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11. IS 1642-1989, ‘Indian standard code of practice for fire safety of rated 70.27% polymorphism. The number of ampli-
buildings (general): details of construction (first revision). Bureau fied products ranged from 2 to 7 for different primers,
of Indian Standards, New Delhi. whereas the percentage of genetic similarity for the
12. IS 10262-1982, Recommended guidelines for concrete mix design. studied primers ranged from 51.4 to 97.0. There were
Bureau of Indian Standards, New Delhi.
two major clusters formed in the genotypes studied.
13. Phan, L. T., Fire performance of high strength concrete: A report
of the state of the art. National Institute of Standards and Techno-
The andrographolide content ranged from 0.69 to
logy, NISTIR 5934, December 1996. 1.85% and the genotype KI-2 had the highest esti-
14. Potha Raju, Shobha, Rambabu. Flexural Strength of fly ash con- mated content. The study demonstrated that simulta-
crete under elevated temperatures. Mag. Concr. Res., 2004, 56, neously morphological, molecular and biochemical
83–88. analysis are useful for characterizing genetic diversity
15. Potha Raju and Janaki Rao, Effect of temperature on residual and defining relationships between kalmegh germ-
compressive strength of fly ash concrete. Indian Concr. J., 2001, plasm. It also gave possible indications to the phyto-
75, 347–352. chemical variation of different genotypes which were
16. Xudong Shi, Teng-Hooi Tan, Kang-Hai Tan and Zhenhai Guo, In- due to the genetic differences.
fluence of concrete cover on fire resistance of reinforced concrete
flexural members. J. Struct. Eng, ASCE, 2004, pp. 1225–1232.
Keywords: Andrographis paniculata, andrographolide
content, genetic diversity, germplasm, RAPD.
ACKNOWLEDGEMENTS. We are grateful to the Department of
Science and Technology, Government of India for funding this research
work. We also thank the Management and Dr V. V. Kutumbarao, Prin- ANDROGRAPHIS PANICULATA (Kalmegh) is an erect grow-
cipal, College of Engineering, GITAM, Visakhapatnam for permission ing annual medicinal herb and well known for its multiple
to conduct various tests in the Concrete Laboratory, Department of health-promoting properties. It is found throughout India
Civil Engineering, GITAM. and other Asian countries namely China, Java, Thailand,
etc. It was credited a wonder drug in 1919 for arresting
Received 26 March 2007; revised accepted 18 December 2008 the spread of the contagious disease ‘global flu’ epidemic.
Kalmegh is also reported to possess antihepatotoxic1, anti-
biotic2, antimalaria3, antihepatitic4, anti-inflammatory5
and anti-snakevenom6 properties to mention a few, besides
its general use as an immune stimulant agent7. It is used
as a wonder drug in traditional Siddha and Ayurvedic
Assessment of intra-specific variability systems of medicine as well as in tribal medicine in India
at morphological, molecular and and several Asian countries for multiple clinical applica-
tions.
biochemical level of Andrographis A recent study conducted at Bastyr University, USA
paniculata (Kalmegh) confirms anti-HIV activity of andrographolide8. It is
widely distributed and exploited as medicinal plant in
almost all regions of India (Himachal Pradesh, Andhra
Shiv Narayan Sharma1,4,
Pradesh, Chhattisgarh, Madhya Pradesh, etc.). It is placed
Rakesh Kumar Sinha2,3,4,*, D. K. Sharma4 and at 17th position among the 32 prioritized medicinal plants
Zenu Jha4 of India with a demand of 2,197.3 tonnes (2005–06) and
1
Division of Genetics, Indian Agricultural Research Institute, annual growth of 3.1%9. Several active components have
New Delhi 110 012, India
2 been identified; two of them are andrographolide and
National Research Centre on Plant Biotechnology,
Indian Agricultural Research Institute, New Delhi 110 012, India neoandrographolide. Andrographolide (the diterpenoid
3
Laboratory of Biophysics, Department of Experimental Physics, lactones) is the main bitter principle found in high con-
Faculty of Science, Palacký University, Olomouc, Czech Republic centrations in the leaves of kalmegh10. Andrographolides
4
Department of Biotechnology, Indira Gandhi Krishi are extracted for their use as drug. The aim of the present
Vishvavidyalaya, Raipur 492 006, India
study was to find out whether phenotypic variations in
In the present investigation, 15 Andrographis panicu- the genotypes grown in different locations are merely
lata genotypes collected from Chhattisgarh and epigenetic or genetic. Furthermore, if the same are gene-
adjoining states were grown in the field for the vari- tically different, then their medicinally active principles
ability studies. Three different variability parameters have to be estimated to find out possible quantitative
namely morphological, molecular and chemotyping of variation, which accord for the variation in medicinal acti-
active ingredient content were employed. Wide varia- vity of the plants collected from different locations. In
tions were recorded with regard to quantitative char- general, genetic diversity can be measured at any func-
acters and RAPD profile. Ten polymorphic RAPD tional level from blueprint (DNA) to phenotype11. In this
primers produced a total of 37 amplicons, which gene- study, possible genetic variation among A. paniculata
genotypes collected from different locations was analysed
*For correspondence. (e-mail: sinharakesh_ag@rediffmail.com) using RAPD (random amplified polymorphic DNA) to
402 CURRENT SCIENCE, VOL. 96, NO. 3, 10 FEBRUARY 2009
RESEARCH COMMUNICATIONS
Table 1. Collection of Andrographis paniculata genotypes taken for the study
Table 2. RAPD markers used for amplification lier12. The quality and quantity of DNA was analysed by
Primer Primer nanodrop and agarose (0.8%) gel electrophoresis. Ex-
tracted DNA was used in subsequent PCR amplifications,
OP A01 OP ACO6 which were performed in a programmable thermocycler
OP A02 OP AC10
(PTC 100, MJ Research Pvt Ltd, USA). Each sample
OP A03 OP AC13
OP A06 OP AG01 (20 μl) was amplified in a reaction mixture containing
OP A08 OP AG03 4 μl (80 ng) of genomic DNA, 1 μl of 1.0 unit of Taq
OP A10 OP AG08 polymerase (Promega Co, USA), 2 μl of 15 pmoles of 10-
OP A11 OP AG09 mer RAPD primer (Operon Technologies Inc, USA), 2 μl
OP A13 OP AG19
of PCR buffer (500 mM KCl, 100 mM Tris-HCl, 1.0%
OP A17 OP AH19
OP AA09 OP AH20 Triton X-100 and 15 mM MgCl2) and 2 μl (100 μm)
OP AA12 OP AI05 dNTP mixture (Promega, USA). The cycling parameters
OP AC01 OP AI09 were 3 min at 94°C for pre-denaturation, 40 cycles each
OP AC04 OP AI14 of 1 min for denaturation, 1 min at 30°C for annealing,
OP AC05
2 min at 72°C for extension and a final extension at 72°C
for 7 min. The mixture was cooled to 4°C and stored at
discriminate molecular variability. Biochemical varia- –20°C until electrophoresis. The 10 μl amplified products
tions were estimated through quantifying andrographolide was separated on 1.5% agarose gel (Pronadisa) in 1×-
contents using HPLC (high performance liquid chroma- TAE buffer. The gels were run for 4 h at 90 V and stained
tography). with ethidium bromide and photographed under UV illu-
The performance of A. paniculata genotypes was mination. Specific amplified products were scored as pre-
evaluated through field experimentation at the Depart- sent (1)/absent (0) depending on decreasing order of their
ment of Biotechnology, Indira Gandhi Agricultural Uni- molecular weights for each DNA sample. Genotypes were
versity, Raipur. Seeds of 15 genotypes were collected grouped by the Dice correlation coefficient and cluster
from different farmer’s fields, government nurseries and analysis by the unweighted pair group method with
forests of Chhattisgarh and adjoining regions (Table 1). arithmetic averages (UPGMA) and phylogenetic tree that
The seeds were sown in small plots in randomized com- summarizes the evolutionary relationship constructed by
plete block design (RBD) in the field. Observations were TREECON software package13. Reproducibility of RAPD
recorded on five randomly selected plants in each geno- banding profiles was determined from duplicate loadings
type. Morphological parameters included seven quantita- of independent, RAPD reaction mixtures and cluster
tive (plant height, number of branches, number of leaves, analysis then performed. To test the discriminatory abi-
leaf length, leaf width, dry weight of whole plant and lity of the primers, 27 random primers (Table 2) were ini-
seed yield per plant) and three qualitative (leaf colour, tially checked, of which 10 primers were polymorphic
flower colour and days required for flowering) characters and were then used for the dominant RAPD grouping and
for exploring the phenotypic diversity. RAPD analysis cluster analysis.
was used for detecting genetic variation and relatedness Fully grown plants of each genotype were screened for
among different genotypes. the active principle (andrographolide) contents. The metha-
Total genomic DNA from the young leaves of the nolic extract of the shade-dried leaves was analysed by
plants was isolated by using the method described ear- HPLC at 223 nm to estimate its concentration following
CURRENT SCIENCE, VOL. 96, NO. 3, 10 FEBRUARY 2009 403
RESEARCH COMMUNICATIONS
Figure 1. Retention time and calibration curve of standard andrographolid (Sigma grade).
the standard protocol14. Leaves at just before flowering of though raised under uniform environmental and cultural
the plants were collected and dried in hot air oven (45°C) conditions. The plant height ranged from 21.44 (AP-5) to
for 4 days followed by grinding. The fine leaf powder 58.67 cm (KI-2) with a mean height of 39.89 cm; number
samples were then used for extracting the androgra- of branches ranged from 18.44 (AP-2) to 33.44 (AP-6)
pholide. HPLC condition was optimized using a ODS C- with a mean value of 22.72, number of leaves ranged
18 column, mobile phase acetonitril : methanol : water from 83.11 (AP-5) to 149.78 (AP-6) with an average of
(65 : 20 : 15), flow rate 1 ml per min, column pressure 111.68, leaf breath ranged from 1.07 (AP-6) to 2.96 cm
88.7 bar and total run time of 10 min at 223 nm. The (KI-2) with a mean value of 2.18, leaf length ranged from
standard andrographolide (Sigma, USA) (22.3 mg) was 5.88 (AP-9) to 7.28 cm (AP-5) with a mean of 6.54, dry
dissolved in 100 ml methanol in which 20 μl of standard weight of plant ranged from 30 (AP-5) to 50 g (KI-2)
solution was injected into the HPLC column for making with a mean value 40.53, seed yield ranged from 2.67
calibration curve (Figure 1). Standard andrographolide (AP-2) to 4.27 (KI-2) with an average of 3.63. All the
took 2.690 min as retention time and peak area corres- quantitative characters were significant at 5% level of
ponded to an amount of 0.222 mg/ml, i.e. 22.2 mg in probability except leaf length. The quantitative data indi-
100 ml (Figure 1). Two gram test sample of dried powder cated that genotype KI-2 gave the best performance. It
was diluted 1000 times in HPLC solvent and 20 μl of it had the maximum plant height (58.67 cm), leaf width
was injected in three replicates. The retention time and (2.96 cm), dry weight of whole plant (50.0 g) and seed
peak area were recorded for calculating the total andro- weight (4.27 g). The qualitative data revealed not much
grapholide content in each genotype to compute an aver- variation in flower colour among the genotypes, but leaf
age content. colour in KI-2 was comparatively distinct (light green)
Out of 15 genotypes collected from Chhattisgarh and compared to others (dark green). The days required for
adjoining states, only 10 (AP-1, AP-2, AP-3, AP-4, AP-5, flowering range from 82 (AP-6) to 158 (KI-2) with mean
AP-6, AP-7, AP-8, AP-9 and KI-2) could be established of 118 days. The genotype KI-2 took maximum number
in field for further studies. In the later stages, KI-2 super- of days (158) for flowering (Figure 3) but showed the
seded all the genotypes. A spectrum of morphological relatively best performance when compared for different
variations was noticed among the genotypes (Figure 2) quantitative characters and growth pattern. The data of
Figure 2. Morphological characterization (quantitative traits) of the Kalmegh genotypes. a, Plant height; b, Number of
leaves; c, Number of branches; d, Leaf width; e, Dry weight of whole plant; f, Seed yield.
Primer code Total no. of bands No. of amplified band(s) Polymorphism (%)
OP AG-3 3 1 33.3
OP A-11 3 2 66.6
OP A-8 8 7 87.5
OP AC-10 5 4 80.0
OP AI-5 2 2 100.0
OP AA-9 3 3 100.0
OP AG-19 4 3 75.0
OP A-10 3 2 66.6
OP AG-1 3 1 33.3
OP AI-9 3 1 33.3
Total number of bands 37 26 70.2
Mean per primer 3.7 2.6
Genotypes AP1 AP2 AP3 AP4 AP5 AP6 AP7 AP8 AP9 KI2
AP1 1.00
AP2 0.62 1.00
AP3 0.62 0.89 1.00
AP4 0.54 0.92 0.86 1.00
AP5 0.59 0.86 0.86 0.84 1.00
AP6 0.56 0.89 0.90 0.97 0.81 1.00
AP7 0.78 0.73 0.73 0.65 0.70 0.68 1.00
AP8 0.59 0.86 0.92 0.95 0.78 0.97 0.70 1.00
AP9 0.51 0.73 0.73 0.81 0.65 0.78 0.68 0.81 1.00
KI2 0.78 0.84 0.73 0.76 0.81 0.73 0.84 0.70 0.68 1.00