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 &
