In vitro Propagation and Andrographolide Analysis of

Hempedu Bumi (Andrographis paniculata Nees)

by

Paritala Vikram

A report submitted in fulfilment of the requirements for the degree of

Masters of Science

Faculty of Agro Based Industry

UNIVERSITI MALAYSIA KELANTAN

2016

i

THESIS DECLARATION

I hereby certify that the work embodied in this thesis is the result of the original

research and has not been submitted for a higher degree to any other University or

Institution.

OPEN ACCESS

EMBARGOES

I agree that my thesis is to be made immediately available

as hardcopy or on-line open access (full text).

I agree that my thesis is to be made available as hardcopy

or on-line (full text) for a period approved by the Post

Graduate Committee.

Dated from until

CONFIDENTIAL

RESTRICTED

(Contains confidential information under the office

Official Secret Act 1972)*

(Contains restricted information as specified by the

organization where research was done)*

I acknowledge that Universiti Malaysia Kelantan reserves the right as follows.

1. The thesis is the property of Universiti Malaysia Kelantan. 2. The library of Universiti Malaysia Kelantan has the right to make copies for the

purpose of research only.

3. The library has the right to make copies of the thesis for academic exchange.

SIGNATURE SIGNATURE OF SUPERVISOR

IC/ PASSPORT NO. NAME OF SUPERVISOR

Date: Date:

ii

ACKNOWLEDGEMENT

First and foremost I would like to express my sincere gratitude to my supervisor

Dr. Mohammed Arifullah for giving me a chance to work under him, for his expert

guidance and encouragement through hardships of my project and helping me to gain

knowledge in various fields of expertise.

I would like to thank Dr. Fatimah Kayat, and Dr. Dwi Susanto for giving

valuable suggestions and guidance in completion of my thesis. I would also like to

thank Faculty of Agro Based Industry, UMK for letting this happen by providing all

necessary chemicals and equipment in labs. I would also like to thank my colleagues

and friends Mr. Ahmed, Ms. Ilfah, Ms. Husna, Ms. Farisya, Ms. Kin Ying, and Ms.

Car Men for their support continuously both in my lab and life. Besides, I would also

like to thank all of the UMK laboratory assistants especially Mr. Muhammad Che Isa

and Mr. Suhaimi Omar for their support in doing the experiments. I also want to

express my gratitude to all my lab students and FYP friends and to one and all whom

either directly or indirectly in making this possible.

Last but not least I would like to thank my real life Gods, my dear parents who

made me possible and what I am and my family members for supporting me in all

kinds of ways to achieve this goal. Thank you all and God bless you all.

iii

TABLE OF CONTENTS

PAGE

DECLARATION i

ACKNOWLEDGEMENTS ii

TABLE OF CONTENTS iii

LIST OF TABLES vii

LIST OF FIGURES ix

LIST OF ABBREIVATIONS xi

ABSTRAK xiii

ABSTRACT xiv

CHAPTER 1 INTRODUCTION

1.1 Medicinal plants 1

1.2 Problem statement 3

1.3 Justification of the study 4

1.4 Objectives of the present study 5

1.5 Scope of the present study 5

CHAPTER 2 LITERATURE REVIEW

2.1 Andrographis paniculata plant and its properties 6

2.1.1 General information 6

2.1.2 Traditional uses and pharmacological activities 9

2.1.3 Phytochemical constituents of A. paniculata 10

2.1.4 Andrographolide 12

2.1.5 Collection and cultivation of A. paniculata 13

2.2 Micropropagation 14

2.2.1 Adventitious shooting 16

2.2.1.1 Direct Adventitious shooting 16

2.2.1.2 Indirect Adventitious shooting 17

iv

2.2.2 Somatic embryogenesis 17

2.2.3 Synthetic seeds 18

2.2.4 Micropropogation studies in A. paniculata 20

2.2.5 Explants used in in vitro regeneration studies 21

2.2.6 Plant growth regulators used in in vitro shoot regeneration studies 23

2.3 Secondary metabolites and their production 24

2.3.1 Callus culture 26

2.3.2 Secondary metabolite studies in A. paniculata 28

2.3.3 Estimation of secondary metabolites using HPLC 30

2.3.4 Antioxidant assay 31

CHAPTER 3 MATERIALS AND METHODS

3.1 Materials 34

3.1.1 Plant materials 34

3.1.2 Apparatus and equipment 34

3.1.3 Chemicals 35

3.2 Methodology 35

3.2.1 Preparation of stock solution 35

3.2.1.1 Preparation of auxins 36

3.2.1.2 Preparation of cytokinins 36

3.2.1.3 Preparation of nutrient media stocks 36

3.2.1.4 Preparation of media 38

3.2.2 Selection of explants 38

3.2.3 Explant sterilization 39

3.2.3.1 Sterilization of the pods 39

3.2.3.2 Sterilization of nodes 40

3.2.4 Secondary metabolite analysis 40

3.2.4.1 HPLC Analysis 40

3.2.4.2 Total phenolic content 41

v

3.2.4.3 Antioxidant activity assay 42

3.3 Experimental protocols 43

3.3.1 Establishment of aseptic shoots 43

3.3.2 Effects of different types of embryonic explants in shoot

organogenesis

43

3.3.3 Adventitious shoot organogenesis from cotyledons and root

decapitated embryonic axes

44

3.3.4 Adventitious shoot organogenesis from leaf explants 45

3.3.5 Shoot regeneration from shoot tips 46

3.3.6 Organogenesis from transverse thin cell layer cultures 47

3.3.7 Shoot elongation 49

3.3.8 Root initiation 49

3.3.9 Acclimatization 50

3.3.10 Induction of somatic embryos 50

3.3.11 Production of synthetic seeds 52

3.3.12 Establishment of callus culture 52

3.4 Experimental design & statistical analysis 53

CHAPTER 4 RESULTS & DISCUSSION

4.1 Micropropogation studies in A. paniculata 55

4.1.1 Adventitious organogenesis from embryonic explants 55

4.1.2 Adventitious organogenesis from leaf explants 67

4.1.3 Multiple shoots regeneration from shoot tips 72

4.1.4 Organogenesis from nodal transverse thin cell layer cultures 76

4.1.5 Shoot elongation of in vitro regenerated shoots 83

4.1.6 In vitro rooting of elongated shoots 86

4.1.7 Acclimatization 89

4.2 Somatic embryogenesis and production synthetic seeds 91

4.2.1 Induction of somatic embryos 91

4.2.2 Studies on synthetic seeds preparation and germination 99

vi

4.3 Secondary metabolite production and analysis 102

4.3.1 Callus culture and estimation of andrographolide 102

4.3.2 Antioxidant study of the callus 107

CHAPTER 5 CONCLUSION

5.1 Conclusion 111

5.2 Research findings 112

5.3 Future suggestions 114

REFERENCES 115

APPENDIX 143

LIST OF PUBLICATIONS 159

LIST OF TABLES

NO. PAGE

3.1 Nutrient composition and preparation of stocks in MS media 37

3.2 Different PGR treatments used to produce shoots from embryo

explants

45

3.3 Different PGR treatments used to produce shoots from leaf

explants

46

3.4 Different PGR treatments used to produce shoots from shoot tips 47

3.5 Different PGR treatments used to produce shoots from tTCL

explants

48

3.6 Different PGR treatments used for shoot elongation 49

3.7 Different PGR treatments used to produce roots from shoots 50

3.8 Different PGR treatments used to induce somatic embryos from

embryo explants

51

3.9 Different PGR treatments used to induce callus from embryo

explant

53

4.1 Effect of different A. paniculata embryonic explants on shoot

regeneration in MS media containing BAP 1.5 mg/L

57

4.2 Effect of diffrenet PGRs on shooting response from root

decapitated embryonic axes of A. paniculata

61

4.3 Effect of diffenet PGRs on shooting response and on multiple

shoot induction from cotyledons of A. paniculata

62

4.4 Effect of different PGRs in shoot induction response from leaf

explants of A. paniculata

70

4.5 Effect of diffenet PGRs on shooting response and on multiple

shoot induction from A. paniculata shoot tips

75

4.6 Effect of different PGRs on growth response of tTCL explants

from A. paniculata node, internode, petiole and hypocotyl

78

4.7 Effect of various PGRs on shoot formation from A. paniculata 79

vii

viii

nodal tTCL explants

4.8 Effect of different growth regulators on in vitro shoot elongation

of A. paniculata

85

4.9 Effect of different PGRs on in vitro root regeneration of A.

paniculata shoots

88

4.10 Data of callus responses of embryo in different plant growth

regulators

94

4.11 Effect of different concentrations and combinations of NAA and

KIN on somatic embryogenesis from A. paniculata embryo

explants

95

4.12 Effect of different PGRs on callus production of A. paniculata

zygotic embryo explants

105

4.13 Amount of andrographolide present in callus produced from

zygotic embryos of A. paniculata on MS media with different

PGRs

106

4.14 Antioxidant IC50 values and total phenolic content of extracts 109

ix

LIST OF FIGURES

NO. PAGE

2.1 Andrographis paniculata plant 8

2.2 Andrographolide 12

3.1 Preparation of different A. paniculata embryonic explants 44

3.2 Preparation of different tTCL explants from in vitro node of A.

paniculata

48

4.1 Growth response of different A. paniculata embryonic explants on

MS media containing BAP 1.5 mg/L

56

4.2 Direct shoot regeneration of A. paniculata cotyledon explants in

different stages on MS media containing BAP 1.5 mg/L

58

4.3 Direct shoot organogenesis from A. paniculata root decapitated

embryo axes explants on MS media containing BAP 1.5 mg/L 59

4.4 Indirect shoot organogenesis from A. paniculata embryonic

explants on MS media containing TDZ 1.0 mg/L

65

4.5 Indirect shoot regeneration from A. paniculata leaves on MS

media containing TDZ 2.0 mg/L

68

4.6 Direct auxiliary shoot regeneration from A. paniculata shoot tips in

MS media containing BAP 1.5 mg/L

72

4.7 Direct shoot regeneration of A. paniculata from nodal tTCLs on

MS media supplemented with BAP 1.0 mg/L

80

4.8 Direct shoot regeneration of A. paniculata from nodal tTCL

explants on MS media with TDZ 3.0 mg/L

81

4.9 Elongation of A. paniculata in vitro regenerated shoots in MS

media with KIN 1.0 mg/L and IBA 0.5 mg/L

86

4.10 In vitro rooting of shoots cultured on different half MS media with

different PGRs

88

4.11 Acclimatization of in vitro grown A. paniculata plants 90

4.12 Formation of somatic embryos on A. paniculata embryo explant in

MS media fortified with NAA 1.5 mg/L+KIN 1.5 mg/L

93

x

4.13 Somatic embryos from A. paniculata embryo explant and their

growth 99

4.14 Synthetic seeds produced from somatic embryos 101

4.15 Callus induced from A. paniculata embryos in media with different

PGRs

104

xi

LIST OF ABBREIVATIONS

g Gram

h Hour

L Litre

cm Centimeter

mg Milligram

ml Milliliter

mm Millimeter

mM Millimoles

MS Murashigae and Skoog

µg Microgram

µl Microliter

BAP Benzylaminopurine

CaCl2 Calcium chloride

DPPH 2, 2-diphenyl-1-picrylhydrazyl

xii

GA Gibberellic acid

HPLC High pressure liquid chromatography

IAA Indoleacetic acid

IBA Indolebutyric acid

KIN Kinetin

NAA Napthaleneacetic acid

NOA Napthaleneoxyacetic acid

PDA Photodiode array

PGRs Plant growth regulators

PIC Picloram

psi Pressure per square inch

SE Standard error

USD United States Dollar

2-iP 2 Isopentenyl adenine

2,4-D 2, 4-Dichlorophenoxyacetic acid

% Percentage

xiii

Perambatan In vitro dan Analisis Andrographolide dalam Hempedu Bumi

(Andrographis paniculata Nees).

ABSTRAK

Andrographis paniculata dan andrographolide memiliki potensi besar dalam bidang

perubatan yang telah meningkatkan permintaan pasaran terhadap tumbuhan ini. Bagi

menepati permintaan tinggi untuk A. paniculata, organogenesis, embriogenesis dan

kalogenesis dikaji dalam penyelidikan ini. Untuk membentuk protokol yang efisyen

penjanaan semula, kajian organogenesis telah menggunakan daun, hujung pucuk,

eksplan embrio dan lapisan sel nipis melintang (tTCL) yang dikulturkan dalam media

Murashige dan Skoog yang mengandungi pelbagai sitokinin secara bersendirian atau

dengan gabungan beberapa auksin. Bilangan pucuk berganda tertinggi telah dijana oleh

eksplan nod tTCL (16 pucuk/eksplan), diikuti eksplan embrio (12 pucuk/paksi embrio

dan 9 pucuk/kotiledon), hujung pucuk (6 pucuk/eksplan) dan daun (6 pucuk/eksplan).

Antara pengawalatur pertumbuhan yang digunakan, didapati thiadiazuron merupakan

PGR yang paling berkesan dalam menghasilkan pucuk berganda bagi kebanyakan

eksplan diikuti oleh benzilaminopurin (BAP) dan kinetin (KIN). Adenin isopentenil 2

(2, IP) didapati paling berkesan untuk merangsang tindak balas organogenik. Pucuk

berjaya memanjang di dalam medium 1.0 mg/L KIN / 0.5 mg/L asid indolbutirik (IBA)

dan menghasilkan akar secara in vitro dalam MS medium separuh kekuatan

mengandungi 0.5 mg/L IBA sahaja dengan purata bilangan akar 7.4 dan akar sepanjang

4.0 cm. Penyesuaian plantlet berakar berjaya dijalankan dengan menggunakan polibeg

dengan 78% kadar kemandirian. Embriogenesis soma telah diaruh menggunakan

eksplan embrio zigot dalam media auksin berbeza secara bersendirian atau gabungan

dengan sitokinin dan peratus embriogenesis tertinggi (75%) dan purata bilangan embrio

(23 embrio/eksplan) didapati di dalam medium 1.5 mg/L asid napthaleneacetic (NAA) /

1.5 mg/L KIN. Embrio soma digunakan untuk menghasilkan biji benih sintetik dengan

menggunakan 3.0% sodium alginat; Walau bagaimanapun kadar percambahan embrio

soma berkurangan daripada 60% kepada 30% akibat pengkapsulan. Kultur kalus yang

terhasil daripada eksplan embrio zigot dengan auksin berbeza atau gabungan sitokinin

dan auksin berbeza menghasilkan pelbagai jenis kalus atau pengakaran berdasarkan

kepekatan dan kombinasi sitokinin. 1.5 mg / L asid Napthaleneoxiacetic (NOA)/ 0.75

mg/L BAP menunjukkan penghasilan jumlah kalus tertinggi manakala 3.0 mg/L NAA

mencatatkan jumlah tertinggi penghasilan akar adventitius. Analisis kandungan

andrographolide dalam kultur kalus dan akar menunjukkan kehadiran andrographolide

tertinggi di dalam kalus rhizogenik dalam 3.0 mg/L NAA (2.4 mg/gm berat kering). A.

paniculata menjadikan kultur embrio tersaur dan realistik bagi penghasilan

andrographolide secara in vitro. Kalus yang dihasilkan di dalam medium 1.5 mg/ L

NOA/ 0.75 mg/L BAP mengandungi lebih kapasiti antioksidan dengan nilai kepekatan

merencat (IC50) sebanyak 20.91 µL dan jumlah kandungan fenolik 0.88 mg/g asid galik

berbanding penghasilan akar dalam 3.0 mg/L NAA. Penghasilan pucuk in vitro secara

organogenesis dalam A. paniculata menggunakan tTCL dan eksplan embrio telah

dilaporkan buat kali pertama dalam kajian ini dan kaedah ini boleh digunakan sebagai

satu protokol yang efisyen bagi perambatan klon A. paniculata.

xiv

In vitro Propagation and Andrographolide Analysis of Hempedu Bumi

(Andrographis paniculata Nees).

ABSTRACT

Andrographis paniculata and andrographolide have huge potential in medical industries

resulting in higher market demand of the plant. To meet the requirement of A.

paniculata, micropropogation by organogenesis, embryogenesis and secondary

metabolte production by callogenesis were studied in the present study. In order to

develop an efficient protocol for propagation, organogenesis studies were carried out

using leaf, shoot tip, embryonic explants and transverse thin cell layers (tTCL) cultured

in Murashige and Skoog media supplemented with various cytokinins either alone or in

combination of auxins. The highest number of multiple shoots were regenerated from

nodal tTCL explants (16 shoots/explant) followed by embryo explants (12

shoots/decapitated embryonic axes and 9 shoots/cotyledon), shoot tips (6

shoots/explant) and leaves (6 shoots/explant). Among the plant growth regulators tested,

thiadiazuron was found to be the most effective hormone in producing multiple shoots

in most of the explants followed by benzylaminopurine and kinetin while 2-isopentenyl

adenine was found to be least effective to induce organogenic response. The shoots

were successfully elongated in kinetin 1.0 mg/L/ indolebutyric acid 0.5 mg/L and rooted

in vitro in half strength MS media containing indolebutyric acid 0.5 mg/L alone with an

average root number of 7.4 and root length of 4.0 cm. Acclimatization of rooted

plantlets was successfully carried out with 78% survival rate using polybags. Somatic

embryogenesis was induced using zygotic embryo explants in media containing

different auxins alone or in combination of cytokinins and the highest embryogenesis

percentage (75%) and average number of embryos (23 embryos/explant) were observed

in media with napthaleneacetic acid 1.5 mg/L/ kinetin 1.5 mg/L. The somatic embryos

were converted into synthetic seeds by encapsulating with 3.0% sodium alginate;

however the germination rate of somatic embryos was reduced from 60% to 30% upon

encapsulation. Callus cultures were established from zygotic embryo explants on

different auxins alone or in combination of cytokinins. Different auxins showed

different kind of callusing or rooting response based on the concentration and

combinations with cytokinins. Napthaleneoxyacetic acid 1.5 mg/L/ benzylaminopurine

0.75 mg/L showed highest amount of callus while napthaleneacetic acid recorded

highest amount of adventitious roots. Analysis of andrographolide content in the callus

and root cultures showed the presence of andrographolide highest in rhizogenic callus

of napthaleneacetic acid 3.0 mg/L (2.4 mg/gm of dry weight) making embryo culture

feasible and realistic for andrographolide production in vitro. Callus produced on media

with napthaleneoxyacetic acid 1.5 mg/L+ benzylaminopurine 0.75 mg/L had more

antioxidant capacity with inhibitory concentration (IC50) value of 20.91 µL and total

phenolic content of 0.88 gallic acid equivalent mg/g compared to roots produced on

napthaleneacetic acid 3.0 mg/L. In vitro organogenesis in A. paniculata using tTCL and

embryo explants is reported for the first time in this study and this method can be used

as an efficient protocol for clonal propagation of A. paniculata.

xv

1

CHAPTER 1

INTRODUCTION

1.1 Background of the study

Medicinal plants are a group of plants used in the treatment of diseases

by humans in traditional and ethno medicinal systems such as Ayurvedic,

Traditional Chinese Medicine, and Unani (Sarker & Nahar, 2007). According to

the World Health Organization (WHO), a medicinal plant can be defined as the

plant as whole or any of its parts or organs that consist of potential substances

with therapeutical aspects or as precursors for synthesis of pharmaceutical

compounds. The phytochemical constituents isolated from medicinal plants are

pharmacologically effective in treating several types of diseases (Tiwari et al.,

2014). Now-a-days, interest in plant-based medicine is reviving due to the

increased realization of the health hazards associated with modern medicine and

also the promising medicinal values and less side effects from herbal medicines

(Sharma et al., 2008). The herbal industries are growing rapidly in the

international market with the current market value estimated to be between USD

40 to 100 billion with an average growth rate of 15 to 20% each year (Aziz et

al., 2004).

2

Andrographis paniculata Burm F. Ness or Hempedu Bumi in Malay is

one such potential medicinal plant vividly used in traditional systems of healing

ailments in most of the South Asia, South East Asia and East Asian countries

along with Scandinavia. In Indian and Oriental medicine A. paniculata has a

long history of therapeutic usage into various diseases such as cold, diarrhea,

fever, inflammation, jaundice, kidney diseases, microbial infections, and snake

bite (Rao, 2006; Woo et al., 2008). It is also been recognized for its therapeutic

efficacy to achieve United Nations millennium development goals (MGDs)

program in control of HIV/AIDS and malaria (Colfer et al., 2006). Several

compounds from different classes including chalcones, chalcone glycosides,

diterpenoids, dimeric diterpenes, flavones, flavone glycosides, flavonoids,

sterols and xanthones were isolated from A. paniculata (Sirisha et al., 2011).

The major secondary metabolite of A. paniculata is andrographolide and it is

responsible for various pharmacological activities of the plant including anti

cancer activity (Jayakumar et al., 2013). A. paniculata preparations are sold

commercially as a medicine in China, India, Malaysia and Thailand. In China

preparations containing andrographolide are sold as over-the-counter (OTC)

(Qiu et al., 2012). A. paniculata is sold for as much as USD 5/kg for quality dry

leaves, while purified andrographolide is sold around USD 100,000/kg

(Satayavivad et al., 2002). The latest pricing by Sigma- Aldrich Corporation in

2013 for the 100 and 500mg packages of andrographolide 98% is USD 36.20

and USD 135.00 respectively. The heavy demand of A. paniculata in

international markets has motivated farmers in India and other countries to start

3

cultivation of this plant (Kataky & Handique, 2010). Priority of A. paniculata is

also recognised by herbal industries of developing countries such as Malaysia

(Rahman, 2012), Thailand (Chuthaputti & Chawapradit, 2008) and Nigeria

(Fasola et al., 2010). It is included in priority herbs under the entry point project

(EPP) in National Key Economic Areas (NKEA) to develop herbal industry in

Malaysia (Rahman, 2012). Field studies on cultivation of the plant are carried by

Malaysian Agricultural Research and Development Institute (MARDI) and

Forest Research Institute of Malaysia (FRIM) (Shukri et al., 2005).

1.2 Problem statement

The major problem encountered in profitable exploitation of A.

paniculata is due to its limited availability. Herbal industries are using wildly

available A. paniculata population to meet the demand of the plant products and

plant biochemicals like andrographolide (Karuppusamy & Kalimuthu, 2010).

The commercial exploitation of the wild plant is now under constrain due to

over-collection, limited availability (Kanjilal et al., 2002) and resulting in a

critical loss of genetic diversity (Jarukamjorn & Nemoto, 2008). In

Devarayanadurga forest of India, A. paniculata has become uncommon due to

various types of human activities (Kamalappa & Ramakrishnappa, 2003) while it

is added to the list of the vulnerable species in Sri Lanka (IUCN, 2007).

The commercialization of A. paniculata culture is largely hampered due

to its limitations of conventional propagation using seeds due to problems such

4

as difficulties in seed procurement (Ghoah, 2004), dormancy, poor germination

rate, scanty and delayed rooting of seedlings (Purkayastha et al., 2008) and

deaths of many young seedlings. While vegetative propagation by stem cuttings

is difficult and considerably slow to meet the large requirement of the plants

(Alagesaboopathi & Ramachandran, 2000; Martin, 2004a).

1.3 Justification of the study

At present, there is extensive research focusing on cultivation of A.

paniculata as the main crop or secondary crop in the existing crops which boosts

the farming as well as herbal industry in Malaysia. So a complete morphogenic

study of different parts of the plant will result in development of an efficient

protocol for micropropagation of the A. paniculata which can be used in clonal

propagation to suffice the agricultural demand of the plant. Synthetic seed

technology was studied in an effort to reduce the long field transplantation time

required by micropropagated plants generally.

The yield of andrographolide is mere 1.0-1.5% from whole plants and for

pharmaceutical industries that depend on andrographolide and other derivatives

of andrographolide. It is troublesome to collect the crude drug from farmers

because of adulteration contamination and not up to the standards, which results

in loss to the companies or high costs of the drug price. In callus culture

technique the industry has a continuous supply and good quality of

andrographolide which results in decrease of the cost of drug production. A new

5

protocol for in vitro production of andrographolide through callus culture will be

developed which might be useful for improved production.

1.4 Objectives of the present study

The objectives of the present study were

To evaluate shooting potential of different explants from A. paniculata

for developing an efficient protocol for micropropagation.

To induce somatic embryos and preparation of synthetic seeds.

To induce callus from embryos to estimate andrographolide content and

antioxidant activity of callus.

1.5 Scope of the present study

The new protocols developed from this study may lead to enhance the

mass propagation of A. paniculata. New protocol for in vitro production of

andrographolide will be developed.

6

CHAPTER 2

LITERATURE REVIEW

2.1 Andrographis paniculata plant and its properties

2.1.1 General information

Andrographis paniculata (Burm. F) Nees (Syn.: Justicia latebrosa Ross.,

J. paniculata Burm F., J. stoicatalam. Ex Stevd.) (Hooker, 1885) belongs to

genus Andrographis family Acanthaceae. The genus consists of 44 plants

distributed in the Tropical Asia, Africa, Brazil and Central America Northward

into Mexico (Ganapaty et al., 2012). Most of them are used medicinally in

various traditional systems of medicine such as A. paniculata, A. alata, A.

echioides, A. lineata and A. serpillifolia. The taxonomic classification of A.

paniculata is depicted below:

Kingdom : Plantae

Subkingdom : Tracheobionta

Superdivision : Spermatophyta

Division : Angiosperma

Class : Dicotyledonae

Sub-class : Gamopetalae

Series : Bicarpellatae

Order : Personales

7

Tribe : Justicieae

Family : Acanthaceae

Genus : Andrographis

Species : A. paniculata (Burm. f) Nees

A. paniculata is annual or perennial erect herb grows to a height of 30-

110 cm in moist and shady places. The stem is dark green in colour, tetrangular

in cross-section with longitudinal furrows and a fibrous or adventitious tap root

system. Leaves are lanceolate, opposite decussate, entire, acute apex, measuring

6.0-10.0 cm length, 3.5-5.0 cm wide green to bottle green in colour (Kumar et

al., 2012a). The inflorescence is spreading racemes with small flowers consist of

small linear calyx, 6mm long corolla divided into upper lip and lower lip. Upper

lip is 3-lobed, oblong, hairy, and rose-purple spotted and the lower lip is 2-lobed,

connate and violet spotted (Fig. 2.1). The reproductive apparatus consists of two

hairy filamentous stamens with dark purple anther and 1 slender ovary. The fruit

is an oblong, longitudinally compressed capsule around 2 centimetres long and a

few millimetres wide, brown in colour when matured and contains yellow brown

seeds (Kumar et al., 2012a).

It is distributed often as isolated patches over a broad range of habitats

like farms, wastelands, dry or wet lands, plains, hill slopes, sea shores along the

road sides in tropical countries. It is a native of India and Sri Lanka

(Balachandran & Govindarajan, 2005) and introduced to Brunei, Hong Kong,

Iran, Indonesia, Malaysia, Thailand, West Indies and in the tropical areas of the

Americas (Correll & Correll, 1982; Hooker, 1885; Valdiani et al., 2012).

8

Currently it is found throughout South and South East Asia including China,

Taiwan, Korea and Japan. In India, A. paniculata is known as “Kalmegh”; in

China as “Chuan-Xin-Lian”; in Japan as “Senshinren”; Malaysia as “Hempedu

bumi”; in Thailand as “Fah Tha Lai”; and in Scandinavian countries as “green

chiretta” (Arifullah et al., 2013).

Figure 2.1: Andrograhis paniculata plant, a) Andrograhis paniculata in vegetative stage

and flowering stage with flower enlarged and b) fruiting stage, Bar=4cm

9

2.1.2 Traditional uses and pharmacological activities

A. paniculata has been used in traditional Asian medicines like Indian

and Oriental medicine for centuries (Rao, 2006; Woo et al., 2008). The herb is

official in Indian Pharmacopoeia (Okhuarobo et al., 2014), Chinese

Pharmacopoeia (Tang, 1992) and in the National list of Essential Drugs A. D.

1999 (List of Herbal Medicinal Products) of Thailand (Pholphana et al., 2004).

Traditionally it is used as anti-inflammatory, antileprotic, antipyretic, blood

purifier, liver stimulant, laxative, and preventive major for malaria, in India (Dey

et al., 2013). In traditional Chinese medicine, A. paniculata is a ‘cold property’

herb and used in treatment of hot conditions such as acute infections including

cough with thick sputum, carbuncle, dysentery, pneumonia, fever, throat

infection, gastroenteritis, pyelonephritis, sores, and snake bites (Chang & But,

1987). In Malaysia, it is extensively used in traditional medicine against

dysentery, diarrhoea, diabetes, cardiovascular diseases and hypertension fever,

inflammation and sore throat (Arifullah et al., 2014). In Thailand the leaves are

used extensively in folklore medicine for the treatment of various diseases such

as the bowels, undiagnosed fever, cholagogues and antihelminthic (Siripong et

al., 1992). In Indonesia this plant is traditionally used for several purposes, like

treating malaria and preventing diabetes mellitus (Susantiningsih et al., 2012). In

Philippines, it is used to cure a number of diseases, such as rheumatism,

hypertension, asthma, diarrhoea, diabetes and other common diseases (Nacpil &