See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/264001885

Production of andrographolide from adventitious root cultures of

Andrographis paniculata

Article in Current Science · January 2009

CITATIONS READS

7 455

1 author:

Poornananda Madhava Naik

Kanara E-Vision PU Science and Commerce College, Chalageri
47 PUBLICATIONS 1,096 CITATIONS

SEE PROFILE

All content following this page was uploaded by Poornananda Madhava Naik on 17 May 2015.

The user has requested enhancement of the downloaded file.

RESEARCH COMMUNICATIONS
19. Bonfini, L., Petra, H., Kay, S. and Van den Eede, G., Review of
GMO detection and quantification techniques. EUR 20384 EN,
2002.
20. Twell, D., Yamaguchi, J. and McCormick, S., Pollen-specific gene
expression in transgenic plants: coordinate regulation of two
different tomato gene promoters during microsporogenesis. Deve-
lopment, 1990, 109, 705–713.
21. Yang, L. et al., Validation of a tomato-specific gene, LAT52, used
as an endogenous reference gene in qualitative and real-time quan-
titative PCR detection of transgenic tomatoes. J. Agric. Food
Chem., 2005, 53, 183–190.
22. Chamberlain, J. S., Gibbs, R. A., Ranier, J. E., Nguyen, P. N. and
Caskey, C. T., Deletion screening of the Duchenne muscular dys-
trophy locus via multiplex DNA amplification. Nucleic Acids Res.,
1988, 16, 11141–11156.
23. Burns, M., Shanahan, D., Valdivia, H. and Harris N., Quantitative
event-specific multiplex PCR detection of Roundup Ready soya
using LabChip technology. Eur. Food Res. Technol., 2003, 216,
428–433.
24. Dainese, E., Angelucci, C., de Santis, P., Maccarrone, M. and
Cozzani, I., A multiplex PCR-based assay for the detection of
genetically modified soybean. Anal. Lett., 2004, 37, 1139–1150.
25. Germini, A., Zanetti, A., Salati, C., Rossi, S., Forré, C., Schmid,
S. and Marchelli, R., Development of a seven-target multiplex
PCR for the simultaneous detection of transgenic soybean and
maize in feeds and foods. J. Agric. Food Chem., 2004, 52, 3275–
3280.
26. Forte, V. T., Pinto, A. D., Martino, C., Tantillo, G. M., Grasso, G.
and Schena, F. P., A general multiplex-PCR assay for the general
detection of genetically modified soya and maize. Food Control,
2005, 16, 535–539.
27. Delano, J., Anna-Mary, S., Erika, W., Margaret, G. and Saad, M.,
Reliable detection and identification of genetically modified
maize, soybean, and canola by multiplex PCR analysis. J. Agric.
Food Chem., 2003, 51, 5829–5834.
28. Wall, E., Lawrence, T., Green, M. and Rott, M., Detection and
identification of transgenic virus resistant papaya and squash by
multiplex PCR. Eur. Food Res. Technol., 2004, 219, 90–96.
29. Yang, L. et al., Qualitative and quantitative PCR methods for
event-specific detection of genetically modified cotton Mon1445
and Mon531. Transgenic Res., 2005, 14, 817–831.
30. Saghai-Maroof, M. A., Soliman, K. M., Jorgensen, R. A. and
Allard, R. W., Ribosomal DNA spacer length polymorphism in
barley, Mendelian inheritance, chromosomal location and popula-
tion dynamics. Proc. Natl. Acad. Sci. USA, 1984, 81, 8014–8019.
31. Lipp, M., Brodmann, P., Pietsch, K., Pauwels, J. and Anklam, E.,
IUPAC collaborative trial study of a method to detect genetically
modified soybeans and maize in dried powder. J. AOAC Int.,
1999, 82, 923–928.
32. Matsuoka, T. et al., A multiplex PCR method of detecting recom-
binant DNAs from five lines of genetically modified maize. J.
Food Hyg. Soc. Jpn., 2001, 42, 24–32.
33. Yang, L., Shen, H., Pan, A., Chen, J., Huang, C. and Zhang, D.,
Screening and construct-specific detection methods of transgenic
Huafan No 1 tomato by conventional and real-time PCR. J. Sci.
Food Agric., 2005, 85, 2159–2166.
ACKNOWLEDGEMENTS. We thank Dr K. C. Bansal, NRCPB, New
Delhi for providing seeds of transgenic tomato lines, viz. 528 and 564
with osmotin gene. We also thank Dr S. K. Sharma, Director, NBPGR,
New Delhi for providing the necessary facilities. M.S., R.C. and R.S.
duly acknowledge the assistance provided under the GEF–World Bank-
aided Capacity Building Project for the Implementation of Cartagena
Protocol on Biosafety.
Received 3 April 2008; revised accepted 13 January 2009
694
Production of andrographolide
from adventitious root cultures of
Andrographis paniculata
N. Praveen1, S. H. Manohar1, P. M. Naik1,
A. Nayeem1, J. H. Jeong2 and H. N. Murthy1,*
1
Department of Botany, Karnatak University, Dharwad 580 003, India
2
Department of Medicinal Resources and Horticulture,
Namdo Provintial College, Jeonnan, Changhung 529 850, South Korea
Adventitious roots were induced directly from leaf seg-
ments of Andrographis paniculata on Murashige and
Skoog (MS) medium with 5.3 μM α-naphthaleneacetic
acid (NAA) and 30 g/l sucrose. Adventitious roots cul-
tured in flasks using MS liquid medium with 2.7 μM
NAA and 30 g/l sucrose showed higher accumulation
of biomass (fresh and dry weight) and andrographolide
within four weeks. Seven-fold increment of fresh bio-
mass was evident in suspension cultures along with
3.5-fold higher andrographolide compared to natural
plants. These results showed a great potentiality of
adventitious root cultures for the production of andro-
grapholide.
Keywords: Adventitious roots, Andrographis pani-
culata, Andrographolide, suspension cultures.
ANDROGRAPHIS PANICULATA Nees (Acanthaceae), com-
monly known as ‘Kalmegh’, has been widely used in
India, Thailand, China and Malaysia for the treatment of
hepatitis1,2. The plant is reported to possess protective
activity against various liver disorders. The primary
medicinal constituents of A. paniculata are andrographolide
and related compounds which are diterpenoids showing
antipyretic, antimalarial, anti-inflammatory, immunostimu-
latory and anticancerous activities3–5.
Plant cell and organ cultures are promising technolo-
gies to obtain plant-specific valuable metabolites6. Cell
and organ cultures have a higher rate of metabolism than
field grown plants because the initiation of cell and organ
growth in culture leads to fast proliferation of cells/organs
and to a condensed biosynthetic cycle7. Further, plant
cell/organ cultures are not limited by environmental, eco-
logical and climatic conditions and cells/organs can thus
proliferate at higher growth rates than the whole plant in
cultivation8. Callus cultures of A. paniculata have been
reported9. However, no andrographolides were detected
in the cultures. In the present study, induction and culture
of adventitious roots of A. paniculata were conducted and
production of andrographolide in adventitious root cultures
was investigated.
Young leaves were collected from field-grown plants
of A. paniculata Nees and were washed thoroughly in
*For correspondence. (e-mail: nmurthy60@yahoo.co.in)
CURRENT SCIENCE, VOL. 96, NO. 5, 10 MARCH 2009

Figure 1. Induction and proliferation of adventitious roots from leaf explants of Andrographis paniculata. a, b, Initia-
tion of adventitious roots from leaf explants (on MS medium supplemented with 5.3 μM NAA). c, d, Proliferation of
adventitious roots in suspension culture.
running tap water, surface sterilized in 0.1% (w/v) mercuric
chloride for 10 min and washed three times in sterile
de-ionized water. The sterilized explants were cut into
5 × 5 mm square segments and cultured onto Murashige
and Skoog10 (MS) agar medium (0.2% w/v gelrite,
Duchefa, The Netherlands) supplemented with 30 g/l
(w/v) sucrose and indoleacetic acid (IAA; 0.5, 2.8, 5.7,
11.4 and 28.5 μM), indole butyric acid (IBA; 0.5, 2.4,
4.9, 9.8 and 24.6 μM) and α-naphthalene acetic acid (NAA;
0.5, 2.7, 5.3, 10.7 and 25.8 μM). The pH of the medium
was adjusted to 5.8 ± 0.2 before autoclaving (121°C for
15 min). The cultures were incubated in the dark at
25 ± 1°C.
Liquid cultures were established by inoculating 0.5 g
(fresh weight, FW) adventitious roots into 250 ml Erlen-
meyer flasks containing 50 ml liquid MS medium sup-
plemented with 2.7 μM NAA and 30 g/l sucrose (w/v).
The flask cultures were under continuous agitation at
100 rpm on orbital shaker at 25 ± 1°C in the dark. The
growth of adventitious roots was recorded at weekly
intervals. Roots were separated from the medium by pass-
ing through a 1 mm stainless steel sieve. Root FW was
measured after rinsing once with sterile water and blot-
ting away surface water, and root dry weight (DW) was
recorded after the roots were dried to a constant weight at
70°C for 2 days.
CURRENT SCIENCE, VOL. 96, NO. 5, 10 MARCH 2009
RESEARCH COMMUNICATIONS
Extraction and HPLC analysis of andrographolide was
done following the method of Jain et al.11. The dried,
powdered root samples (500 mg) were extracted in a mix-
ture of dichloromethane and methanol (1 : 1) by cold
maceration. The extract was filtered and solvent removed
under vacuum. The extract was washed 2–3 times with
toluene and then dissolved in methanol. The androgra-
pholide fractions were analysed using HPLC (Waters)
with XTerra RP18 column (150 mm × 3 mm, 5 μm). The
mobile phase was acetonitrile : water (70 : 30, v/v), flow
rate 1 ml/min, column temperature 26°C, and detector
wavelength 230 nm. Andrographolide standard was obtai-
ned from ChromaDex (Laguna Hills, CA, USA).
Leaf explants developed protuberances in two weeks
from the cut ends (Figure 1 a). These protuberances
developed into adventitious roots directly without callus
induction phase in another two weeks (Figure 1 b). NAA
was the only phytohormone responsible for the induction
of adventitious roots from the leaf explants, among the
three phytohormones tested (Table 1). MS medium sup-
plemented with 5.3 μM NAA induced maximum number
of roots (14.0 roots per explant).
Explants cultured in vitro may be involved in organo-
genesis and develop shoots or roots depending on the
morphogenetic potentiality of the cells. There are three
distinct stages during organogenesis, namely dedifferen-
695
RESEARCH COMMUNICATIONS
Table 1. Effect of various plant growth regulators on adventitious root induction from leaf
explants of Andrographis paniculataa

Concentration Percentage of responding Mean number of

Phytohormone (μM) explants roots per explantb

NAA 0.5 0 0
2.7 41.6 4.8 ± 1.4
5.3 91.6 14.0 ± 3.6
10.7 75.0 12.3 ± 4.9
26.8 58.3 7.1 ± 1.7
IAA 0.5 0 0
2.8 0 0
5.7 0 0
11.4 0 0
28.5 0 0
IBA 0.5 0 0
2.4 0 0
4.9 0 0
9.8 0 0
24.6 0 0
a
Explants were cultured on MS medium for four weeks.
b
Mean values with standard error of 12 replicate of three independent experiments.

Figure 2. a, Time profile of root growth (500 mg of inoculum was cultured in 50 ml of medium). b, Kinetics of produc-
tion of andrographolide in flask-scale cultures of adventitious roots of Andrographis paniculata (500 mg of inoculum was
cultured in 50 ml of medium).

tiation, induction of organogenesis pathway and devel-
opment of organs12. Specific phytohormones supplemented
exogenously can trigger the process of differentiation and
induction pathways. For example 2,4-dichlorophenoxy-
acetic acid (2,4-D) initiated callus formation and IBA was
responsible for adventitious root development from the
callus in Panax notoginseng13. However, in the current
experiments NAA is more potent in triggering induction
of adventitious roots from leaf explants of A. paniculata.
Time profile of growth of adventitious roots in suspen-
sion culture is depicted in Figure 2 a. The fresh and dry
biomass of adventitious roots increased slowly and
reached a maximum of 70.73 g/l FW and 7.615 g/l DW
respectively, at the end of 5 weeks. A seven-fold increase
was evident when compared to the initial inoculum fresh
biomass (Figure 1 c and d). These results suggest that ad-
ventitious root cultures of A. paniculata are promising for
large-scale biomass production in suspension cultures.
Similarly, adventitious root suspension cultures have
been proved to be efficient for biomass accumulation in
P. notoginseng13 and Echinacea purpurea14.
The HPLC profile of andrographolide extracted from
the adventitious roots is depicted in Figure 2 b. The results
suggest that andrographolide content increased gradually
during the course of culture and its concentration was opti-
mum after 4 weeks of culture (72.86 mg/g DW), with fur-
ther decrease in its level. Thus, cultivation of adventitious
roots for 4 weeks was found optimal for both biomass
accumulation and production of andrographolide.
HPLC analysis of andrographolide from natural plants
(whole plant extract) was 20.68 mg/g DW, while the andro-
grapholide content in the adventitious roots was
72.86 mg/g DW. Thus 3.5-fold higher andrographolide
concentration is evident in the adventitious roots. Similar
to the present observation higher accumulation of bioac-
tive compounds in the adventitious roots of Echinacea
purpurea has been reported when compared to natural
plants14.

696 CURRENT SCIENCE, VOL. 96, NO. 5, 10 MARCH 2009


In conclusion, in the present study we have success-
fully induced adventitious roots from the leaf explants of
A. paniculata. The adventitious roots were cultured in flask-
scale suspension cultures using MS medium supplemented
with 2.7 μM NAA and 30 g/l sucrose. Adventitious root
cultures showed higher biomass as well as andrographolide
accumulation capabilities. Our study demonstrates the
possibilities of production of andrographolides for com-
mercial purposes in a large scale using bioreactor cultures.
1. Sharma, A., Singh, R. T., Sehgal, V. and Handa, S. S., Antihepato-
toxic activity of some plants used in herbal formulation.
Fitoterapia, 1991, 62, 131–138.
2. Tang, W. and Eisenbrand, G., Chinese Drugs of Plant Origin,
Springer-Verlag, Berlin, 1992, pp. 97–103.
3. Mishra, P., Pal, N. L., Guru, P. Y., Katiyar, J. C. and Srivastava
Tandon, J. S., Antimalarial activity of Andrographis paniculata
(Kalmegh) against Plasmodium berghei NK 65 in Mastomys
natalensis. Int. J. Pharmacogn., 1992, 30, 263–274.
4. Saxena, S., Jain, D. C., Bhakuni, R. A. and Sharma, R. P., Chem-
istry and pharmacology of Andrographis species. Indian Drugs,
1998, 35, 458–467.
5. Kumar, R. A., Sridevi, K., Kumar, N. V., Nanduri, S. and
Rajagopal, S., Anticancer and immunostimulatory compounds
from Andrographis paniculata. J. Ethnopharmacol., 2004, 92,
291–295.
6. Verpoorte, R., van der Heijden, R., ten Hoopen, H. J. G. and
Memelink, J., Metabolic engineering of plant secondary metabo-
lite pathways for the production of fine chemicals. Biotechnol.
Lett., 1999, 21, 467–479.
7. Dornenburg, H. and Knorr, D., Strategies for improvement of sec-
ondary metabolite production in plant cell cultures. Enzyme Mi-
crob. Technol., 1995, 17, 674–684.
8. Rao, S. R. and Ravishankar, K. A., Plant cell cultures: chemical
factories of secondary metabolites. Biotechnol. Adv., 2002, 20,
101–153.
9. Butcher, D. N. and Connolly, J. D., An investigation of factors
which influence the production of abnormal terpenoids by callus
cultures of Andrographis paniculata Nees. J. Exp. Bot., 1971, 22,
315–322.
10. Murashige, T. and Skoog, F., A revised medium for rapid growth
and bioassays with tobacco tissue cultures. Physiol. Plant., 1962,
15, 474–497.
11. Jain, D. C., Gupta, M. M. and Saxena, S. K., LC analysis of hepa-
toprotective diterpenoids from Andrographis paniculata. J.
Pharm. Biomed. Anal., 2000, 22, 705–709.
12. Klerk, G. J. D., Arnholdt-Schmitt, B., Lieberei, R. and Neumnann,
K. H., Regeneration of roots, shoots and embryos: physiological,
biochemical and molecular aspects. Biol. Plant., 1997, 39, 53–66.
13. Gao, X., Zhu, C., Jia, W., Gao, W., Qiu, M., Zhang, Y. and Xiao,
P., Induction and characterization of adventitious roots directly
from leaf explants of Panax notoginseng. Biotechnol. Lett., 2005,
27, 1771–1775.
14. Wu, C. H., Murthy, H. N., Hahn, E. J. and Paek, K. Y., Large-
scale cultivation of adventitious roots of Echinacea purpurea in
airlift bioreactors for the production of chichoric acid, chlorogenic
acid and caftaric acid. Biotechnol. Lett., 2007, 29, 1179–1182.
ACKNOWLEDGEMENTS. This work was partially funded by the
University Grants Commission (Special Assistance Programme),
Department of Science and Technology (FIST Programme), Council of
Scientific and Industrial Research and National Medicinal Plant Board,
New Delhi, India.
Received 24 December 2007; revised accepted 23 December 2008
CURRENT SCIENCE, VOL. 96, NO. 5, 10 MARCH 2009
View publication stats
RESEARCH COMMUNICATIONS
Climatic influence on radial growth of
Pinus wallichiana in Ziro Valley,
Northeast Himalaya
Santosh K. Shah1, Amalava Bhattacharyya1,* and
Vandana Chaudhary2
1
Birbal Sahni Institute of Palaeobotany, 53, University Road,
Lucknow 226 007, India
2
Department of Science and Technology, New Mehrauli Road,
New Delhi 110 016, India
An attempt has been made here to study the climatic
influence on variation of tree-ring width (radial growth)
of Blue Pine (Pinus wallichiana A.B. Jackson) growing
in five different sites in and around Ziro Valley, Arun-
achal Pradesh, Northeast Himalaya. The site chrono-
logies have been evaluated to assess inter-site differences
through several statistical analyses, viz. correlation
matrices, principal component and hierarchical cluster
analysis. Analysis of tree growth–climate relationship
suggests that the pre-monsoon precipitation (December–
April) is a significant factor influencing the growth of
Blue Pine in all these sites.
Keywords: Blue Pine, climatic influence, radial growth,
tree ring.
BLUE Pine (Pinus wallichiana), a large evergreen conifer
tree, is found all along the Himalayas from west Kashmir
to east Arunachal Pradesh1, at altitude generally ranging
from 1800 to 3900 m. Typical habitats are mountain screes
and glacier forelands and appears as a pioneer species,
but it also forms old growth forests as a primary species
in mixed forests with deodar (Cedrus deodara), spruce
(Picea smithiana) and fir (Abies pindrow) in the temperate
belt. In some places beyond 3000 m, it reaches the tree-
line and is associated with birch (Betula utilis) and juniper
(Juniperus macropoda)2. Blue Pine growing in several
sites of the western and central Himalaya has been found
suitable for various environmental and ecological applica-
tions in tree-ring analyses3–9, but no such analyses are
available from the northeastern part of the Himalaya.
In this paper an attempt has been made to analyse tree
rings of P. wallichiana growing in and around Ziro Valley,
Lower Subansiri District, Arunachal Pradesh, Northeast
Himalaya, towards understanding its dendroclimatic poten-
tial from a new geographical region which is greatly
influenced by the southwest monsoon.
Tree-ring samples of Blue Pine were collected from
five forest sites characterized by open pine mixed broad
leaved forest in and around Ziro Valley. These sites are
Dobya (DOB), Hari (HAR), Hong (HON), Michi-Rant
(MIC) and Raga (RAG; Figure 1). All these sites are
*For correspondence. (e-mail: amalava@yahoo.com)
697