 18.3 5
38 35.1 2
70
65
42 1.88
39 00.8 9
60
55
52
52
%T
50
45
40
37 48.2 0
35 8 8.0 7
79 8.52
35 62.7 5
35 45.5 2
35 24.5 4
35 02.7 033 67.0 7
77 2.68
23 22.7 2
35
30
(C-H) oop
12 60.2 4
(O-H)
17 16.0 0
14 55.7 5
11 57.7 0
25
17 46.8 5
20
10 20.1 6
(C-H )
28 53.6 4
(C=O)
15
10
(C-O) stretching
29 23.8 1
(C-H) sp
5
0.0
4000.0
3600
3200
2800
2400
Figure 4.1
2000
1800
1600
cm-1
1400
1200
Infrared spectrum of compound PPM-1
1000
800
600
400.0
max = 242.50 nm
53
53
max = 308.50 nm
max = 329.50 nm
Figure 4.2
UV-Vis spectrum for compound PPM-1 in CHCl3
(CH2)
54
CH3
54
Figure 4.3
H-NMR spectrum of PPM-1 in Deuterated-chloroform (CDCl3)
55
55
Figure 4.4
13
C-NMR spectrum of PPM-1 in deuterated-chloroform (CDCl3)
CH2
APT
CH2
CH2
DEPT
135
56
CH3
56
13
Figure 4.5
13
APT, DEPT 135 and C NMR spectra of PPM-1 in Deuterated-chloroform (CDCl3)
L. .
PPM1 HMBC
ppm
10
..
,' l
--
:-- 30
' I .
I:-
40
I:-
50
1'-
60
i'-
70
1'-
80
E- 90
100
110
120
130
140
150
8.5 8.0
0.5 0.0
Figure 4.6
7.5
7.0 6.5
6.0
5.5
5.0
4.5
4.0 3.5
3.0 2.5 2.0
1.5
HMBC spectrum of PPM-I in deuterated-chloroform (CDCh)
1.0
ppm
PPM1 HMQC
ppm
f- 10
1- 20
..
1r
=---
Vl
".k:.
r 30
_,.
f- 40
00
1- 50
r 60
1- 70
'
r 80
I
r 90
6.5
6.0
2.0
5.5
5.0
4.5
4.0
3.5
3.0
2. 5
1.0
0.5
0. 0
-0.5
1.5
Figure 4.7
HMQC spectrum ofPPM-1 in deuterated-chloroform (CDCh)
-1 .0
ppm
PPMl.
COSY
ppm
-1
o- c:::;, @
0'
ail
8
9.0
8.5
8.0
Figure 4.8
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
COSY spectrum ofPPM-1 in deuterated-chloroform (CDCb)
1.0
0.5
0.0
ppm
4.5.2 PPC-5
PPC-5 (10.1 mg) was isolated as yellow liquid. Its molecular structure was
characterized using spectral data and comparison with literature values.
-1
The IR spectrum (Figure 4.10) showed a broad band at 3455.24 cm indicating the
3
presence of OH (hydroxyl) group in this compound. C-H sp in the compound is

3
-1
clearly shown by the presence of strong C-H stretching (sp ) at 2957.67 cm . A strong
-1
band at 1740.12 cm indicated the probability of presence of C=O (carbonyl) group.

-1
-1
Umbrella bending of C-H showed at bands 1436.11 cm , 1373.17 cm , 978.85 cm

-1
and 946.24 cm . Finally, the absorption band at 1047.80 cm
-1
-1
was assigned to C-N
group. It is probably from a tertiary amine (R3N) since there is no N-H stretching
bands are observed in the spectrum. The IR characteristics bands are shown in Table
4.6.
Table 4.6
Infra-red characteristics bands for compound PPC-5

-1
Wavenumber (cm )
Assignments
3455.24
O-H stretching
2957.67
-C-H sp stretching
1740.12
C=O stretching
1436.11, 1373.17, 978.85, 946.24
C-H umbrella bending
1047.80
C-N group
PPC-5 was analyzed by UV-Vis spectroscopy (Figure 4.11) in the wavelength ranging
from 200 nm to 500 nm. Six absorbance peaks were obtained from the spectrum,
where the maximum length (max) were 435.50 nm, 316.00 nm, 302.00 nm, 245.00
nm, 233.50 nm and 216.00 nm respectively (Table 4.6). The solvent cut off point of
the CHCl3 solvent was max 245.00 nm. max 435.50 nm, 316.00 nm, 302.00 nm
indicated the to * transitions.
60
Table 4.7 UV-Vis absorbance data analysis of PPC-5

Number of peak
Wavelength (nm)
Absorbance
435.50
0.1616
316.00
0.2463
302.00
0.2675
The H NMR (Figure 4.12, 4.13 and Table 4.8) data of PPC-5 disclosed a methyl
triplet (J= 7.2 MHz) at 1.281, two proton siglet at 2.067 and 3.512. It also
13
afforded a methylene quartet (J= 7.2 MHz) at 4.143. The C NMR (Figure 4.14 and
Table 4.8) spectrum of PPC-5 exhibited signals for 6 carbon atoms. Two of which
were assigned to methyl (CH3) groups (14.20 ppm and 21.05 ppm) where the signal at
21.05 ppm most likely belongs to CH3 attached adjacent to the carbonyl (C=O) carbon
at 171.17 ppm. HMBC and HMQC spectra further supported this since there is
correlation between protons ( 4.143) attached to this carbon with carbonyl (171.17
ppm). One signal to a RCH2-N bonded carbon at 50.89 ppm, one to an oxygenated
carbon (60.41 ppm) and. At c 77.02 ppm, there is a triplet peak observed which
indicated CHCl3 solvent. Apart from that, there is a strong singlet at 77.22 ppm which
mostly represented RCH-O bonded carbon.
A methyl signal at 1.281 in the H NMR spectrum which corresponded to the
13
NMR signals at 14.20 ppm and 21.05 ppm showed HMBC (Figure 4.15 and Table
4.8) correlation to the C-3 (50.89 ppm) and confirmed the attachment position for the
1
methyl group. Further, another methyl group at 2.067 in the H NMR which
13
corresponded to the C NMR signal at 21.05 ppm showed HMBC correlations to the
C-3 (50.89 ppm) and C-5 (171.17 ppm) respectively and confirmed the attachment
position for the methyl group.
In addition, HMBC correlation H-4 and C-1 which corresponded to C-3 and C-5,
permitted assignment of the methylene group with the C-1 methyl group. The carbon
resonance associated with this group is found at 60.41 ppm in the HMQC spectrum
(Figure 4.16). The methyl group is coupled to an allyl group at 4.12 as evidenced by
the strong cross peak in the COSY (Figure 4.17) spectrum.
All the data were combined and the predictable structure of the compound was only
partially determined. It was due to insufficient data to predict the complete structure of
the isolated compound, PPC-5. The obtained data was insufficient because the
quantity of PPC-5 was very little. However, few fragments of the compound were
1
13
suggested based on the spectra obtained using H NMR,
C NMR, HMBC, HMQC
and COSY. Figure 4.9 shows the suggested fragments for PPC-5.
Table 4.8
13
1D-NMR ( H and C NMR) and 2D-NMR assignments for PPC-5

PPC-5
C#
1H mult.,
13
HMBC
COSY
#H
1
14.20 (CH3)
1.26 t, 3H
21.05 (CH3)
2.07 s, 3H
50.89 (RCH2-N)
3.51 s, 2H
60.41 (CH2-O)
4.12 q, 2H
77.22 (RCH-O)
171.17 (C=O)
NR2
4
H H
COSY
Figure 4.9
H-1, H-2
H4, H1
H-2, H-4
1
H3C
H-4
2
CH3
HMBC
Suggested fragments from PPC-5
100.0
95
90
45 5.61
85
80
60 6.41
97 8.85 94 6.24
75
70
29 57.6 7
65
14 36.1 1
(C-H) sp
60
(C-H)
umbrella
55
64
63
%T
50
34 55.2 4
45
40
(O-H)
NR2
H
3
C
35
30
13 73.1 7
20
15
10
1
H3C
4 O
C
HH
10 47.8 0
(C-H)
umbrella
25
CH3
(C=O)
(C-N)
12 34.9 6
17 40.1 2
5
0.0
4000.0
3600
3200
2800
2400
2000
Figure 4.10
1800
1600
cm-1
1400
Infrared spectrum of PPC-5
1200
1000
800
600
400.0
NR2
H
3
C
1
H3 C
4 O
HH
64
64
max = 302.00 nm
max = 316.00 nm
max = 435.50 nm
Figure 4.11
UV-Vis spectrum of PPC-5
C
O
CH3
NR2
H
3
C
1
H3C
4 O
HH
CH3
64
65
Figure 4.12
H NMR spectrum of PPC-5 in deuterated chloroform (CDCl3)
NR2
3
C
1
H3C
3
4
CH3
C
HH
64
66
Figure 4.13
H NMR spectrum of PPC-5 in deuterated chloroform (CDCl3) (Expansion 1)
77.22
NR2
H
3
C
1
H3C
4 O
HH
CH3
64
67
1
3
Figure 4.14
13
C NMR spectrum of PPC-5 in deuterated chloroform (CDCl3)
2
4
C-1/H-4
NR2
H
3
C
3
1
H3C
64
68
C-4/H-4
4
HH
O
C
CH3
C-5/H-4
Figure 4.15
C-4/H-2
C-5/H-3
C-5/H-2
HMBC spectrum of PPC-5 in deuterated chloroform (CDCl3)
C-2/H-2
64
69
C-1/H-1
3
4
C-3/H-3
C-4/H-4
NR2
H
3
C
1
H3C
2
CH3
4
C
O
H H
Figure 4.16
HMQC spectrum of PPC-5 in deuterated chloroform (CDCl3)
43
1
2
64
70
3
4
NR2
H
3
C
1
H3 C
4 O
HH
Figure 4.17
CH3
COSY spectrum of PPC-5 in deuterated chloroform (CDCl3)
4.5
Antimicrobial Test
The antimicrobial activity of methanol crude extract of Peperomia pellucida has been
screened using agar diffusion method. Eight bacteria were used for the test and the
bacteria were Klebsiella pneumonia (gram negative), Escherischia coli (gram
negative), Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio mimicus, Bacillus
cereus (gram positive), Micrococcus sp. (gram positive) and Streptococcus uberis
(gram positive) (Tshikalange et al., 2005; Krittika et al., 2007; Tan & Charles, 2011;
Govindappa et al, 2011). Table 4.9 shows antimicrobial result for MeOH crude extract
of Peperomia pellucida.
The crude extract has showed that it has different level of inhibition to Bacillus
cereus, Micrococcus sp. and Streptococcus uberis. MeOH crude extract of Peperomia
pellucida were inactive against Klebsiella pneumonia (gram negative), Escherischia
coli (gram negative), Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio mimicus.
71
Table 4.9
Antimicrobial result for MeOH crude extract of Peperomia pellucida
Bacteria
100
mg/ml
Inhibition Zone (mm)

50
25
12.5
6.25
mg/mL mg/mL mg/mL mg/mL
DMSO
Escherischia coli
Vibrio
alginolyticus
Vibrio
parahaemolyticus
Vibrio mimicus
Bacillus cereus
Micrococcus sp.
8
7
11
Klebsiella
pneumonia
Streptococcus
uberis
- = no inhibition
According to research by Lee et al., (2011), the growth of Escherischia coli was
inhibited at 3.125 mg/mL, Klebsiella sp. and Vibrio alginolyticus was controlled at
6.25 mg/mL and Vibrio parahaemolyticus was inhibited at 12.5 mg/mL by MeOH leaf
extract of Peperomia pellucida was found. By comparing the results with the present
study done, the previous study by Lee et al., (2011) found to have better antimicrobial
activity. It may be because the test was done on unfractioned methanol leaf extract of
Peperomia pellucida. Currently, there is no research done regarding antimicrobial
activity on MeOH crude extract with same extraction method.
4.6
Free Radical Scavenging Assay-Antioxidant Activity
Methanol extract of Peperomia pellucida for antioxidant activity was using

diphenylphenylhydrazine (DPPH) free scavenging method (Ebrahimzadeh et al.,
2008; Nan et al., 2009; Govindappa et al., 2011). The concentration of quercetin was 1
mg/ml and the concentration of the extract samples was 5 mg/ml. The absorbance was
and methanol extract have IC50 is 6.41 +/- 1.34 mg/ml which
measured at 517 nm
means that the antioxidant activity over 50%. IC50 of methanol extract was determined
based on three readings and average was taken into account. Quercetin has IC 50 value
which is 1.67 +/- 0.06. Figure 4.18 shows free radical scavenging activity by MeOH
extract of Peperomia pellucida.
According to research done on Peperomia pellucida leaf extract by Ganiyat et al.,

(2011), methanol leaf extract of Peperomia pellucida was found to have moderate
antioxidant activity where inhibition percentage was from 51 % - 99 % at 1.0 0.0625
mg/mL. By comparing the results, the previous study by Ganiyat et al., (2011) found
to have better antioxidant activity. It is may be because methanol leaf extract was
plant sample were
used, different preservation method of plant sample and the
collected at different place. Currently, there is no research done regarding antioxidant
activity on methanol crude extract with same extraction method.
Figure 4.18
Free radical scavenging activity by MeOH extract of

Peperomia pellucida
4.7
Total Phenolic Content
Polyphenols were determined spectrophotometrically using Folin-Ciocalteu reagent

based on a colorimetric oxidation or reduction reaction (Ebrahimzadeh et al., 2008;
Suhanya et al., 2009; Mutee et al., 2010; Govindappa et al., 2011). Gallic Acid was
MeOH extract of
used as standard in this determination. Total phenolic content in
Peperomia pellucida is represented by GAE value. GAE value for MeOH extract of
was 3.5 mg/g sample. Figure 4.19 shows gallic acid standard
Peperomia pellucida
curve graph.
Figure 4.19
Gallic acid standard curve graph
4.8
Determination of Powder Sample Colour with Different Chemical

Reagents
Different colour produced when powdered air dried Peperomia pellucida plant was
mixed with different chemical reagents. Observation was done under ordinary white
light. Results are tabulated in Table 4.10. This observation contributes to the
identification of pharmacognostical and stardardization of the drug in the crude form
(Pulak, 2011).
Table 4.10
Colour Characteristics of Powdered Peperomia pellucida

with different chemical reagents
Sample
Powdered Plant
Powdered Plant + 50% HNO3
Powdered Plant + 1N HCl
Powdered Plant + 50% H2SO4
Powdered Plant + 10% FeCl3
Powdered Plant + Iodine
Powdered Plant + 5% KOH
Powdered Plant + MeOH
Colour Produced
Pale green
Orange
Light brown
Yellowish brown
Brownish green
Dark reddish brown
Brown
Brown
CHAPTER 5
CONCLUSION
In this section summary of the research been concluded. Besides, recommendations

regarding the studies were suggested as well to assist the research in the future.
5.1
Conclusion
Extraction was done based on solvent polarity extraction where three different
solvents were used. The solvents used for extraction were DCM, EtOAc and MeOH.
MeOH extract yield was only 10.48%.
Isolation and purification of chemical compounds were done using TLC and CC. TLC
profiling on MeOH extract was done to identify the presence of chemical compounds.
Then, DCVC was used for chemical compound isolation and six combined fractions
were obtained. The solvent system used to elude the fractions were CHCl3,
CH3COCH3 and MeOH by increasing gradient. Few potential fractions were selected
for further isolation and purification of pure chemical compounds. Five pure
compounds were isolated from MeOH crude extract of Peperomia pellucida.
Structural identification of isolated pure compounds was done by characterization

using FT-IR, UV-Vis, 1D and 2D NMR techniques. The obtained spectra compared
with previous literature review where PPM-1 and PPC-5 were partially characterized.
Based on phytochemical studies done on MeOH crude extract of Peperomia

pellucida, it showed that MeOH crude extract contains high level of alkaloid, saponin
and also contains flavones compound. There is no terpenoid or steroid presence in
MeOH crude extract.
Antimicrobial assay done MeOH crude extract showed that it has different level of
inhibition to Bacillus cereus, Micrococcus sp. and Streptococcus uberis bacteria. It
was inactive against Klebsiella pneumonia, Escherischia coli, Vibrio alginolyticus,
Vibrio parahaemolyticus and Vibrio mimicus bacteria.
Antioxidant assay showed that MeOH crude extract of Peperomia pellucida have IC50
which is 6.41 1.34 mg/ml compared to standard (quercetin) which has IC50 which is
1.67 0.06 mg/ml. While, MeOH crude extract have GAE value of 3.5 mg/g sample
based on total phenolic content assay done on it. Different colour produced when
powdered air dried Peperomia pellucida plant was mixed with different chemical
reagents and observation was done under ordinary white light.
5.2
Future Recommendations
1. Chlorophylls are removed first before further isolation and purification of
chemical compounds since Peperomia pellucida contains high level of
chlorophylls and presence of chlorophylls disables purification process.
2. Focus on essential oil extraction of Peperomia pellucida and main components
of Peperomia pellucida can be identified using GC/MS.
3. Focus on purification of active compounds based on antioxidant evaluation of
Peperomia pellucida extracts.
4. Bioassay such as antioxidant, antimicrobial and cytotoxicity assay must be
done on isolated pure compounds to determine its biological activity
properties.
77
REFERENCES
Abulude, F. O., Ogunkoya, M. O., & Akinjagunla, Y. S. 2010. Phytochemical
screening of leaves and stem of Cashew tree (Anacardium occidentate).
Electronic Journal of Environmental, Agricultural & Food Chemistry, 9(4), 815819.
Akinnibosun, H. A., Akinnibosun, F. I., & German, B. E. 2008. Antibacterial activity
of aqueous and ethanolic leaf extracts of Peperomia pellucida (L.) H. B. & K.
(Piperaceae) on three-gram negative bacteria isolates. Science World Journal,
3(4), 33-36.
Alam, K., Moizur, R. & Shariful, I. 2008. Antipyretic activity of Peperomia pellucida
leaves in rabbit. Turkey Journal Biology, 32, 37-41.
Alam, K., Moizur, R. & Shariful, I. 2008. Neuropharmacological effects of
Peperomia pellucida leaves in mice. DARU, 16(1), 35-40.
Alam, K., Moizur, R. & Shariful, I. 2010. Isolation and bioactivity of a xanthone
glycoside from Peperomia pellucida. Life Science and Medicine Research, 1, 14.
Arrigoni-Blank, M. d. F., Elena, G. D., Elaine, M. F., Angelo, R. A., Marcio, R. A. &
Murilo, M. 2004. Anti-inflammatory and analgesic activity of Peperomia
pellucida (L.) HBK (Piperaceae). Journal of Ethnopharmacology, 91, 215-218.
Arrigoni-Blank, M. F., Ricardo, L. B. O., Sandra, S. M., Paulo, d. A. S., Angelo, R.
A., Jeane, C. V., Socrates, C. d. H. C. & Arie, F. B. 2002. Seed germination,
phenology and antidermatogenic activity of Peperomia pellucida (L.) H. B. K.
BMC Pharmacology, 2(12), 1-8.
Aja, P. M., Okaka, A. N. C., Onu, P. N., Ibiam, U. & Urako, A. J. 2010
Phytochemical composition of Talinum triangulare (water leaf) leaves. Pakistan
Journal of Nutrition, 9(6), 527-530.
Ates, D. A., & Erdogrud, O. T. 2003. Antimicrobial activities of various medicinal
and commercial plant extracts. Turkey Journal Biology, 27, 157-162.
Aziba, P. I., Adedeji, A., Ekor, M. & Adeyemi, O. 2001. Analgesic activity of
Peperomia pellucida aerial parts in mice. Fitoterapia, 72, 57-58.
Ayoola, G. A., Coker, H. A. B., Adesegun, S. A., Adepoju-Bello, A. A., Obaweya, K.,
Ezennia, E. C. & Atangbayila, T. O. 2008. Phytochemical screening and
antioxidant activities of some selected medicinal plants used for malaria therapy
in South Western Nigeria. Tropical Journal of Pharmaceutical Research, 7(3),
1019-1024.
Bayma, J. d. C., Mara, S. P. A., Adolfo, H. M., Alberto, C. A. & Williams, C. C.
2000. A dimeric ArC2 compound from Peperomia pellucida. Phytochemistry, 55,
779-782.
Chan, E. W. C., Lim, Y. Y., Wong, L. F., Lianto, F. S., Wong, S. K., Lim, K. K., Joe,
C. E. & Lim, T. Y. 2008. Antioxidant and tyrosinase inhibition properties of
leaves and rhizomes of ginger species. Food Chemistry, 109, 477-483.
Cera, G. M. D. P., Ramos, N. R. B. & Siazon, N. I. T. 2011. In vitro evaluation of
potential chemotherapeutic and chemopreventive biocompounds in P.pellucida
(L.) Kunth on HCT 116 cancer cell line. Journal of Pharmacology, 143.
Chen, I.-N., Chang, C.-C., Ng, C.-C., Wang, C.-Y., Shyu, Y.-T. & Chang, T.-L.
2008. Antioxidant and antimicrobial activity of Zingiberaceae plants in Taiwan.
Plant Foods Hum Nutrition, 63, 15-20.
De, S., Dey, Y. N. & Ghosh, A. K. 2010. Phytochemical investigation and
chromatographic evaluation of the different extracts of tuber of Amorphaphallus
paeoniifolius (Araceae). International Journal on Pharmaceutical and
Biomedical Research, 1(5), 150-157.
th
Donald, J. A. 2008. Burgers medicinal chemistry and drug discovery. 6 Edition. 1:

New York: John Wiley and sons, Inc., Publication.
Ebrahimzadeh, M.A., Hosseinimehr, S. J., Hamidinia, A. & Jafari, M. 2008.
Antioxidant and free radical scavenging activity of Feijoa sellowiana fruits peel
and leaves. Pharmacologyonline, 1, 7-14.
Egwuche R. U., Odetola A. A., & Erukainure O. L. 2011. Preliminary investigation
into the chemical properties of Peperomia pellucida L.. Research Journal of
Phytochemistry, 5(1), 48-53.
Ganiyat K. O., Onocha, P. A., & Olaniran, B. B. 2011. Phytochemical, toxicity,
antimicrobial and antioxidant screening of leaf extracts of Peperomia pellicuda
from Nigeria. Advances in Environmental Biology, 5(12), 3700-3709.
Gislene, G. F. N., Juliana, L., Paulo, C. F., Giuliana, L. S. 2000. Antibacterial activity
of plant extracts and phytochemicals on antibiotic-resistant bacteria. Brazilian
Journal of Microbiology, 31, 247-256.
Gordon, M. C., & David, J. N. 2007. Drug Discovery and development from natural
products: the way forward. NAPRECA, 56-59.
79
Govindappa, M., Sadanandda, T. S., Channabasava, R., Jeevita, M. K., Pooja, K. S. &
Vinay, B. R. 2011. Antimicrobial, antioxidant activity and phytochemical
screening of Tecoma stans (L.) Juss ex Kunth. Journal of Phytology, 3(3), 68-76.
Hatil, H. El-K. & Mohammed, Y. El-A. 2010. Antibacterial activity and
phytochemical screening of ethanolic extracts obtained from selected Sudanese
medicinal plants. Current Research Journal of Biological Sciences, 2(2), 143146.
Himal, P. C., Yogol, N. S., Sherchan, J., Anupa, K. C., Mansoor, S. & Thapa, P. 2008.
Phytochemical and antimicrobial evaluations of some medicinal plants of Nepal.
Kathmandu University Journal of Science, Engineering and Technology, 1(5),
49-54.
Houghton, P. J., & Raman, A. 1998. Laboratory handbook fractionation of natural
st
extracts. 1 Edition. Chapman & Hall.
Igbinosa, O. O., Igbinosa, E. O., & Aiyegoro, O. A. 2009. Antimicrobial activity and
phytochemical screening of stem bark extracts from Jatropha curcas (Linn).
African Journal of Pharmacy and Pharmacology, 3(2), 58-62.
Joy, R. B., Donald, L. W., Craig, C. S., Kendra, L. K., Fulcher, R. G., Nancy, J. E.,
David, D. B. & Russell, F. B. 2008. Antimicrobial activity of native and
naturalized plants of Minnesota and Wisconsin. Journal of Medicinal Plants
Research, 2(5), 98-110.
Kai, M., Klaus, H. V., Sebastian, L., Ralf, H., Andreas, R. & Ulf-Peter, H. 2007.
Determination of DPPH radical oxidation caused by methanolic extracts of some
microalgal species by linear regression analysis of spectrophotometric
measurements. Sensors, 7, 2080-2095.
Karge, H. G. 2004. Molecular sieves. Weitkamp, J. Science and Technology.
Springer.
Khan M.R., & Omoloso, A.D. 2002. Antibacterial activity of Hygrophila stricta and
Peperomia pellucida. Fitoterapia, 73, 251-254.
Krittika, N., Natta, L. & Orapin, K. 2007. Antibacterial effect of five Zingiberaceae
essential oils. Molecules, 12, 2047-2060.
Kumaran, A. & Joel, K. R. Antioxidant and free scavenging activity of an aqueous
extract of Coleus aromaticus. Food Chemistry, 97, 109-114.
Lee S. W., Wee, W., Yong, F. S. J., & Syamsumir, D. F. 2011. Characterization of
anticancer, antimicrobial, antioxidant properties and chemical composition of
Peperomia pellucida leaf extract. Acta Medica Iranica, 49(10), 670-674.
Leland, J. C., Ara, K., Peter, B. K., Sara, L. W., James, A. D. & Harry, L. B. 2006.
nd
Natural products from plants. 2 Edition. Boca Raton: CRC Press.
Leosvaldo, S. M. V., Marcelo, J. P. F., Maria, I. S. S., Davyson, L. M., Vicente, P. E.

& Maria, A. C. K. 2006. Unusual chromenes from Peperomia blanda.
Phytochemistry, 67, 492-496.
Lidiane, G. F., Baldoqui, D. C., Kato, M. J., Bolzani, V. da S., Guimares, E. F.,
Cicarelli, R. M. B., & Maysa F. 2008. Trypanocidal tetrahydrofuran lignans from
Peperomia blanda. Phytochemistry, 69, 445-450.
Lixin, Z. 2005. Drug discovery and therapeutic medicine. Arnold, L. D. Natural
Products. Totowa: Humana Press.
Marisi, G. S., Felippe, A. P. V. de, Guimares, E. F., Kato, M. J., Ellena, J., &
Doriguetto, A. C. 2006. 2-Hydroxy-4,6-dimethoxyacetophenone from leaves of
Peperomia glabella. Journal of Brazil Chemistry Society, 17(7), 1205-1210.
Mohamad, Y. & Wong, C. 1999. A short botanical survey of Brunei plants and some
preliminary results of antimicrobial screening. Natural products, 1.
Murkam, M. M., Lim, Y. M., Amran, M., Nashriya, M. Nordin, H. L. & Abdul, M. A.
2012. Non-cytotoxic and antitumour-promoting activities of Garcinia acid esters
from Garcinia atroviridis Griff, ex. T. Anders (Guttiferae). Journal of
Ethnopharmaclogy, 1(1).
Mutee A.F., Salhimi, S. M., Yam, M. F., Lim, C. P., Abdullah, G. Z., O.Z., Ameer, O.
Z., Abdulkarim, M. F. & Asmawi, M. Z. 2010. In vivo anti-inflammatory and in
vitro antioxidant activities of Peperomia pellucida. International Journal of
Pharmacology, 6(5), 686-690.
Nabavi, S. M., Ebrahimzadeh, M. A., Nabavi, S. F., Hamidinia, A. & Bekhradnia A.
R. 2008. Determination of antioxidant, phenol and flavonoid content of Parrotia
persica mey. Pharmacologyonline, 2, 560-567.
Nan, W., Kuang, F., Fu, Y.-J., Zu, Y.-G., Chang, F.-R., Chen, Y.-H., Liu, X.-L., Yu,
K., Wei, L. & Gu, C.-B. 2009. Antioxidant activities of extracts and main
components of Pigeonpea [Cajanus cajan (L.) Millsp.] leaves. Molecules, 14,
1032-1043.
Neung, J. J., Ashik, M., Jeong, Y. M., Ki-Chang, J. Dong-Sun, L., Kwang, S. A. &
Somi, K. C. 2011. Cytotoxic activity of -caryophyllene oxide isolated from jeju
guava (Psidium cattleianum sabine) leaf. Records of Natural Products, 5(3),
242-246.
Nguyen, H. L., Nguyen, H. B., Tae-Geum, K. & Moon-Sik, Y. 2010. Tissue culture
and expression of Escherichia coli heat-labile enterotoxin B subunit in transgenic
Peperomia pellucida. Protein Expression and Purification, 72, 82-86.
Paola, D. L. L., Yoni, F., Catalina, M. v. B., Arnaldo, L. B., Jorge, D. C. & Ana, P. D.
A. 2007. Composition of the essential oil of two peperomia from Peru: P.
Galioides and P. Chalhuapuquiana. Rev. Latinoamer. Quim, 35(1), 7-12.
Pulak, M. 2011. Phytochemical, pharmacognostical and physiochemical

Standardization of Peperomia pellucida (L.) HBK. stem. International Journal
of Comprehensive Pharmacy, 2(8).
Pulak, M. & Arun, K. K. V. 2011. Establishment of quality parameters and
pharmacognostic evaluation of leaves of Peperomia pellucida (L.) HBK.
International Journal of Pharmacy and Pharmaceutical Sciences, 3(5).
Raphael, I. 2006. The origin and the nature of natural products. Selected topics in the
chemistry of natural products. World Scientific Publishing Pte Ltd.
Ritson, P. & Duvita, S. N. 2007. Analisis fitokimia dan uji bioaktivitas daun kaca
(Peperomia pellucida (L.) Kunth). Jurnal Kimia Mulawarman, 5(1), 5-8.
Sangeetha, S. Marimuthu, P., Sarada, D. V. L. & Ramasamy, K. 2010. Isolation of
antimicrobial compound from Sphaeranthus indicus against human pathogens.
International Journal of Biotechnology and Biochemistry, 6(4), 569-577.
Sanjay, P., Nirav, G. Ashok, S. & Anand, S. 2009. In-vitro cytotoxic activity of
Solanum nigrum extract against Hela cell line and Vero cell line. International
Journal of Pharmacy and Pharmaceutical Sciences, 1(1), 38-46.
Satyajit, D. S. 2006. Natural products isolation. Zahid Latif & Alexander, I. G.
nd
Methods in Biotechnology. 2 Edition. Totowa: Humana Press.
Sayeed, A. 2007. Introduction to plant constituents and their tests. Journal of
Pharmacognosy.
Shahid-Ud-Daula, A. F. M. & Mohammad, A. B. 2009. Phytochemical screening,
plant growth inhibition and antimicrobial activity studies of Xylocarpus
granatum. Malaysia Journal Pharmaceutical Sciences, 7(1), 9-21.
Silverstein, R. M., Webster, F. X. & Kiemle, D. J. 2005. Spectrometric identification
of organic compounds. New York: John Wiley & sons, Inc. Publication.
Su, X., Li, N., Ning, M.-M., Zhou, C.-H., Yang, Q.-R., & Wang, M.-W. 2006.
Bioactive compounds from Peperomia pellicuda. American Chemical Society
and America Society of Pharmacognosy.
Suhanya, P., Azizi, J., Ramanathan, S., Ismail, S., Sreenivasan, S., Said, M. I. M., &
Mansor, S. M. 2009. Evaluation of antioxidant and antibacterial activities of
aqueous, methanolic and alkaloid extracts from Mitragyna speciosa (rubiaceae
family) leaves. Molecules, 14, 3964-3974.
Surya, H. & John, B. B. 2001. Initial studies on alkaloids from Lombok medicinal
plants. Molecules, 6. 117-129.
Susie, O. S., Nelia, P. C.-M. & Isidro, C. S. 2000. Acute oral toxicity of freeze-dried
aqueous extract of Peperomia pellucida (L.) HBK (ulasimang bato) in mice. Acta
Medica Phillipina.
Tan, K. L. & Charles, S. V. 2011. Antioxidant, antibacterial and cytotoxic activities of

essential oils and ethanol extracts of selected South East Asian herbs. Journal of
Medicinal Plants Research, 5(21), 5284-5290.
Tshikalange, T. E., meyer, J. J. M. & Hussein, A. A. 2005. Antimicrobial activity,
toxicity and isolation of a bioactive compound from plants used to treat sexually
transmitted diseases. Journal of Ethnopharmacology, 96. 515-519.
APPENDIX A
Free Radical Scavenging Assay-Antioxidant Activity Assay

MeOH Crude extract
Concentration
Exp 1
Inhibition, %
Exp 2
Exp 3
Average
(mg/ml)
Standard
Deviation
0.00000
0.0000
0.0000
0.0000
0.0000
0.0000
0.15625
0.9725
0.9725
0.2363
0.7271
0.4251
0.31250
4.1860
4.1860
1.8061
3.3927
1.3741
0.62500
12.8281
13.0530
12.4967
12.7926
0.2798
1.25000
24.7250
24.5968
24.0449
24.4555
0.3614
2.50000
39.8056
42.1832
43.2021
41.7303
1.7429
5.00000
68.5990
67.9896
69.1337
68.5741
0.5724
79
Quercetin
Inhibition, %
Concentration
Exp 1
Exp 2
Exp 3
Average
(mg/ml)
Standard
Deviation
0.00000
0.0000
0.0000
0.0000
0.0000
0.0000
0.15625
30.0309
28.9150
29.2051
29.3837
0.5790
0.31250
56.7131
47.8680
51.2553
51.9455
4.4627
0.62500
82.5445
75.8505
77.5728
78.6559
3.4759
1.25000
87.0766
86.7901
83.9336
85.9334
1.7378
2.50000
87.1384
87.0281
86.0993
86.7553
0.5708
5.00000
87.2158
87.6777
86.4282
87.1072
0.6318
85
CURRICULUM VITAE
Name
Address
Phone Number
Email
Date of Birth
Place of Birth
Nationality
Race
Religion
Gender
Education
: Nirosa a/p Raman

: PT 25920, Jalan Kasturi 6, Kasturi Heights,
71800 Putra Nilai, Negeri Sembilan
: 0176729916
: nivetha_nishara@yahoo.com.my
th
: 26 December 1989
: Selangor
: Malaysian
: Indian
: Hindu
: Female
: 2009-2012
Bachelor of Science (Chemical Sciences)
Universiti Malaysia Terengganu
2007-2008
Sijil Pelajaran Tinggi Malaysia (STPM)
SMK (L) Bukit Bintang, Petaling Jaya, Selangor
2002-2006
Sijil Pelajaran Malaysia (SPM)
SMK Dato Mohd Said, Nilai, Negeri Sembilan
Awards
: Deans List (Semester I 2011/2012)
Others
: 2012
Secretariat of the Student Representative Council
2011
University Representative in Student Leadership
Convention 2011
2011
University Representative in UM Tamil Parliamentary
Style Debate
2010-2011
Fasilitator of 2010 and 2011 UMT New Students
Orientation Week
2010
Public Relation Exco of the Baratham Cultural Club
2010
Protocol Bureau of Appreciation Dinner Programme
by Baratham Cultural Club
2010
UMT Representative for 2010 MASUM Athletics
Tournament
2010
Committee Member for Annual Grand Meeting of
Sekretariat Rakan Integriti Mahasiswa UMT
2010/2011
2007
Participant of National Service Programme

Herb

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ISOLATION OF CHEMICAL COMPOUNDS

NIROSA A/P RAMAN

FAKULTI SAINS DAN TEKNOLOGI

UNIVERSITI MALAYSIA TERENGGANU

ISOLATION OF CHEMICAL COMPOUNDS FROM

A PITA report submitted in partial fulfillment of

DEPARTMENT OF CHEMICAL SCIENCES

JABATAN SAINS KIMIA

KIM 4998/ 4999

oleh NIROSA A/P RAMAN, No. matrik: UK20028 telah

keperluan memperolehi Ijazah SARJANA MUDA SAINS (SAINS KIMIA), Fakulti

ISOLATION OF CHEMICAL COMPOUND FROM METHANOL EXTRACT

PEMENCILAN SEBATIAN KIMIA DARIPADA EKTRAK METANOL

RESULTS AND DISCUSSIONS

Introduction to Natural Products

deoxyribonucleic acid (DNA).

Translucent green, heart

Dot like seeds attached to

Picture of Peperomia pellucida with descriptions

Significance of the study

The general aim of this study is to utilize Peperomia pellucida as it is an invasive

Currently, there is no detailed scientific paper on its bioactive properties. In Malaysia

Objectives of the study

The objectives of the studies are:

Phytochemical profile from genus Piperaceae is classified by occurrence of alkaloids,

chromenes, several nor/seco-compounds

prenylated hydroquinones (Leosvaldo et al., 2006). Species of Peperomia are well

Traditional Uses of Peperomia pellucida

Biological Activity Studies On Peperomia pellucida

Evalution of anti-inflammatory effect was done using the carrageenan-induced rat

Determination of in vitro antibacterial, antifungal and cytotoxic activities was done on

Previous Chemical Compounds Isolated from Peperomia pellucida

characterized using liqud chromatography/electrospray-mass spectroscopy, NMR ( H,

C, JMOD, COSY, NOESY, HMBC, and HMQC).

OCH3 OCH3 OCH3

CH3 OCH3 OCH3 OH

OCH3 OCH3 OCH3 OCH3

OCH3 OCH3 OCH3 OCH3 OCH3 OCH3

OCH3 OCH3 OCH3 OCH2O

Previous Phytochemical Screening on Peperomia pellucida

Analysis of methanol extract from Peperomia pellucida leaves by using GC/MS

Other than that, phytochemicals such as alkaloids, flavonoids, carbohydrates,

Chemical Variation in Genus Piperceae

2.5.1 Peperomia glabella

2.5.2 Peperomia blanda

3,4,5,3-tetramethoxy-7,7-epoxylignan (26), 4-hydroxy-3,4,5,3,5-pentamethoxy-

7,7-epoxylignan (27), 4,5,4,5-dimethylenedioxy-3,3-dimethoxy-7,7-epoxylignan

Plant Collection and Preparation of Plant Samples

Approximately 5 kg of whole fresh Peperomia pellucida plants were collected at a

Schematic Diagram of Overall Extraction Procedure

3.2.1 Chemical reagents used for crude cold extraction

Phytochemical screenings is a qualitative method to verify the presence of

300 mg MeOH crude extract was dissolved in 15 ml ammoniacal chloroform

Schematic Diagram of Alkaloid Test

Transparent solution with faint trace of precipitate

Mostly transparent solution with some precipitate

Translucent solution with definite precipitate

Immediate precipitate formation

Large amount of precipitate formed immediately

Source: Houghton & Amala (1998)

Qualitative Scale for Triterpenoid/Steroid Test