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ASIAN JOURNAL OF
SCIENCE AND TECHNOLOGY

Asian Journal of Science and Technology

Vol. 5, pp.091-094, September, 2010

RESEARCH ARTICLE
ANTIMICROBIAL ACTIVITY AND PHYTOCHEMICAL ESTIMATION OF
MICROPROPAGATED Andrographis paniculata (Burm.f) NEES
Ankita Kataky* and Handique, PJ.
Department of Biotechnology, Gauhati University, Guwahati-781 014, Assam, India
Received 8th August, 2010; Received in revised form; 24th August, 2010; Accepted 30th August, 2010; Published online 4th September, 2010

Antimicrobial activity of eight-months old micropropagated plantlets of Andrographis paniculata was evaluated with various
organic and aqueous extract against gram negative (Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa),
gram positive (Staphylococcus aureus and Bacillus subtilis) bacteria and the fungal pathogen (Candida albicans). Agar well
diffusion method was used to assess the antimicrobial activity of Andrographis paniculata. Chloroform extract showed strong
inhibitory activity with all the microbes tested. Out of the five microbial test organisms Staphylococcus aureus was the most
susceptible. The minimal inhibitory concentration (MIC) of the chloroform extract ranged from 15.625g/ml to 31.5g/ml.
Phytochemical test reveals the presence of carbohydrates, proteins, flavonoids, phenolics, saponin and alkaloids in the dried
powder.

Key words: Antimicrobial activity, Andrographis paniculata, bacteria, fungi, MIC, phytochemical test.
Copy Right, AJST, 2010 Academic Journals. All rights reserved.

INTRODUCTION
Antimicrobials of plant origin have enormous therapeutic
potential. They are effective in the treatment of infectious
diseases while simultaneously mitigating many of the side
effects that are often associated with synthetic
antimicrobials. The beneficial medicinal effects of plant
materials typically result from the combinations of
secondary metabolites such as alkaloids, steroids, tannis,
and phenol compounds, flavonoids, resins fatty acids
gums which are capable of producing definite
physiological action on body (Joshi et al., 2009).
Nowadays multiple drug resistance has developed due to
the indiscriminate use of commercial antimicrobial drugs
commonly used in the treatment of infectious disease. In
addition to this problem, antibiotics are sometimes
associated with adverse effects on the host including
hypersensitivity, immune-suppression and allergic
reactions. This situation forced scientists to search for new
antimicrobial substances. Giving the alarming incidence
of antibiotic resistance in bacteria of medical importance,
there is a constant need for new and effective therapeutic
agents. Therefore, there is a need to develop alternative
antimicrobial drugs for the treatment of infectious diseases
from medicinal plants (Agarwal et al., 1996).
A. paniculata is in demand in terms of its medicinal
properties. It has been used for centuries in Asia to treat
gastro-intestinal tract and upper respiratory infections,
fever, herpes, sore throat, and a variety of other chronic
and infectious diseases (Mishra et al., 2007).
*Corresponding Author: ankita_lon03@rediffmail.com

Mostly the leaves and roots have been traditionally used

over the centuries for medicinal purposes in Asia and
Europe as a folklore remedy for a wide spectrum of
ailments or as an herbal supplement for health promotion.
The Indian Pharmacopoeia narrates that it is a
predominant constituent of at least 26 Ayurvedic
formulations, Zhang (1978). In Traditional Chinese
Medicine, it is an important cold property herb used to
get rid the body heat, as in fevers, and to dispel toxins of
the body, Deng (1997). In Scandinavian countries, it is
commonly used to prevent and treat common cold
(Caceres et al., 1997). In Thailand, this plant was selected
by the Ministry of Public Health as one of the medicinal
plants to be included in The National List of Essential
Drugs A.D. 1999 (List of Herbal Medicinal Products)
(Pholphana et al., 2004; Kanokwan and Nobuo, 2008).
So far antimicrobial activities of Andrographis
paniculata are reported with aqueous, methanol, ethanol,
hexane and chloroform extracts (Prajjal et al., 2003; Xu et
al., 2006; Mishra et al., 2009; Bobbarala et al., 2009).
However, the antimicrobial activities of ethyl acetate,
acetone, DMSO and petroleum ether with in vitro raised 8
months old plants were not made yet. Thus, an attempt
was made to evaluate the antimicrobial potential of the
micropopagated plants with common human pathogenic
microbes.

MATERIALS AND METHOD

Plant extract preparation
The field established micropropagated plants of A.
paniculata were collected from the green house of

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Asian Journal of Science and Technology, Vol. 5, pp.091-094, September, 2010

Department of Biotechnology, Gauhati University,

Assam, India. 10g of dried plant material (leaf, stem and
roots) was extracted with various organic (chloroform,
acetone, ethyl acetate, DMSO, petroleum ether) and
aqueous solvents in a soxhlet extractor until the solvent
was colorless. The extract was collected and filtered
through eight layers of muslin cloth. The filtrate was
evaporated to dryness in vacuo and weighed.
Microbial strains tested
Five microbial strains were used from the Microbial Type
Culture Collection (MTCC) Chandigarh, India. They were
gram negative Klebsiella pneumoniae (MTCC 432),
Escherichia coli (MTCC 739), Pseudomonas aeruginosa
(MTCC 443), gram positive Staphylococcus aureus
(MTCC 96) and Bacillus subtilis (MTCC 441) bacteria
and the fungal pathogen Candida albicans (MTCC 227).
Antimicrobial assay
Antimicrobial activity was determined by the well
diffusion method (Shanab et al., 2004) with minor
modifications. Petri plates containing 25 ml of Nutrient
agar/Potato dextrose medium were seeded with a 24 hours
culture of the microbial strains. Wells (6 mm diameter)
were cut into the agar and 100l of the plant extracts were
tested in a concentration of 50g/ml. The inoculum size
was adjusted so as to deliver a final inoculum of
approximately 106 colony-forming units (CFU)/ml.
Incubation was performed at 37C for 24 hours for
bacterial strains and 72 hours at 28C - 30C for the
fungal pathogen, C. albicans. Microbial growth was
determined by measuring the diameter of zone of
inhibition in millimeters (mm). For each bacterial and
fungal strains control were maintained where pure
solvents were used. Broad spectrum antibiotics viz.
ampicillin, tetracycline and fluconazole at 1mg/ml
concentrations were used as standards.
Minimal inhibitory concentration (MIC)
Minimal inhibitory concentration (MIC) was done by two
fold serial dilution. 2000g of the plant extract was
dissolved in 1ml of the solvent which gives maximum
zone of inhibition in the antimicrobial sensitivity test. The
test tubes were taken and labeled as per concentrations
taken. 2ml nutrient broth was added to all the test tubes.
Serial dilution of the plant extract was done by
transferring 2ml from each test tube to obtain two fold
dilutions (1000g/ml, 500g/ml, 250g/ml, 125g/ml,
62.5g/ml, 31.25g/ml, 15.625g/ml, 7.81g/ml,
3.90g/ml and 1.95g/ml). 50l of the test organism(s)
were added to all the test tubes except the negative
control. The tubes were then incubated at 37C for 24
hours for bacterial cultures and 28C for yeast for 2-3
days. MIC was determined by choosing the lowest
concentration in which no growth occurs.
Phytochemical estimation
The A. paniculata extracts were subjected to various
biochemical tests to determine the total carbohydrate
(Sadasivam and Manickam, 1991a), total protein
(Sadasivam and Manickam, 1991b) and total phenolics
(Sadasivam and Manickam, 1991c), total flavonoid
(Pourmorad et al., 2006), total saponin (Obadoni and
Ochuko, 2001; Edeoga et al., 2005) and total

alkaloid, Harborne (1973) present in the crude extract. All

the data expressed as mean standard deviation (SD),
where n = 5.

RESULT AND DISCUSSION

Antimicrobial assay
The antimicrobial activities of the various extracts of
Andrographis paniculata (leaves, stems and roots)
exhibited different degrees of antimicrobial activity
against the test organisms. The traditional healers use
primarily water as a solvent. The aqueous extract in the
present study showed inhibitory effect against all the gram
positive, gram negative bacteria (except P. aeruginosa)
and the fungal pathogen C. albicans. The antimicrobial
activities of the plant extracted in different solvents varied
greatly because there are many factors influence the active
compounds present in the plant. Here the polarity of the
extracting solvents are different and greatly influenced the
antimicrobial properties. This may be due to the better
solubility of the active compounds in organic solvents (De
Boer et al., 2005; Parekh et al 2005). In almost all the test,
the crude chloroform extract showed better inhibition
against all the tested microbial strains, indicating active
ingredients in plant material could be extracted into
chloroform. However, highest antimicrobial activity was
observed against B. subtilis and C. albicans. Ethyl acetate
and acetone extracts showed inhibitory effects against all
the gram positive bacteria but no inhibitory effect was
observed with gram negative bacteria and the fungal
pathogen C. albicans. Whereas, the DMSO extract
showed inhibitory effect against the gram positive bacteria
S. aureus. Petroleum ether extract does not have any
inhibitory effects on the microorganisms tested. However,
no inhibition was observed in control, which proves that
solvents could not act as antimicrobial agents (Table 1).
Amongst the gram positive and gram negative bacteria,
gram positive bacterial strains were more susceptible to
the extracts as compared to the gram negative bacteria.
Generally gram negative bacteria are more resistant than
gram positive bacteria (Rabe and van Staden, 1997;
Kelmanson et al., 2000). This could be due to several
possible reasons; one is the presence of the double
membrane surrounding each bacterial cells. Their outer
membrane excludes certain drugs and antibiotics from
penetrating the cell, partially accounting for why gram
negative bacteria are generally more resistant than gram
positive bacteria (Yao and Moellering, 1995; Tortora and
Funke, 2001; Lotfipour et al., 2008). Secondly the basis of
their differences in susceptibility might be due to the cell
wall composition of gram positive and gram negative
bacteria (Grosvenor et al., 1995). The minimal inhibitory
concentration (MIC) values with respect to chloroform
extract for Klebsiella pneumoniae, Escherichia coli,
Psuedomonas
aeruginosa,
Bacillus
subtilis,
Staphylococcus aureus and Candida albicans were found
to be 15.6255g/ml to 31.5g/ml, 15.6255g/ml to
31.55g/ml, 15.6255g/ml, 15.6255g/ml, 15.6255g/ml
and 15.6255g/ml respectively (Table 2).
Phytochemical estimation
The phytochemical estimation was done for analyzing
primary and secondary metabolites. The A. paniculata

Ankita Kataky and Handique., Antimicrobial activity and phytochemical estimation of micropropagated
Andrographis paniculata (Burm.f) nees

088

Table 1. Antimicrobial activity of A. paniculata and the standard antibiotics.

Plant extract
Aqueous leaf
Aqueous stem
Aqueous roots
Chloroform leaf
Chloroform stem
Chloroform roots
Ethyl acetate leaf
Ethyl acetate stem
Ethyl acetate roots
Acetone leaf
Acetone stem
Acetone roots
DMSO leaf
DMSO stem
DMSO roots
Pet ether leaf
Pet ether stem
Pet ether roots

K. pneumoniae
120.344
120.216
140.297
120.283
160.295
100.329

Ampicillin
Tetracyclin
140.218
Fluconazole
Data reported as mean SD, where n = 5

E.coli
70.428
80.179
100.173
140.241
150.313
100.134

170.426
160.315

P.aeruginosa

80.444
120.215
100.163

150.167

S.aureus
120.447
120.568
100.328
150.335
180.001
150.006
140.357
80.303
70.356
130.416
160.374
150.055
150.141
160.332
140.187

B.subtilis
120.084
130.373
120.363
220.071
240.219
180.055
150.152
80.327
100.626
100.283
130.365
120.251

170.376

170.283

C.albicans
80.227
120.336
120.311
340.297
370.503
240.305

210.403

Table 2. Minimal inhibitory concentrations of the chloroform extract against the 6 test organisms

Tested Organisms
K. pneumoniae
E. coli
P. aeruginosa
S. aureus
B. subtilis
C. albicans

Minimal Inhibitory Concentration (g/ml)

Chloroform Leaf
Chloroform Stem
31.5
15.625
15.625
15.625
31.5
31.5
15.625
15.625
15.625
15.625
15.625
15.625

Chloroform Roots
31.5
31.5
31.5
15.625
15.625
15.625

Table 3: Phytochemical constituents of A. paniculata

Plant material
Phytochemicals (mg/g)
Leaf
Stem
Total carbohydrates
58.000.002
57.00 0.007
Total proteins
0.56 0.006
0.37 0.003
Total phenolics
0.50 0.002
0.70 0.012
Total flavonoids
4.35 0.007
3.68 0.006
Total saponin
274.600.015
179.200.133
Total alkaloids
598.800.002
299.000.017
Data reported are the mean of 5 replications SD, where n = 5

extracts were subjected to various biochemical tests to

determine the active constituents present in the crude
extract and it is depicted in (Table 3). The biochemical
estimation results revealed the presence of carbohydrates,
proteins, phenolics, flavonoids, saponins and alkaloids.
Flavonoids protect plants from attacks by microbes and
insects. Flavonoids have been referred to as natures
biological response modifiers because of strong
experimental evidence of their inherent ability to modify
the bodys reaction to allergens, viruses and carcinogens.
They show anti-allergic, anti-inflammatory (Yamamoto
and Gaynor 2008), antimicrobial (Cushnie and Lamb,
2005), anti-neoplastic, anti-viral, anti-thrombic (Ayoola et
al., 2008) and anti-cancer activity. Phenols, the aromatic
compounds with hydroxyl groups are widespread in plant
kingdom. They occur in all parts of the plant. Phenols are

Leaf/stem
60.00 0.002
0.78 0.005
2.50 0.002
6.82 0.005
200.600.007
622.600.002

Roots
54.00 0.004
0.25 0.005
3.20 0.004
3.41 0.001
175.450.004
268.400.003

said to offer resistance to diseases and pest in plants

(Sadasivam and Manickam c, 1991). Saponin protects the
plant against microbes and fungi. Alkaloids usually have
marked the physiological action on human or animals
(Adedapo et al., 2009). Whereas, carbohydrates and
proteins determine the nutritive value of A. paniculata.
Therefore, it was reasonable to determine the total primary
and secondary metabolite content in the plant extract.
In the present investigation, the screening of
micropropagated plantlets of A. paniculata with
chloroform, ethyl acetate, DMSO and acetone extracts
gives better response than the methanol, ethanol or hexane
extracts in the preliminary studies. The phytochemical
estimation of Andrographis paniculata revealed that the
antibacterial activity is due to the presence of the
secondary metabolites. And carbohydrates and proteins

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Asian Journal of Science and Technology, Vol. 5, pp.091-094, September, 2010

give the nutritive value of A. paniculata. These results

indicated the possibility of using Andrographis paniculata
for medicinal uses and food preservation.

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