Part 1

CHEMICAL CONSTITUENTS OF LOCAL HERB:
ANDROGRAPHIS PANICULATA
By
KUNG ZHEN YANG
A project report submitted to the Department of Chemical Science,
Faculty of Science,
UniversitiTunku Abdul Rahman
in partial fulfillment of the requirement for the degree of
Bachelor of Science (Hons) Chemistry
JANUARY 2013

ii
ABSTRACT
CHEMICAL CONSTITUENTS OF LOCAL HERB:
Kung Zhen Yang
2 ent-labdane diterpenoid type compounds were isolated from the aerial part
of Andrographis paniculata which was collected from Pulau Pinang, Malaysia.
The compounds were obtained via methanolic extract of the plant and were
subjected to a series of chromatographic methods, mainly column
chromatography (CC) and preparative centrifugal thin-layer chromatography
(CTLC). The isolated compounds were later characterized using spectral
analysis including 1D and 2D NMR (Nuclear Magnetic Resonance), IR
(Infrared Spectroscopy), MS (Mass Spectrometry), UV-Vis (Ultraviolet-
visible light Spectroscopy), and Polarimetry. Some common physical
experiments, such as melting point experiment was also conducted. The 2
isolated compounds were identified as ent-labdane diterpenoid
andrgorapholide and 14-deoxy-11,12-didehydroandrographolide with
literature data supporting the validity of the compound characterization and
structural elucidation.

iii
ABSTRAK
BARANGAN KIMIA DARIPADA HERBAL TEMPATAN:
Kung Zhen Yang
Andrographis paniculata yang dikumpul dari Pulau Pinang, Malaysia telah
dijalankan proses perasingan dan 2 jenis ent-labdan dwiterpenoid produk
semula jadi telah diasingkan daripada bahagian atas tanah herbal itu. Produk
semula jadi itu diperolehi daripada ekstrak methanol dan ekstrak tersebut telah
dijalankan process perasingan secara bersiri menggunakan cara-cara
kromatografi terutamanya kromatografi turus dan kromatografi sentifugal
lapisan nipis. Produk semula jadi yang telah diperolehi daripada process
perasingan telah dijalankan pencirian and penyelidikan dengan mengguankan
analisis spektrum termasuk 1D and 2D NMR (Nuclear Magnetic Resonance),
IR (Infrared Spectroscopy), MS (Mass Spectrometry), UV-Vis (Ultraviolet-
visible light Spectroscopy), dan Polarimetri. Eksperimen fizikal seperti
eksperimen takat lebur juga dijalankan. Kedua-dua produk semula jadi yang
telah diasing dikenal pastikan sebagai dwiterpenoid jenis ent-labdan
andrografolid dan 14-deoxy-11,12-didihydroandrografolid dengan
sokongan data kesusasteraan.

iv
ACKNOWLEDGEMENTS
I am hereby to express my utmost appreciation to those who had helped and
provided me assistance for the possibility of completing this project. First and
foremost, I would like to express my greatest gratitude to my project
supervisor, Assoc. Prof. Dr Lim Tuck Meng for his exemplary and
unconditional guidance, monitoring, advice, patience and time throughout the
course of the completion of this report. His noble sacrifices and the passion
towards education will always be remembered.
In addition, I would like to express my gratefulness towards Dr. Sim Kooi
Mow and Dr. Lim Chan Kiang for their generous help on the organic
chemistry concepts and the experimental preparation. I would like to also
thank Universiti Tunku Abdul Rahman (UTAR), for the good environment
and facilities, and of course the helpful laboratory officers in UTAR, for their
cooperation and help throughout the project.
I would like to express my special thanks to Eileen Goh Ching Yee for the
great assistance to ease my struggle during the compilation of the report. I
would like to thank Heng Zu Wai for his generosity in providing useful
resources for the project. I would like to also thank my bench mates Tay Vui
Kit, Goh Wee Sheng and Ooi Yan Jie for their kindness, advice and
encouragement throughout the process of the project.

v
I am truly grateful to the residents of Taman Perwira, who had provided me to
the source of sample collection. Finally, an honourable mention goes to my
family and also friends for their understanding, support and constant
encouragement in completing the project.
Any omission in this brief acknowledgement does not mean lack of gratitude.

vi
DECLARATION
I hereby declare that the project report is based on my original work except for
quotations and citations which have been duly acknowledge. I also declare that
it has not been previously or concurrently submitted for any other degree at
UTAR or other institutions.
(KUNG ZHEN YANG)
Date:

vii
APPROVAL SHEET
This project report entitled “CHEMICAL CONSTITUENTS OF LOCAL
HERB: ANDROGRAPHIS PANICULATA” was prepared by KUNG ZHEN
YANG and submitted as partial fulfilment of the requirements for the degree
of Bachelor of Science (Hons) Chemistry at Universiti Tunku Abdul Rahman.
Approved by:
____________________ Date: ____________
(Assoc. Prof. Dr. LIM TUCK MENG)
Supervisor
Department of Chemical Science
Faculty of Science
University of Tunku Abdul Rahman

viii
FACULTY OF SCIENCE
UNIVERSITY OF TUNKU ABDUL RAHMAN
Date: ____________
PERMISSION SHEET
It is hereby certified that KUNG ZHEN YANG (ID No: 11ADB03081) has
completed this final year project entitled “CHEMICAL CONSTITUENTS
OF LOCAL HERB: ANDROGRAPHIS PANICULATA” supervised by
Assoc. Prof. Dr. LIM TUCK MENG from the Department of Chemical
Science, Faculty of Science.
I hereby give permission to my supervisor to write and prepare manuscripts of
these research findings for publishing in any form, if I do not prepare it within
six (6) months from this data, provided that my name is included as one of the
authors for this article. The arrangement of the name depends on my
supervisor.
Yours truly,
_______________
(KUNG ZHEN YANG)

ix
TABLE OF CONTENTS
Page
ABSTRACT ii
ABSTRAK iii
ACKNOWLEDGEMENTS iv
DECLARATION vi
APPROVAL SHEET vii
PERMISSION SHEET viii
TABLE OF CONTENTS ix
LIST OF TABLES xv
LIST OF FIGURES xvi
LIST OF ABBREVIATIONS xxi

x
CHAPTER 1 INTRODUCTION Page
1.1 Overview 1
1.2 Evolution of Natural Products 1
1.3 Natural Products/ Secondary Metabolites 3
1.4 Terpenes 6
1.5 Terpenoid Chemistry 10
1.5.1 Biosynthesis, Enzymes, and Coenzymes 10
1.5.2 The C-15 Pyrophosphate 11
1.5.3 Linear Terpenoids Synthesis 14
1.5.4 Cyclic Terpenoids Synthesis 16
1.6 Plant of Interest 20
1.6.1 Andrographis paniculata 20
1.6.2 Classification of Andrographis Paniculata 22
1.7 Objectives of Research 23

xi
CHAPTER 2 LITERATURE REVIEW Page
2.1 Overview 24
2.2 Elucidation of Andrographolide 24
2.3 Known Andrographolide Analogue Compounds 28
2.4 New Andrographolide Analogue Compounds 34
2.5 Other Chemical Constituents: Flavonoids 42
2.7 Bioactivty of AngrographisPaniculata 47
2.7.1 Hepatoprotective Effects 48
2.7.2 Antimicrobial, Antiviral, and Antiparasitic Effects 49
2.7.3 Disease and Infection Treatment 51

xii
CHAPTER 3 METHODOLOGY Page
3.1 Introduction 52
3.2 Apparatus, Materials, Chemicals and Instruments 52
3.3 Plant Material Collection 54
3.4 Preparation of Plant Material 54
3.5 Extraction from Raw Plant Materials 55
3.6 Separation and Isolation of Compounds from Crude Product 58
3.6.1 Thin-Layer Chromatography (TLC) 58
3.6.2 Column Chromatography (CC) 59
3.6.3 Centrifugal Thin Layer Chromatography (CTLC) 59
3.6.3.1 Preparation of the Sorbent for CTLC 59
3.7 Characterization on Pure Compounds Obtained 61
3.7.1 Nuclear Magnetic Resonance Spectroscopy (NMR) 61
3.7.2 Infrared (IR) Absorption Spectroscopy 61
3.7.3 Mass Spectrometry (MS) 62
3.7.4 Ultra Violet-Visible Light Spectrometry (UV-Vis) 62
3.7.5 Polarimetry 62

xiii
CHAPTER 4 RESULTS AND DISCUSSION Page
4.1 Overview 64
4.2 Isolation of Compounds from Crude Extract 65
4.2.1 Filtration 65
4.2.2 Column Chromatography (CC) 65
4.2.3 Centrifugal Thin Layer Chromatography (CTLC) 66
4.3 Characterization and Structural Elucidation of Compounds 69
4.3.1 Characterization of Compound 1: KZY001 69
4.3.2 Characterization of Compound 2: KZY002 83

xiv
CHAPTER 5 CONCLUSION Page
5.1 Conclusion 94
5.2 Future Studies 95
REFERENCES 96

xv
LIST OF TABLES
Table Page
1.1 Further classification of terpenoids based on the number of
carbons and number of isoprene units
7
3.1 Sources and purity of organic solvents used in the project 53
3.2 Sources of chemicals used in the project 53
3.3 List of instruments used in the project and its manufacturer 53
4.1 The IR assignments for KZY001 71
4.2 Summary of spectral data for KZY001 80
4.3 The IR assignments for KZY002 83
4.4 Summary of spectral data for KZY002 91

xvi
LIST OF FIGURES
Figure Page
1.1 Origins of the main secondary metabolites in relation to the
basic metabolic pathway
4
1.2 Structure of isoprene 6
1.3 The proposed mechanisms that can occur between 2 isoprene
units in the process of forming terpenoids
8
1.4 Formation of geraniol via 2 isoprene units and 1 hydroxyl
group. The red arrow dotted line show a head-to-tail linkage
between the isoprene building blocks, while the introduction
of a hydroxyl group to the structure will often allow the
functionality of the terpenoid (shown in green arrow and
green dotted line).
9
1.5 Formation of acetyl-CoA from glycolysis during
photosynthesis
11
1.6 The formation of acetyl-CoA anion due to the loss of α-
hydrogen
12
1.7 Nucleophilic attack of acetyl-CoA anion to acetoacetyl-CoA
to form a dithiolester
13

xvii
1.8 Transformation of the dithiolester compound into isopentyl
pyrophosphate, the building block of terpenoids.
35
1.9 Isomerism of isopentenyl pyrophosphate to prenyl
pyrophosphate
14
1.10 The possible orientations for geranylgeranyl pyrophosphate,
which leads to the formation of labdane and cembrane, both
are the major cyclic classes under the diterpenoid family
17
1.11 The mechanism involved in the synthesis of different classes
of terpenoids from the C-5 pyrophosphate
19
1.12 Various views on the appearances of Andrgraphis paniculata 22
2.1 1,2,5,6-tetramethylnapthalene (left) and 1,5-dimethyl-2-
naphthol (right) formed after the selenium dehydrogenation
on andrographolide.
26
2.2 Suggested partial structure of andrographolide after selenium
dehydrogenation experiment
26
2.3 The proposed and the refined structure of andrographolide
suggested by Cava and collaborators
27
2.4 The conclusive structure of andrographolide with its absolute
stereochemistry.
28
2.5 The major andrographolide analogue compounds from
Andrographis paniculata.
29
2.6 Minor diterpenoids, diterpenoidglucosides and diterpenoid
dimers from Andrographis paniculata.
31
2.7 Structure of the 23-carbon terpenoid, identified as14-deoxy-
15-isopropylidene-11,12-didehydroandrographolide.
34

xviii
2.8 The new diterpenoids discovered by various authors. 36
2.9 Recently discovered diterpenoids that were isolated from
40
2.10 Dihydroskullcapflavone, an example of 2’-oxygenated
flavanone.
42
2.11 Flavonoids isolated from Andrographis paniculata. 43
2.12 New flavonoids from Andrographis paniculata discovered in
the 21st
century
45
2.13 The polyphenols that are found in Andrographis paniculata. 46
3.1 Extraction flowchart of the axial part of Andrographis
paniculata
57
3.2 The set-up of CTLC 60
4.1 The circular bands were separated from each other in the Si
gel, which each of them can be collected when they move the
end edge of the rotor via centrifugal forces during the
separation via CTLC.
67
4.2 The isolation and separation of compounds from the aqueous
layer and CHCl3 crude, after the partition extraction on the
MeOH extract of Andrographis paniculata.
68
4.3 IR spectrum of KZY001 obtained from KBr thin-film
(C5H5N).
70

xix
4.4 The structure of a 5 membered α,β-unsaturated-γ-lactone, in
which the oxy-methylene signal at δC76.3 in 13
C NMR
spectrum was associated.
72
4.5 Partial structure constructed from the correlations that were
observed in HMBC spectrum by the protons of the two
tertiary methyl C-18 and C-20.
74
4.6 Refined partial structure of KZY001 after observed
correlations of the exo-methylene at C-17.
74
4.7 Partial homocyclic ring formed when correlations in HMBC
spectrum were observed made from the td splitting proton (δH
2.03) and multiplet signal of proton (δH 1.32).
75
4.8 Refined partial structure of KZY001 showed an ent-labdane
type structure after the hydroxyl group being added into the
structure.
76
4.9 The lactone position in KZY001. 77
4.10 The partial structure A was later refined to give proposed
structure B of KZY001.
78
4.11 Structure of andrographolide 78
4.12 The hypothetical biosynthesis mechanism of
andrographolide.
79
4.13 13
C NMR spectrum for KZY001 (100 MHz, CD3OD) 81
4.14 1
H NMR spectrum for KZY001 (400 MHz, CD3OD) 82
4.15 IR spectrum of KZY002 obtained from KBr thin-film
(CHCl3).
84

xx
4.16 The observed correlation at C-9 confirmed the previous
assigned structure and revealed the possible position of
suspected olefinic group at C-11 and C-12.
86
4.17 Correlations from HMBC that revealed the partial lactone
structure from the proton (δH 6.15) attached to C-12.
87
4.18 The presence of the lactone was confirmed when correlations
of proton signals at δH 7.18 and 4.81 were observed form the
HMBC spectrum.
88
4.19 Structure of 14-deoxy-11,12-didehydroandrographolide 88
4.20 The hypothetical biosynthesis mechanism of 14-deoxy-11,12-
didehydroandrographolide
89
4.21 13
C NMR spectrum for KZY002 (100 MHz, CHCl3) 92
4.22 1
H NMR spectrum for KZY002 (400 MHz, CHCl3) 93

xxi
LIST OF ABBREVIATIONS
° Unit degree
1-D NMR 1 Dimensional Nuclear Magnetic Resonance
2-D NMR 2 Dimensional Nuclear Magnetic Resonance
ATP Adenosine Triphosphate
br Broad
C Carbon atom
C5H5N Pyridine
CC Column Chromatography
Co-A Coenzyme A
CTLC Centrifugal Thin Layer Chromatography
d Doublet
dd Doublet of a doublet
DEPT Distortionless Enhancement by Polarisation Transfer
DNA Deoxyribonucleic acid
dt Double of a triplet
EA Ethyl acetate, CH3COOC2H5
EM Electromagnetic

xxii
ent Enatiomeric
eV Electron voltage
exo Exocyclic
Fr. Fraction
H Hydrogen atom
Hex. n-Hexane
HMBC Heteronuclear Multiple Bond Correlation
HMQC Heteronuclear Multiple Quantum Correlation
i.d. Internal Diameter
IR Infrared spectroscopy
J coupling constant
L Litre
m.p. Melting point
MS Mass spectrometry
NADPH Nitcotinamide Adenine Dinucleotide Phosphate
s Singlet
Si Silica
t Triplet
td Triplet of a doublet

xxiii
TLC Thin Layer Chromatography
TMS Trimethylsilane, (CH3)3SiH
UV Ultraviolet
v/v Volume per volume
x Multiply
α Alpha
δC
13
C NMR chemical shift
δH
1
H NMR chemical shift
λ Wavelength

1
CHAPTER 1
INTRODUCTION
1.1 Overview
In the early sections of this chapter, a brief introduction on the natural product
chemistry and its application are being discussed. Terpenes, being the largest
group amongst the other classes of secondary metabolites, are highlighted and
discussed with explanation on the mechanisms and their synthetic pathway
included. This is followed by a description on the plant of interest and its
classification. The objectives of this research are provided at the end of this
chapter.
1.2 Evolution of Natural Products
About 4.54 billion years ago, our planet Earth, being created in the Solar
System, had been the habitat of living organisms from then until today. Less
than a billion years later after its emergence, cyanobacteria (now in the form
of stromatolites) first emerged onto the surface of Earth as the first sign of life.
They were then aquatic organisms, which triggered off the main important
processes, such as photosynthesis and vegetable decomposition. This

2
indirectly contributes towards the structure complexity of organisms and
shaping of Earth’s atmosphere (Riding, 2000). These organisms had
extensively grown in great numbers, and through a series of evolution stages
over the billions years ahead, the organisms managed to evolve into plants and
other living organisms, which happened to spread across the continents of the
Earth. The process of evolution and creating new chemistry did not stop there,
in fact, the process has been going on till today that leads to the unlimited
ways of creating new species and new sophisticated products by the plants and
other living organisms (Ikan, 2008).
Ever since the emergence of the human evolution and civilisation, these
natural resources of Earth had been the main supply for the basic needs of
human. Throughout the ages, humans have relied heavily on the Nature to
provide the main food source and medicinal suppliesfor the treatment
ofdiseases and illness, and astonishingly, it is still practiced in modern times
today. Plants particularly, are the main choice of medication due to their
known healing properties since it was discovered by the Egyptians, Romans,
Chinese and Indians (Ikan, 2008). Medicinal practitioners later extracted these
biologically active chemicals from various plants to be used in treating a broad
spectrum of illnesses ranging from light fever to serious respiratory illness like
asthma. These extracted compounds are purely natural and are not induced by
any of the man-made methods, and hence, the beginning usage of the term
“natural products”.

3
1.3 Natural Products/ Secondary Metabolites
In plant biology, the vital pathway for life is referred to as basic or normally
known as the primary metabolism, in which it includes primary metabolites
such as carbohydrates, proteins, lipids and nucleic acid that a plant synthesises
for the need to survive.
However, plant often undergoes another pathway simultaneously, referred to
as the secondary metabolism. This pathway (seen in Figure 1.1) produces a
wide variety of chemical compounds that offer no contribution towards the
plants’ growth and other primary function of the plant. The secondary
metabolites, however, possess a much obvious ecological role, such as acting
as an attractant for pollination, involved indefence mechanism towards
predators like insects and parasitic plants and possible use for human needs in
providing flavour, fragrance, oils, as well as medicine (Hajnos, Sherma and
Kowalska, 2008)
Though our generation today uses a vast variety of natural products for
industrial and pharmaceutical uses, a large gap still exists in conclusive
understanding of their actual role in Nature. Plants, with the capability of
creating these complicated natural products in their system are often
perplexing and sometimes, untraceable, which is the reason why scientists and
chemists today are still trying very hard to solve the puzzle behind the
formation of these secondary metabolites.

4
Figure 1.1: Origins of the main secondary metabolites in relation to the basic
metabolic pathway (Adapted from Hajnos, Sherma and Kowalska, 2008).
With a strong scientific curiosity and the help of advanced spectroscopic
methods, pain-staking natural products studies and research have been
aggressively pursued over the years, which resulted in the findings of the
distinctive structures that these natural products somehow, exhibited. This

5
eventually leads to the classification and categorization of these natural
products into 12 major classes of natural products: polyphenols, carotenoids,
glucosinolates, polysaccharides, lectins, terpenes, alkaloids, polyacetylenes,
allium compounds, chlorophyll, capsaicinoids and bealains (Tiwari, Brunton,
Brennan, 2013).
Each class of the secondary metabolites mentioned has different range of
biochemical activities and physiological effects that can be used for
medication development, industrial uses and pharmaceutical designs. These
secondary metabolites are of high value than their primary metabolites, which
are regarded as the model for drug discovery which greatly helped in
medicinal drugs synthesis, increase the efficiency of treatment and forever
change the world of medicine.

6
1.4 Terpenes
Out of the 12 classes of secondary metabolites, terpenes caught the most
attention as almost all living organisms have the tendency of producing
terpene as their secondary metabolites. This is mostly due to the capability of
terpenes that can contribute greatly to the essential physiological functions of
the plants (Tiwari, Brunton, Brennan, 2013). It happens to be the largest group
among the classes of natural products, and is suspected to have consisted over
36000 terpene structures, which most of them have not been investigated in
terms of functionality (Buckingham, 2007 in Tiwari, Brunton, Brennan, 2013).
Figure 1.2: Structure of isoprene (IUPAC name: 2-methylbuta-1, 3-diene).
Terpenes, known for their common name, terpenoids and isoterpenoids, are
defined as the chemical product in which their carbon skeletal structure is
made up of 5 carbon isoprene, or 2-methylbuta-1,3-diene units (see Figure
1.2). Since isoprene consists of 5 carbons, thus the carbon numbers in any
terpenoids that is built using isoprene are always in the multiple of five, which
can be used to further classify them accordingly.

7
Due to thetremendous amount of varied structures studied over the years,
terpenoids are later sub-divided into groups that based on the number of
carbons they possessed, for the ease of characterization. Terpenoids that have
15 carbon atoms are classified as sesquiterpenoids, while terpenoids that are
classified as diterpenoids contain 20 carbon atoms. Higher terpenoids, which
consist of triterpenoid (30 carbons) and tetraterpenoid (40 carbons) are further
divided into steroids and carotenoids respectively. Polyisoprenoids on the
other hand, refer to the terpenoids that have 40 carbon atoms and above. The
Table 1.1 below shows the sub-grouped terpenoids that are classified by the
number of carbons and isoprene units that a compound has.
Table 1.1: Further classification of terpenoids based on the number of carbons
and number of isoprene units (Adapted from Charles, 2003).
Name No. of isoprene units No. of carbon atoms
Hemiterpenoids 1 5
Monoterpenoids 2 10
Sesquiterpenoids 3 15
Diterpenoids 4 20
Sesterterpenoids 5 25
Triterpenoids
(Steroids)
6 30
Tetraterpenoids
(Carotenoids)
8 40
Polyisoprenoids > 8 > 40

8
It is of no surprise to know the ability of isoprene transforming into various
kinds of structures via the second metabolism pathway, as the diene in the
isoprene is known for its ability to both donate and accept electrons, which
allows the possibility of simultaneous linkage of one isoprene unit to another.
In 1887, Otto Wallach proposed the Isoprene Rule, which denotes the
formation of terpenoids are mainly due to the head-to-head (does not occur in
nature), tail-to-tail, head-to-tail linkages between 2 isoprene units as shown in
Figure 1.3 (Christmann, 2010).
Figure 1.3: The proposed mechanisms that can occur between 2 isoprene units
in the process of forming terpenoids (Christmann, 2010).

9
Often, the linkage process goes on with the multiple additions of isoprene
units to that terpenoid, via the head-to-tail and tail-to-tail mechanism. When
the number of carbons for that particular terpenoid is sufficient enough for
folding, cyclic rings are often formed through the twisting of the carbon bonds,
which creates the finalized structure of the terpenoid. Upon the introduction of
oxygen atoms to the compound as substituents, terpenoid gains its
functionality as a secondary metabolite for the plant itself. Figure 1.4 below
shows how the linkage of the isoprene units and hydroxyl group generates
geraniol, a common terpenoid extracted for the use of perfume making and
beverage flavouring (Charles, 2003).
Figure 1.4: Formation of geraniol via 2 isoprene units and 1 hydroxyl group.
The red arrow dotted line show a head-to-tail linkage between the isoprene
building blocks, while the introduction of a hydroxyl group to the structure
will often allow the functionality of the terpenoid (shown in green arrow and
green dotted line).
Geraniol

10
1.5 Terpenoid Chemistry
1.5.1 Biosynthesis, Enzymes and Coenzymes
Once a living organism produces a particular chemical product for its own
needs, it is said to have undergone a biosynthesis (Christmann, 2010).
Biosynthesis is the main process of biochemical reactions that occurs in plants
and animals alike, in which scientists can use it as an outline and assist one in
medicinal drugs development as biosynthesis itself contains vital information
on how the living organism synthesize its natural products, which can be
mimicked by chemist in the lab, and further mass produce to benefit all.
Unlike the chemical reaction that uses chemical catalyst in the laboratory,
biosynthesis involves natural protein catalysts known as enzymes, which
similarly speeds up the biochemical reaction by lowering the enthalpy
barricade of the system, and selectively reacts with the reactant to produce
specific products with the controlled molecular orientation. Enzymes are the
ones that produce the distinctive products in some of the plants and are
controlled by the DNA of the plant possess. Different DNA will translate
different enzymes, which then produce different natural products among the
different plant species.
Sometimes, enzymes alone are unable to synthesize chemical products if
coenzymes are absent in the system. These coenzymes usually bounded to the

11
enzymes, and provide additional assistant, in terms of oxidizing or reducing
ability towards the reaction. In terpenoid chemistry, 3 main coenzymes: ATP,
NADPH, CoA are frequently utilized in the process of terpenoids synthesis.
These coenzymes sometimes take part in the reaction and bind to the reactant
prior to the next biochemical reaction.
1.5.2 The C-15 Pyrophosphate
As mentioned briefly in section 1.4, the synthesis of terpenoids involves the
repetitive linkage of isoprene units into long chain and cyclic rings following
the Isoprene Rule. In this section, focus is on the synthesis of the natural
occurring isoprene unit in plants and further explains the mechanism of this
amazing building block that is responsible for all the terpenoid syntheses.
Figure 1.5: Formation of acetyl-CoA from glycolysis during photosynthesis
(Adapted from Charles, 2003).

12
It is known generally that a plant absorbs CO2 and H2O to synthesize glucose
for its basic growth and needs, but however, one might not have noticed the
astonishing biological pathway after that. During photosynthesis, sunlight is
absorbed to facilitate the breakdown of glucose in which the process is known
as glycolysis. Glucose undergoes a series of degradation until its final form,
phosphoenol pyruvate. The phosphate group will be detached through
hydrolysis to form a free pyruvate, which later accepts a unit of CoA after
decarboxylation to form acetyl-CoA. Figure 1.6 shows the pathway to the
formation of acetyl-CoA.
Figure 1.6: The formation of acetyl-CoA anion due to the loss of α-hydrogen
(Modified from Charles, 2003)
The carbonyl functional group, C=O distorts the electron cloud of the α-
carbon (carbon besides the C=O group), causing the α-hydrogen (H that is
attached to α-carbon) to be acidic, and tends to be deprotonated from the
acetyl-CoA to form anionic acetyl-CoA (Wade, 1999). This nucleophilic anion
can attack the carbonyl group of another acetyl-CoA and remove its CoA unit
to form acetoacetyl-CoA, in which it is attacked by an anion again to form a
hydroxyl dithiolester compound as seen in Figure 1.7 in the next page.

13
Figure 1.7: Nucleophilic attack of acetyl-CoA anion to acetoacetyl-CoA to
form a dithiolester (Modified from Charles, 2003).
The two CoA units of the dithiolester compound were later removed by
hydrolysis and reduced by NADPH successively to yield mevalonic acid,
which the latter was phosphorylated by 3 equivalents of ATP to form a
triphosphate. The tertiary phosphate group was removed through
decaboxylation as it is prone to elimination, thus forming isopentenyl
pyrophosphate (Figure 1.8), which is the main building block for terpenoid
synthesis we had discussed so far (Romano and Conway, 1996).

14
Figure 1.8: Transformation of the dithiolester compound into isopentyl
pyrophosphate, the building block of terpenoids.
1.5.3 Linear Terpenoids Synthesis
The formation of linear and cyclic terpenoids that are synthesized by the plants
can be explained and ascribed to isopentenyl pyrophosphote. Isopentenyl
pyrophosphate often converts to its isomer, prenyl pyrophosphate (shown in
Figure 1.9) which can be hydrolysed to remove the pyrophosphate group and
form C5 class terpenoids, hemiterpenoids.
Figure 1.9: Isomerism of isopentenyl pyrophosphate to prenyl pyrophosphate
(Modified from Charles, 2003).

15
The allylic hydrogen of isopentenyl pyrophosphate is removed upon the
addition of a base and forms an anion. This anion can act as a nucleophile and
via SN2 (head-to-tail linkage) attack the allylic pyrophosphate of the prenyl
pyrophosphate, an isomer of isopentenyl pyrophosphate, leading to the
formation of geranyl pyrophosphate, which is the main component for
monoterpenoid synthesis. The anion of isopentenyl pyrophosphate, constantly
being replaced in the system, further undergoes nucleophilic attack on the
geranyl pyrophosphate to increase its carbon atoms to a C-15, C-20
pyrophosphate compound and so on, which eventually contributes to the
synthesis of the classes of terpenoids (Christmann, 2010).

16
1.5.4 Cyclic Terpenoids Synthesis
Most of the times, these linear carbon pyrophosphates are bend and twisted
under the influence of the enzymes that plants possess to form cyclic
terpenoids. These bond twisting and bending contribute to the almost infinite
possibilites of orientation, again creating wide sub-classes of terpenoids. The
orientated structure is further complicated with the addition of other
substituents and chemical transformations that lead to an almost unlimited
forms of terpenoids, which cannot be fully discussed in this report.
Nevertheless, we will still be looking at a particular class of cyclic terpenoids
that is associated to the plant of interest of this project, which is the
diterpenoid labdane.
Cyclic terpenoids are often made possible through the carbocation-electron
transfers, due to the presence of electron rich C=C in the carbon
pyrophosphate itself, which can donate electrons to the electron deficient
carbocation and hence forming bonds and complete the cyclic linkages
(Reilley, 1964). In Figure 1.10, the geranylgeranyl pyrophosphate, a 20
carbon pyrophosphate, is seen to have twisted in two possible orientations
(under the influence of different enzymes in the plants) that results in two
different classes of the diterpenoid family, the labdane and cembrane type
terpenoids, which are being synthesised during the biochemical reaction. The
italicized term solely refers to the specific class of the diterpenoid family.

17
Figure 1.10: The possible orientations for geranylgeranyl pyrophosphate,
which leads to the formation of labdane and cembrane, both are the major
cyclic classes under the diterpenoid family (Adapted from Charles, 2003)

18
One should always note that this carbocation-electron transfer process is
similar in any terpenoid synthesis and is applicable to the other types of ring-
forming reaction that occurs in the synthesis of hemiterpenoids,
monoterpenoids, etc. Most of the reactions are initiated by heterolysis of
pyrophosphate group, epoxidation and deprotonation as shown in Figure 1.11.
Upon the heterolysis of the C-O of the geranylgeranyl pyrophosphate, the
reaction of ring-forming is initiated, forming the electron poor carbocation at
the 1-carbon (denoted as “C+” in Figure 1.10), where it is subsequently being
filled by the electron rich C=C to form a cyclic skeleton of cembrane, a 14-
membered ring diterpenoid.
Epoxidation, as mentioned, can also occur to initiate the biosynthesis. This is
clearly shown by the synthesis of labdane in Figure 1.10 where the C-14 and
the C-15 can be epoxidised and protonated to form an oxonium ion (oxygen
cation with 3 bonds). The oxonium ion is torsionally strained and tends to
open its rings. The ring-opening process directs the resultant hydroxyl group
to the 14-carbon, which is thermodynamically preferred.
The positive charge, on the other hand, positions itself on the C-15 and gives a
stable tertiary carbocation structure. C-14 is not the choice for the position of
the positive charge as its secondary carbocation structure is not as stable as the
tertiary carbocation (Reilley, 1964). Electron transfers from the C=C initiate a
series of ring-closing reaction, which eventually forms the diterpenoid labdane,
which is found abundantly during the investigations of this project.

19
Figure 1.11: Mechanism involved in the synthesis of different classes of terpenoids from the C-5 pyrophosphate (Adapted from Charles, 2003)

20
1.6 Plant of Interest
1.6.1 Adrographis paniculata
Andrographis paniculata, under the family of Acanthaceae, is a herbaceous
herbal plant which grows abundantly throughout across tropical climatic
countries like Malaysia, Thailand, Indonesia, and India. Though its centre of
origin, believed to be South India and Sri Lanka, where the population and
diversification of the plant are observed, the shrubs are widely cultivated in
sub-tropical countries like China, Mauritius and the Eastern and Western Indies
(Mishra, Sangwan, and Sangwan, 2007).
Generally known as the “King of Bitter”, the plant also gains its numerous
common names in different countries: Kalmegh (कालमेघ), Hempedu Bumi,
Chuanxinlian (穿心莲), in which all the common names literally describe the
extreme bitter taste that the plant possesses. The shrubs of Andrographis
paniculata are often found spreading in shady and moist area, isolated patches,
slopes along low hills, farms, and even near the drains and along the road side
(Niranjan, Tewari and Alok, 2010).
Depending on the soil condition and where the shrubs are grown, the perennial
plant may grow from 30 cm to 110 cm tall. The dark-green coloured stems of
the plant are found to be squared and slim, with occurring longitudinal seams
and wings alternately around the surface of the stems. The hairless spear-
shaped leaves grow to 8 cm long and 2.5 cm wide, but prone to shrinking
during the flowering period. The flowers of the plant are relatively small and

21
have distinctive white petals with dark purple staining. The fruit is seen to be a
small capsule that grows along the stem and “pops” to spread small yellow-
brown seeds during propagation (Kumar et al., 2012).
The bitter leaves and stems of the plants are frequently cut, dried, grinded and
capsulized for consumption among the Indians and Chinese. It is widely used
for Ayervedic and household remedies as the bitter taste the plant possesses is
believed to have a strong association to heat and toxic removal, in which
common sickness like cold, flu and fever can be treated upon consumption.
Further medicinal application and biological effects of Andrographis
paniculata are explained later in the next chapter (Akhar, 2011)

22
1.6.2 Classification of Andrographis Paniculata
Figure 1.12: Appearances of Andrgraphis paniculata.
Kingdom Plantae - Plants
Subkingdom Viridaeplantae – Green plants
Infrakingdom Streptophyta– Land plants
Division Tracheophyta – Vascular plants
Subdivision Spermatophytina – seed plants
Infradivision Angiospermae – Flowering plants
Class Magnoliosida – Dicotyledons
Superorder Asteranae
Order Lamiales
Family Acanthacecae - acanthacées
Genus
Andrographis – Wall. ex Nees – false
waterwillow
Species
Andrographispaniculata (Burm. f.) Wall. ex.
Nees
Retrieved 0ctober 15, 2013, from the Integrated Taxonomic Information
System (ITIS) (http://www.itis.gov).

23
1.7 Objectives of Research
The objectives of this research are:
1. To extract and isolate chemical constituents from the aerial part of
2. To identify ad characterize the structure of the pure compounds
obtained via spectroscopic analyzes.

24
CHAPTER 2
LITERATURE REVIEW
2.1 Overview
In accordance to the project title of the research, this review would concentrate
on the compounds isolated from Andrographis paniculata, particularly
andrographolide and its analogue constituents, with flavonoids and
polyphenols. Health benefits of the plant and its application were also
discussed at the end of this chapter.
2.2 Elucidation of Andrographolide
Andrographolide [1], the major constituent of Andrographis paniculata, was
found to have a unique bitter taste and has led scientists to study and research
thoroughly for it over a century now. It was reported that Boorsma was the first
to have isolated a colourless, neutral, bitter crystalline compound from the
plant in 1896. The compound was later characterized by a Dutch chemist,
Gorter in 1911 (Matsuno, 2013). Gorter later named the compound as
andrographolide and stated that the molecular formula of andrographolide was
likely to be C20H30O5. Based on the acetylation experiment he conducted,
andrographolide was proposed to have a tri-hydroxy-lactone function with one
of the hydroxyl groups being tertiary (Chakravarti and Chakravarti, 1952).

25
Guha-Sircar and Moktader 1939, however, were unsuccessful in reproducing
the triacetylandrographolide stated in Gorter's report, but the team managed to
prove the presence of methylenedioxy-group function and onehydroxy-group
in andrographolide (Guha-Sircar and Moktader 1939, as cited in Chakravarti
and Chakravarti, 1952). The statement, however, was not true as it was rejected
by a team of scientists lead by Paist and further agreed with Gorter's previously
mentioned observations, with new evidence on the presence of a α,β-
unsaturated lactone function (Paist, et al., 1941 as cited in Chakravarti and
Chakravarti 1952).
Upon hydrogenation in weak acid (acetic acid) solution with platinum dioxide
(Adam's catalyst) as catalyst, andrographolide was transformed to two isomeric
compounds, identified as deoxytetrahydroandrographolides, which signify the
presence of two double bonds and a readily-liable eliminated hydroxy-group,
agreeing to Gorter's statement (Schwyzer, Biswas and Karer, 1951 as cited in
Chakravarti and Chakravarti 1952).
The structure of andrographolide was later being further defined using
selenium dehydrogenation, in which 1,2,5,6-tetramethylnapthalene and 1,5-
dimethyl-2-naphthol (seen in Figure 2.1) were afforded after the reaction
which indicated the presence of bicyclic diterpenoid lactone, with the
additional indication of a hydroxy-group at the C-3 shown in Figure 2.2
(Schwyzer, Biswas and Karer, 1951 as cited in Cava, et al., 1962).

26
Figure 2.1: 1,2,5,6-tetramethylnapthalene (left) and 1,5-dimethyl-2-naphthol
(right) formed after the selenium dehydrogenation on andrographolide.
Figure 2.2: Suggested partial structure of andrographolide after selenium
dehydrogenation experiment (Modified from Cava, et al., 1962).
With all the previous data and the present information obtained, Cava, et al.
(1962) had proposed the structure of andrographolide for the first time (Figure
2.3, (a)). The proposed structure, however, was later found to be incorrect due
to the wrong placement of the hydroxy-group on C-12 instead of C-14 and the
unassigned stereochemistry at C-3 and C-4. The team, with its collaborators

27
later refined the structure of andrographolide and reported (Figure 2.3, (b)) the
structure without the confirmation on the stereochemistry of the hydroxyl
group on C-14 (Cava, et al., 1965).
Figure 2.3: The proposed and the refined structure of andrographolide
suggested by Cava and collaborators (Modified from Cava, et al., 1962; 1965).
Due to the multiple failuresin attempting to determine the absolute
configuration of the C-14 of andrographolide, X-Ray Crystallographic analysis
was conducted by Fujita, et al. (1984) to assign the previously unresolved
configuration of andrographolide. The experiment was conducted with the help
of previous known structural data to determine the absolute stereochemistry of
C-14 of andrographolide, and eventually establish the absolute configuration of
andrographolide shown in Figure 2.4.
(a) (b)

28
Figure 2.4: The conclusive structure of andrographolide with its absolute
stereochemistry.
2.3 Known Andrographolide Analogue Compounds
With the conclusive structural elucidation of andrographolide, other analogue
compounds having the same carbon skeleton, i.e. ent-labdane bicyclic
diterpenoid can now be deduced and concluded as the difference is often,
shown only on the substituents attached to the analogue compounds.
Over the course of the years, the other major constituents (Figure 2.5) from
Andrographis paniculata have been isolated successively by various authors
and identified them as 14-deoxyandrographolide [2],14-deoxy-11-
oxoandrographolide [3], 14-deoxy-11,12-didehydroandrographolide [4],
neoandrographolide [5], andrographanin [6], 19-β-glucosyl-14-
deoxyandrographiside [7], 19-β-glucosylandrographiside [8] (Balmain and
Connolly, 1973; Fujita, et al., 1984; Matsuda, et al., 1994).
1
2
3 5
4 6
7
8
9
10
11
12
13
14
15
16
17
1819
20

30
Figure 2.5: The major andrographolide analogue compounds from
Matsuda and his team in 1994 had successfully isolated, characterized and
refined the structure of isoandrographolide [9] that was first proposed by Cava,
et al. (1965). On top of that, the ent-labdane type diterpenoids, diterpenoid
glucosides and diterpenoid dimers found were isolated and identified by
Matsuda, et al (1994) as well. The compounds were identified as 14-epi-
isoandrographolide [10], 14-deoxy-12R-methoxyandrographolide [11], 14-
deoxy-12S-methoxyandrographolide [12], 14-deoxy-12R-hydroxyandro-
grapholide [13], 14-deoxy-12S-hydroxyandrograp-pholide [14], 14-deoxy-11-
hydroxyandrographolide [15], 14-deoxy-11,12-didehydro-andrographiside [16],
6’-acetylneoandrographolide [17]. A strange occurring diterpenoid dimers,
identified as isomers bisandrographolide A [19] and B [20] and another pair of
isomers C [21] and D [22] were also reported but unfortunately, the absolute
configuration of the linkage (at C-12 and C-15’) was not able to be assigned till
today.
[8]

31
Jantan and Waterman (1994), on the other hand, have reported a rare ent-14β-
hydroxy-8(17),12-labdadien-16,15-olide-3β,19-oxide [18] whereby the 2
hydroxy-groups of C-3 and C-4 undergo epoxidation to form an unstable
epoxide substituent which was not found abundantly. Figure 2.6 below shows
the structures of the isolated minor diterpenoids, diterpeneglucosides, and
diterpenoid dimer previously mentioned.
[9] [10]
[11]
[12]

32
[13] [14]
[15] [16]
[17]
[18]
3
4

33
Figure 2.6: Minor diterpenoids, diterpenoidglucosides and diterpenoid dimers
from Andrographis paniculata.
[19], [20]
[21], [22]
12
15’

34
2.4 New Adrographolide Analogue Compounds
Upon entering the 21st
century, new compounds from Andrographis paniculata
are still being discovered by various scientists. Reddy, et al. (2003) had
successfully isolated and characterized an uncommon 23-carbon terpenoid
(Figure 2.7) from the plant. The compound is somewhat similar to that of
compound [3] in the previous section 2.2, with the only difference is the
presence of isopropylidene substituent on the new compound at C-15. The new
compound was later identified by Reddy and fellow scientists as 14-deoxy-15-
isopropylidene-11,12-didehydroandrographolide [23].
Figure 2.7: Structure of the 23-carbon terpenoid, identified as14-deoxy-15-
isopropylidene-11,12-didehydroandrographolide.
[23]

35
Reddy, et al. (2005) on the other hand, happened to isolate and propose the
structure of new bisandrographolide ether [24] that does not exhibit anti-HIV
activity and poses cytotoxic properties. Chen, et al. (2006) have successfully
isolated and characterized 9 new ent-labdane diterpenoids [25] – [33] from the
aerial part of Andrographis paniculata that was collected in Fujian Province,
China. Zhang, et al. (2006), in the same year, was able to isolate three new
diterpenoids identified as 19-norandrographolide A-C [34] – [36] from the
plant that was collected in Jiangxi Province, China. It seems to appear that the
plant tends to create new products when it is at a different location and
environment, with soil composition being a major role in this occurrence.
This phenomenon is observed again in the reports of Chen, et al (2008)
whereby three new diterpenoid stereoisomer compounds, namely 7S-hydroxy-
14-deoxyandrographolide [37], 7R-hydroxy-14-deoxyandrographolide [38],
and 12S,13S-hydroxyandrographolide [39] were isolated and characterized
using advanced computational power. At the same time, Chen’s team members
had collaborated in Zhou, et al. (2008) and successfully isolated two new
diterpenoid glucosides: 3-O-β-D-glucosyl-14-deoxyandrographolide [40], 3-O-
β-D-glucosyl-14-deoxy-11,12,-didehydroandrographolide [41], using
spectroscopic and chemical methods. Figure 2.8 shows the new diterpenoid
compounds that were mentioned previously in the discussion.

37
[30]
[29]
[31]
[32]
[34]
[33]

38
[35]
[36]
[39]
[40]
[37]
[38]

39
Figure 2.8: The new diterpenoids discovered by various authors.
It is still believed that new natural products are still being synthesized by
Andrographis paniculata at different parts of the world, where soil
composition is the main factor as it is different from one another, thus the
variety of the secondary metabolites can be seen from the plant. In 2009,
andrographlocatone [42], a rare seven membered ring diterpene compound
which was not normally seen in natural products, was discovered by Wang and
fellow chemists using spectral approach and X-ray diffraction analysis for
confirmation (Wang, et al., 2009).
Ma, et al. (2009) at the same year had isolated a new eipenoid and a new
diterpenoid compound where it was identified as 17β-epoxy-3.19-dihydroxy-
11,13-ent-labdatrien-15,16-olide [43] and 3,7,19-trihydroxy-8,11,13-ent-
labdatrien-15,16-olide [44] respectively. Xu, Chou and Wang (2010) have
managed to discover a rare tetrahydroxyditerpene, which was not seen in the
[41]

40
plant for so many years of discovery. It was then identified as (13R, 14R)
3,13,14,19-tetrahydroxy-ent-labda-8(17),11-dien-16,15-olide [45].
The discovery did not stop there as new compounds were still being discovered
by various scientists in the recent years. In the most recent publication, four C-
8 and C-12 diastereoisomers of andrographolide were determined by Hu, et al.
(2012) and were identified as (8S,12S)-isoandrographolide [46], (8S,12R)-
isoandrographolide [47], (8R,12R)-isoandrographolide [48], and (8R,12S)-
isoandrographolide [49]. Figure 2.9 below shows the recent discovered natural
products that were isolated from Andrographis paniculata.
[42]
[43]

41
Figure 2.9: Recently discovered diterpenoids that were isolated from Andrographis
paniculata.
[44]
[45]
[46] [47]
[49][48]

42
2.5 Other Chemical Constituents: Flavonoids
Apart from being rich in diterpenoids asmentioned in the previous sections,
Andrographis paniculata has been reported to be the source of flavonoids as
well, especially 2’-oxygenated flavonoids (seen in Figure 2.9) as reported by
Govindachari et al. (1969).
Figure 2.10:Dihydroskullcapflavone, an example of 2’-oxygenated flavanone.
Jalal, et al. (1979) were one of the first groups to study the flavonoids isolated
form Andrographis paniculata. Using cultures of the plant, the team was able
to extract three new flavonoids that were identified as 5-hydroxy-7,8,2’-
trimethoxyflavone [50], 5,2’-hydroxy-7,8-dimethoxyflavone [51], 5 -hydroxy-
7,8-dimethoxyflavone (7-O-methylwogonin) [52]. Gupta, et al. (1982), on the
other hand, used petrol to soak the roots of the plant and obtained 2 new
flavonoids, namely 5 -hydroxy-7,8-dimethoxyflavanone [53] and 5 -hydroxy-
3,7,8,2’-tetramethoxy-flavone [54]. Japanese chemists led by Kuroyanagi were

43
successful in isolating 6 flavone glucosides which were identified as
andrographidine A [55], B [56], C [57], D [58], E [59], F [60] from methanolic
extract of roots of the plant (Kuroyanagi, et al. 1987). The flavonoids
mentioned above are shown structurally in Figure 2.10.
[50] [51]
[54]
[53][52]
[55]
[56] [57]

44
Figure 2.11: Flavonoids isolated from Andrographis paniculata.
In the past decade (2000 – 2010), 3 minor flavones (Figure 2.11) were
discovered and isolated by Reddy, et al. (2003) and Rao, et al. (2004) , which
were later characterized as 5-hydroxy-7,2’,6’-trimethoxyflavone [61], 2(S)-
5,7,2’,3’-tetramethoxyflavanone [62], 5-hydroxy-7,2’,3’-trimethoxy-flavone
[63]. In the most recent publication, Chen, et al. (2014) have successfully
isolated and elucidated another new flavone glucoside which was identified as
7,8-dimethoxy-2’-hydroxy-5-O-β-D-glucopyranosyl-oxyflavone [64] and is
also shown in Figure 2.11.
Similar fashion to the main constituents of Andrographis paniculata, i.e. ent-
labdane diterpenoids, new flavonoids are still being synthesized by the plant till
[58]
[59]
[60]

45
today which makes the study of these compounds necessary as the new
compounds might have possible health benefits or become the lead to new drug
discovery.
Figure 2.12: New flavonoids from Andrographis paniculata discovered in the
21st
century (Adapted from Reddy, et al., 2003; Rao, et al., 2004; Chen, et al.,
2014).
[61]
[64]
[63]
[62]

46
2.6 Other Chemical Constituents: Polyphenols
Polyphenols, being the least number of compounds isolated, were also found in
the leaves, roots and shoots of Andrographis paniculata. Among them were
cinnamic acid [65], caffeic acid [66], ferulic acid [67], and chlorogenic acid
[68] that were first discovered by Satyanarayana and his team (Satyanarayana
et al., 1978 as cited in Rao, et al. 2004). Figure 3.2 below shows the structure
of the polyphenols that were isolated and proposed by Satyanarayana and
collaborators.
Figure 2.13: The polyphenols that are found in Andrographis paniculata.
[62] [63]
[64]
[65]

47
2.7 Bioactivity of Andrographis Paniculata
As mentioned in section 1.1, Andrographis paniculata is an Ayuverdic and
Traditional Chinese Medicine herb that is commonly used for treating illnesses
and is regarded to have “blood purifying” and “cooling” property that can be
used on a series of diseases, such as cold, skin inflammation, scabies, boils and
even fever. The aerial parts of the plant are often grinded and capsulized (due
to its extreme bitter taste) or extracted for its juice upon consumption. In some
cases, the leaves are eaten fresh by the users or to be bashed up and applied
onto the inflamed, erupted or itchy skin (Akbar, 2011).
Although the common uses of this plant are mainly for treating cold and
seasonal fever, numerous pharmaceutical and bioactivity studies have shown
that the plant does more than it is thought to be. The diterpenoids and the
flavonoids (discussed in sections 2.2 – 2.5) studied showed positive bioactivity
towardsa wide spectrum of illness and diseases. Various bio-action and
bioactivities of the plant will be discussed accordingly in the subsequent
sections.

48
2.7.1 Hepatoprotective Effects
CCl4, a common substance found in fire extinguishers, is a hepatotoxin (toxic
to liver) which is responsible for many liver damage and cancer cases. A CCl4-
induced liver damage experiment was conducted by Handa and Sharma (1990)
as well as Rana and Avadhoot (1991), have discovered that the ethanolic
extract from the leaves of Andrographis paniculata has a significant
hepatoprotective (liver protection) effect, making the plant to be the choice of
hepatoprotective agent.
This effect is also observed in the reports of Shukla, et al (1992) and Visen, et
al. (1993), where andrographolide, the major constituent of the plant,
significantly prevents the undesired actions of paracetamol-induced effect (low
bile flow, low bile salts and bile acid production), protects the hepatocytes and
making it a better hepatoprotctive agent than Silymarin, a commercial
hepatoprotective drug.
Apart from andrographolide, other constituents in the extract of Andrographis
paniculata also found to participate in the hepatoprotective activity as well. As
reported in Choudhury and Poddar (1985), it was solely the extract of the plant
that showed significant hepatoprotective effect when a higher concentration of
CCl4-induced microsomal lipid peroxidation in vitrowas introduced. This was
further confirmed by Handa and Sharma (1990) whereby an andrographolide-

49
free methanol extract and methnol extract of the plant showed inhabitation
towards the hepatotoxins relatively in high percentage.
Studieswere also done by Trivedi and Rawal who had shown that the aqueous
extract of the plant was able to prevent the increase of unwanted enzymatic
activities under the influence of hepatotoxin substances, such ashexachloro
cyclohexane (BHC), which indicates the strong hepatoprotective action by the
plant, that can be orally consumed without any side effects (Trivedi and Rawal,
2000).
Hepatoprotective activity by Andrographis paniculata seems promising as
researches have showed numerous success of the plant inhibiting various
hepatoxins, indicating its versatility against a broad range of liver diseases,
which can be issued to patients with liver illness of various sources.
2.7.2 Antimicrobial, Antiviral, and Antiparasitic Effects
Various researches have been conducted to investigate the activity of
Andrographis paniculata towards bacteria, viruses and parasites. Thai
biologists in 1990 reported that even in low concentration of crude powder
product suspended in water, the extract showed in vitro antibacterial activity
against Salmonella, Shigella, Escherichia coli, gram A Sterptococci and

50
Staphylococcus aureus, which are the ones causing various diseases and illness
(Leelarasamee, Trakulsomboon and Sittisomwong, 1990). Singha, Roy, Dey
(2003) have also reported the antibacterial activity exhibited by the plant and
concluded it was contributed by the combined action of the andrographolide
constituents and the arabinogalactan proteins.
In addition, Malaysian scientists from University of Malaya had found
andrographolide, neoandrographolide and 14-deoxy-11,12-didehydro-
andrgrapholide, the major constituents of the plant to be virucidal (virus
destroyer) against herpes simplex virus 1 (HSV-1) without being cytotoxic
towards the user even at the virucidal dosage (Wairt, et al., 2005). It was also
reported that the chloroform and methanol extracts of the plant significantly
inhibited malarial parasites growth for 24 hours and 48 hours respectively even
when the extract was used in a very low concentration (Najib, et al. 1999).
Although all the researches showed significant and predominant results, in
vitro and in vivo experiments do not reflect to the relevant clinical uses and the
concentration of the pure isolated compounds used may not be financially
feasible. More research has to be done in order to modify the existing natural
product in order to enhance the bioactivities of Andrographis paniculata
towards diseases.

51
2.7.3 Disease and InfectionTreatment
The obvious benefits of Andrographis paniculata towards diseases have led
scientists to study the plant for its effect on infectious diseases. It was reported
in Chang and But (1987) that ethanol extract tablets and andrographolide and
neoandrographolide containing tablets have cured up 80 percent of acute
dysentery and gastroenteritis cases, which makes the utilization of the plant far
more efficient than the commercial chloramphenicol and furazolidine.
Cases of leptospirosis, tuberculous meningitis, pulmonary tuberculosis and
acute pyelonephritis were also reportedly being cured by the plant’s
constituents in Chang and But’s report, without any significant adverse side
effects. It was also reported by Chang and But (1987) that a staggering 10
cases of viper bites were cured in a week using Andrographis paniculata as the
main component in a formulation, which increases the plant’s potential in
antidote development for poisonous snake and insect bites.
Extract of Andrographis paniculata with the commercial combination of
Eleutherococcus senticosus (also known as Kan Jang in China) has proved to
be beneficial towards uncomplicated upper respiratory tract infections (URTIs)
as the combination significantly improved patients’ headache, nasal and throat
symptoms with faster recovery and lower requirement for standard medications.
The efficiency of treatment cannot be solely accredited to Andrographis

52
paniculata as a synergistic combination of the two herbs has bought out the
effect rather than if any of the individual herb could do (Melchior, et al. 2000;
Spasov, et al. 2004).

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