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Phytochemical analysis of Andrographis paniculata extract and its

antimicrobial activity

Article  in  World Journal of Microbiology and Biotechnology · January 2009

DOI: 10.1007/s11274-009-0146-8

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World J Microbiol Biotechnol (2010) 26:85–91
DOI 10.1007/s11274-009-0146-8
ORIGINAL PAPER
Phytochemical analysis of Andrographis paniculata extract
and its antimicrobial activity
Soma Roy Æ Kiranmayee Rao Æ Ch. Bhuvaneswari Æ
Archana Giri Æ Lakshmi Narasu Mangamoori
Received: 30 December 2008 / Accepted: 31 July 2009 / Published online: 11 August 2009
Ó Springer Science+Business Media B.V. 2009
Abstract The present study describes the phytochemical
profile and antimicrobial activity of Andrographis panicu-
lata. For the present investigation, two samples of A. pan-
iculata extracts, obtained by extraction in chloroform and
chloroform ? HCl, respectively, were compared for their
antimicrobial activity and further subjected to GC-MS
analysis to find out the nature of the compounds responsible
for the antimicrobial activity. The antibacterial activities
were assessed by measuring the diameter of the inhibition
zones, MIC and MBC values. Compared to the chloro-
form ? HCl extract, the chloroform extract showed better
antimicrobial activity against all the nine pathogenic bac-
terial strains tested. The chloroform extract was observed to
be active against the opportunistic and pathogenic gram-
negative bacteria, indicating its potential application related
to noscomial infections. GC-MS results revealed phenols,
aromatic carboxylic acids and esters in the chloroform
extract to be the molecules responsible for the antimicrobial
activity of A. paniculata. This is the first report on analysis
of antimicrobial components from A. paniculata, and our
results confer the utility of this plant extract in developing a
novel broad spectrum antimicrobial agent.
S. Roy
Biotechnology Department, Chaitanya Bharathi Institute
of Technology, Hyderabad, India
e-mail: royksi@yahoo.co.in
K. Rao
Ch. Bhuvaneswari
A. Giri (&)
L. N. Mangamoori
Center for Biotechnology, Institute of Science and Technology,
Jawaharlal Nehru Technological University, Hyderabad, Andhra
Pradesh 500 085, India
e-mail: archanagiriin@yahoo.co.in
Keywords Andrographis paniculata

Antimicrobial activity
Gram-positive bacteria

Gram-negative bacterial
GC-MS
Introduction
The increase in prevalence of multiple drug resistance has
slowed down the development of new synthetic antimi-
crobial drugs, and has necessitated the search for new
antimicrobials from alternative sources. In general, bacteria
have the genetic ability to transmit and acquire resistance
to drugs used as therapeutic agents. One way to prevent
antibiotic resistance is by using new compounds which are
not based on the existing synthetic antimicrobial agents
(Shah 2005). Phytochemicals from medicinal plants
showing antimicrobial activities have the potential of fill-
ing this need, because their structures are different from
those of the more studied microbial sources, and therefore
their mode of action may too very likely differ (Fabricant
and Fansworth 2001). There is growing interest in corre-
lating the phytochemical constituents of a medicinal plant
with its pharmacological activity (Prachayasittikul et al.
2008; Nogueira et al. 2008; Costa et al. 2008; Al-Bayati
and Al-Mola 2008; Chen et al. 2008; Pesewu et al. 2008;
Turker and Usta 2008). Screening active compounds from
plants has lead to the discovery of new medicinal drugs
which have efficient protection and treatment roles against
various diseases, including cancer (Kumar et al. 2004;
Sheeja and Kuttan 2007) and Alzheimer’s disease (Muk-
herjee et al. 2007).
Andrographis paniculata, a member of Acanthaceae
(Acanthus) family has been used for centuries as a
medicinal herb for treatment of upper GI tract and upper
respiratory infections, fever, herpes and other chronic
123
86
diseases. It has a broad range of pharmacological effects.
The primary medicinal component of A. paniculata is an-
drographolide, which is a diterpene lactone. Androgra-
pholide has been reported for its anti-cancer (Sheeja and
Kuttan 2007), anti-HIV (Calabrese et al. 2000), cardio-
protective (Yoopan et al. 2007) and hepatoprotective
(Trivedi et al. 2007) properties among others. The other
active components include 14-deoxy-11,12-dihydroandro-
grapholide (andrographolide D), homoandrographolide,
andrographosterin and stigasterol (Siripong et al. 1992).
Prior to this study, the antimicrobial activity of
A. paniculata has been tested and reported. Leelarasamee
et al. (1990) reported the absence of antibacterial activity
for the aqueous extract of A. paniculata. Zaidan et al.
(2005) in their study on in vitro screening of five local
medicinal plants for antibacterial activity using disc dif-
fusion method, found water extract of A. paniculata to
posses potential antibacterial activity towards both gram-
positive and gram-negative microorganisms. Xu et al.
(2006) have investigated the antimicrobial activity using
A. paniculata (methanolic and aqueous) extracts and
authentic andrographolide against nine human bacterial
pathogens. Their results indicated methanolic extracts of
A. paniculata to be active against only two of the patho-
gens, while authentic andrographolide did not show any
activity. They concluded that the observed antimicrobial
activity was due to other active principle(s) present in the
extracts that were used in the investigation.
The objective of the present study was to identify the
active components responsible for the antimicrobial
activity in A. paniculata. In vitro antimicrobial activity of
A. paniculata extracts using chloroform and chloro-
form ? HCl as the solvent extraction systems were
investigated. For the identification of compounds respon-
sible for antimicrobial activity, the extracts were subjected
to GC-MS analysis. Chloroform was the choice of solvent
in the present investigation because, earlier reports have
shown A. paniculata chloroform extracts to be effective
against the malarial parasite (Najib Nik et al. 1999). In this
study, HCl has been used in combination with chloroform,
as the plant metabolites are known to be extracted at higher
concentrations in the acidic pH range.
Materials and methods
Plant material
Andrographis paniculata plants were obtained from the
fields of Andhra Pradesh India. The aerial parts of the plant
were washed thoroughly under running tap water and dried
under shade. They were then finely ground to a powder in
an electric blender.
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World J Microbiol Biotechnol (2010) 26:85–91
The solvents used for the extraction procedure in the
present study were chloroform and chloroform ? 1 M HCl (at
2 ml/100 ml). About 25 g of dried plant powder was extracted
using 250 ml of the extraction solvents with continuous
shaking on a rotary shaker at 150–180 rev/min for 48 h. The
filtrates were concentrated using a rota-vapour, at 45°C and
stored away at 4°C in air tight containers for further use.
Test organisms
The extracts of A. paniculata were screened against a total
of nine pathogenic bacterial clinical isolates. The patho-
genic microbial strains used were obtained from Global
Hospitals, Hyderabad, India. The pathogenic strains used
for testing the antimicrobial activity of A. paniculata
included five gram-negative bacteria (Escherichia coli,
Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmo-
nella typhimurium, Enterobacter cloacae), and four gram-
positive bacteria (Staphylococcus aureus, Bacillus subtilis,
Enterobacter faecalis, Staphylococcus epidermidis). All
the microbial cultures were maintained on Mueller Hinton
Agar (MHA) slants at 4°C with a subculture period of
15 days. The microbial stock solution with a concentration
of 108–109 colony forming units (c.f.u.)/ml was maintained
using surface viable counting technique (Miles and Misra
1938). Each time, a 2-h-old microbial stock suspension was
prepared and the experimental conditions (temperature and
aeration) were maintained constant before the antimicro-
bial assay was carried out.
Antimicrobial assay
The agar well diffusion method was followed for estimat-
ing the antimicrobial activity of A. paniculata extract
(Okeke et al. 2001). About 100 ll of standardized micro-
bial stock suspension (1 9 108) was thoroughly mixed
with molten Mueller–Hinton agar medium and poured into
sterile Petri plates. In each Petri plate, three 8 mm wells
were made using a sterile cork borer no. 4. Then, 100 ll of
each of the extracts containing 250 lg were added into two
of the wells, respectively. As the control, 100 ll of a broad
spectrum antibiotic amikacin, containing 5 lg was added
to the third well. The plates were then incubated overnight
at 37°C for allowing bacterial growth. After incubation, the
diameter of the zones of inhibition were measured and
tabulated for each test microorganism.
Minimum inhibitory concentration (MIC)
and minimum bactericidal count (MBC)
of A. paniculata extracts
The minimum inhibitory concentration (MIC) was deter-
mined by broth dilution method (Chattopadhyay et al.
World J Microbiol Biotechnol (2010) 26:85–91 87

Table 1 Susceptibility pattern of human pathogens to Andrographis paniculata extracts

Microorganism Inhibition zone diameter (mm)a,b MIC (mg/ml) MBC (mg/ml)
CHCl3 extract CHCl3 ? HCl extract Amikacin
(250 lg/100 ll) (250 lg/100 ll) (5 lg/100 ll)

Escherichia coli (G -ve) 25 ± 0.5 12 ± 0.5 17 ± 0.5 0.25 0.5

Pseudomonas aeruginosa (G -ve) 28 ± 0.5 13 ± 0.4 16 ± 0.5 0.25 0.75
Klebsiella pneumoniae (G -ve) 22 ± 0.3 16 ± 0.5 18 ± 0.5 0.5 1.0
Salmonella typhimurium (G -ve) 18 ± 0.4 10 ± 0.5 18 ± 0.3 0.75 1.0
Enterobacter cloacae (G -ve) 30 ± 0.5 28 ± 0.3 30 ± 0.5 0.25 0.5
Staphylococcus aureus (G ?ve) 15 ± 0.5 14 ± 0.5 24 ± 0.4 1.0 1.5
Bacillus subtilis (G ?ve) 26 ± 0.5 20 ± 0.4 23 ± 0.5 0.25 0.5
Enterobacter faecalis (G ?ve) 35 ± 0.5 30 ± 0.5 25 ± 0.3 0.25 0.5
Staphylococcus epidermidis (G ?ve) 20 ± 0.3 16 ± 0.5 30 ± 0.5 0.5 0.75
a
Mean value ± SD, n = 3 (the zone of inhibition (in millimeter) including disc of 8 mm in diameter)
b
Statistical analysis data are expressed as means ± SD

1998a). Twofold serial dilutions (0–2,000 lg/ml) of the
crude extracts, with appropriate antibiotic as control were
prepared in Mueller–Hinton broth (Chattopadhyay et al.
1998b). A direct suspension of microorganisms was pre-
pared in 5 ml sterile distilled water from a 24-h-old sus-
pension in Mueller–Hinton broth. The turbidity of the
suspension was adjusted to match 0.5 McFarland standard
using a spectrophotometer (Shimadzu UV 2450 UV–Vis
Spectrophotometer) at 625 nm, which corresponds to
2.4 9 108 c.f.u./ml. For broth dilution tests, 0.1 ml of
standardized suspension of bacteria (108 c.f.u./ml) was
added to each tube at a final concentration of 0–2,000 lg/
ml, and incubated at 37°C. The lowest concentration of the
tube which did not show any visible growth after macro-
scopic evaluation was considered as the MIC. The dilu-
tions, starting from the tube that did not show any visible
growth, were streaked on to Mueller–Hinton agar plates.
These plates were then incubated overnight at 37°C and
observed for visible growth. The tube containing the lowest
concentration of the extract, which when streaked on to the
plate, did not show any visible growth after 24 h, was
considered as the minimum bactericidal count (MBC). All
the assays were performed in triplicates.
GC-MS analysis
For GC-MS analysis, the samples were injected into a
HP-5MS capillary column (30 m length 9 0.25 m i.d 9
0.25 lm film thickness), Agilent Technologies, USA GC-
MS model, consisting of 6890 N Gas Chromatograph
coupled with 5,973 insert MSD [Mass Selective Detector].
The injector was set at 250°C and the detector at 280°C.
The stepped temperature program-was as follows: held
at 50°C for 2 min, then, from 50 to 280°C at the rate of
10°C/min, held for 5 min. The total run time was of
30 min. The GC-MS interface temperature was at 280°C.
The injection volume was 1 ll. The solvent delay was
2 min and injected in a split ratio of 1:10. The MS scan
range was from 35–6,000 Da. Compound identification
was obtained by comparing the retention times with those
of authentic compounds and the spectral data obtained
from library data of the corresponding compounds.
Results and discussion
The results from the present study showed that the two
extracts (chloroform and chloroform ? HCl) of A. pan-
iculata, displayed antimicrobial activities against all the
nine human pathogens tested. As seen from Table 1, both
the extracts exhibited broad spectrum of activity. When the
two crude extracts were compared with each other and with
that of standard antibiotic amikacin, the chloroform extract
was seen to have greater potential compared to that of the
chloroform ? HCl extract.
Chloroform extract antimicrobial results showed the
diameter of inhibition zones ranging from 15 to 35 mm, with
the highest inhibition zone observed against E. faecalis
(35 mm), followed by E. cloacae (30 mm) P. aeruginosa
(28 mm) and E. coli (25 mm). Least inhibition zone was
observed against S. aureus (15 mm). Though the inhibition
zone observed against S. typhimurium was only 18 mm, it is
noteworthy when comparing it with that of the control result.
Out of the 9 pathogenic strains tested, 7 strains showed
inhibition zones comparable with that of the control (ami-
kacin) used. The chloroform extract antimicrobial activity
seen against all the tested gram-negative opportunistic and
pathogenic bacteria is very encouraging and important con-
sidering the role of gram-negative bacteria in noscomial
infections leading to increased morbidity and mortality rates.

123
88
The chloroform ? HCl extract antimicrobial activity
results showed diameter of inhibition zones ranging from
10 to 30 mm, with the highest zone of inhibition shown
towards E. faecalis (30 mm). Though both the extracts
have shown antimicrobial activity, the response shown by
chloroform extract (without HCl) was higher when com-
pared to the chloroform extract with HCl, indicating that
the pH of the extraction solvent did not have any effect on
the extraction of antimicrobial phytochemicals.
The MIC is interpreted as the lowest concentration that
inhibits visible microbial growth and expressed in terms of
mg/ml, whereas the minimum bactericidal concentration
(MBC) is interpreted as the lowest concentration that can
completely remove the microorganisms. The MIC values
were evaluated after 24 h of incubation and MBC values
were obtained from further analysis of the MIC results. The
MIC values of most of the test organisms ranged from 0.25
to 0.75 mg/ml while that of MBC values ranged between
0.5 and 1.0 mg/ml. The exceptions were S. aureus and S.
typhimurium, both of which have shown higher values for
MIC as well as MBC, and this supports the present study
result of both the organisms exhibiting highest resistance to
the plant extracts in terms of antimicrobial activity. Even
though the MIC and MBC values were high for a few
resistant organisms, A. paniculata continues to be a
promising source for the extraction of antimicrobial com-
pounds taking into account the crude state of the extract
and the lower concentration of the compounds responsible
for the activity in the samples.
The extract was seen to be active against many
opportunistic as well as pathogenic microorganisms like
E. coli (Crohn’s disease and ulcerative colitis), B. subtilis
(food poisoning), E. faecalis (endocarditis), P. aerugin-
osa (nosocomial infections) and K. peumoniae (urinary
tract infections and pulmonary infections). Among the
microorganisms used, S. aureus and S. typhimurium
(typhoid/enteric fever) are potentially pathogenic.
S. aureus in particular can cause a range of infections,
from minor skin infections to life threatening meningitis,
toxic shock syndrome, endocarditis and septicemia.
Hence the crude extract of A. paniculata in chloroform
can be used for further purification and preparation of
new antimicrobials for the more resistant type of
microorganisms.
GC-MS analysis
This report is the first of its kind to analyse the chemical
constituents responsible for the antimicrobial activity of A.
paniculata. GC-MS phytochemical screening results of
chloroform and chloroform ? HCL extracts showed 25
and 39 retention peaks, respectively.
123
World J Microbiol Biotechnol (2010) 26:85–91
Table 2 gives the retention times and the relative per-
centages of the compounds present in the respective
extracts.
Some of the main components were seen to be present in
both the extracts, but differed in their relative amounts,
indicating their role in antimicrobial activity. Some of the
GC-MS peaks remained unidentified because of lack of
authentic samples and library data of the corresponding
compounds.
The components listed in Table 2 can be divided into
four main groups, the contributions to which were observed
to be different in the two extracts. The first group (I)
consists of the aromatic compounds, comprising of phe-
nols, aromatic carboxylic acids and esters. The second
group (II) consists of the aliphatic compounds, represented
by acidic compounds of aliphatic series, which included
the saturated and unsaturated mono, dicarboxylic and hy-
drocarboxylic acids. The third group (III) consists of the
aliphatic hydrocarbons, which had n-alkanes and alkenes.
The fourth group (IV) consists of the terpenoid based
compounds.
As can be seen from Table 3, the relative percentage of
total aromatic compounds is more prominent in the chlo-
roform extract, where it is 31.56%, as compared to only
7.57% in the chloroform ? HCl extract. In contrast, the
total percentage of aliphatic compounds was only 12.53%
in the chloroform extract, compared to 21.32% seen in the
chloroform ? HCl extract. In case of total aliphatic
hydrocarbons, the amounts present in both the extracts did
not exhibit much variation, being 30.36% in the chloroform
extract and 24.19% in the chloroform ? HCl extract.
As antimicrobial activity is more pronounced in the chlo-
roform extract, it could be reasonably argued that the
aromatic compounds comprising of phenols, aromatic
carboxylic acids and esters are responsible for the antimi-
crobial activity of A. paniculata. Chloroform ? HCl
extract, which showed less antimicrobial activity, was seen
to extract huge amount of aliphatic compounds, and hence
these compounds can be interpreted as not to have a key
role in antimicrobial action. The amount of aliphatic
hydrocarbons found to be extracted in more or less similar
amounts in both the extracts, indicates that though they
form a major part of the components, their role in anti-
microbial activity is not evident, and may be a part of the
cuticular waxes of plant leaves and stems, of which the
main components are higher alkanes. The presence of
terpenoid-based compounds in both the extracts is pre-
sumably due to andrographolide, the primary medicinal
value component of A. paniculata, which is chemically a
diterpene lactone. Previous report (Xu et al. 2006) has
already reported andrographolide not to be responsible for
the observed antimicrobial activity in A.paniculata.
World J Microbiol Biotechnol (2010) 26:85–91 89

Table 2 Chemical compositions of Andrographis paniculata extracts by GC-MS analysis

Name of compounda Groupb Retention % in Andrographis % in
time (min) paniculata Andrographis
CHCl3 extract paniculata CHCl3
extract ? HCl

1,4 Dichlorobenzene I 7.664 0.564

1-Dodecene III 10.480 1.29 0.39
2-Tetradecene III 13.295 1.63 0.68
Pentadecane III 14.677 1.21
Eicosane III 14.677 3.66
Phenol, 2,4,di-tert-butylphenol, 2,4 bis(tert butyl)-phenol I 14.862 1.85
I 14.863 5.74
Benzofuranone I 15.250 0.62
1-Hexadecene, Cetene, III 15.789 0.92
a-Hexadecene, hexadecylene 15.790 2.52
Diethyl pthalate, diacyl ester I 15.874 18.96 4.54
1,2-benzenedicarboxylic acid
Heptadecane III 17.020 0.91
Pentadecane III 17.189 0.565
Heptacosane III 17.661 0.90
1-Hexadecene, Cetene, III 18.048 0.83
a-Hexadecene, hexadecylene
5-Octadecene III 18.049 1.86
Octadecane III 18.116 0.61
Neophytadiene 18.537 1.95
IV 18.538 3.61
2-Pentadecanone, 6,10,14,trimethyl IV 18.605 1.82 1.33
hexafarnesyl, 6,10,14,trimethyl-2-pentadecanone
Neophytadiene IV 18.790 0.43
1,2-Benzenedicarboxylic acid, I 18.874 0.46
isobutyl ester, Isobutyl pthalate 18.875 1.35
Neophytadiene 18.976 1.26
IV 18.992 0.71
Pentadecanoic acid, methyl ester II 19.397 1.30
Hexadecanoic acid, palmitic acid, palmitinic acid, II 19.768 9.43
hexadecanoic acid, hexadecoic acid, pentadecane 19.801 12.0
carboxylic acid, cetylic acid, coconut oil fatty acid
Hexadecanoic acid,palmitic acid ethyl ester, ethyl palmitate, II 20.071 2.10
hexadecanoic acid ethyl ester, ethyl palmitate 20.072 3.10
Unknown 21.251 4.01
21.285 3.81
9-Octadecanoic acid, oleic acid II 21.470 4.24
Octadecanoic acid, stearic acid II 21.639 1.68
5-Eicosane III 21.925 0.64
Tricosane III 22.852 1.06
Eicosane II 22.853 1.58
Unknown 22.628 1.64
Unknown 23.679 2.05
Tetracosane III 23.679 2.48
Pentacosane III 24.488 2.45
Eicosane III 24.488 2.69
Unknown 24.825 8.10

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90 World J Microbiol Biotechnol (2010) 26:85–91

Table 2 continued
Name of compounda Groupb Retention % in Andrographis % in
time (min) paniculata Andrographis
CHCl3 extract paniculata CHCl3
extract ? HCl

Di-n-octyl pthalate I 24.909 6.86

Unknown 24.909 11.21
Hexacosane III 25.263 3.17 2.74
Unknown 25.567 4.44
Heneicosane III 26.123 2.79
Eicosane, 5-hydroxy-7,8-dimethyl flavone III 26.123 2.94
26.629 2.45
Unknown 26.645 5.51
Eicosane III 27.101 2.15
Nonacosane III 27.117 2.63
Unknown 27.842 7.54
Unknown 27.843 9.27
Octadecane III 28.280 2.59
Nonacosane III 28.281 2.33
Unknown 29.022 2.0
Nonadecane III 29.696 1.88
a
Compounds listed in order of their elution from HP-5MS column
b
I Aromatic compounds, II aliphatic compounds, III aliphatic hydrocarbons, IV terpenoid compounds

Table 3 Group composition of GC-MS analysis components extract showed better inhibitory action against the gram-
Group % In Andrographis %In Andrographis negative bacteria, and this highlights its future to be
paniculata paniculata exploited as a potentially powerful antimicrobial agent
CHCl3 extract CHCl3 ? HCl extract against the gram-negative bacteria that have made treating
noscomial infections increasingly difficult. The antimicro-
Aromatic compounds 31.56 7.57
bial activity results were also comparable to that of the
Aliphatic compounds 12.53 21.32 antibiotic (amikacin) used as a standard reference. GC-MS
Aliphatic hydrocarbons 30.36 24.19 analysis of the extracts indicated that phenols, aromatic
Terpenoid compounds 6.69 4.41 carboxylic acids and esters are the active antimicrobial
principles present in A. paniculata. The results also indi-
Phenols and phenolic acids, among the simplest bioactive cated that chloroform was a suitable organic solvent for
phytochemicals, are known to be toxic for microorganisms. extraction of the active compounds responsible for anti-
The mechanisms thought to be responsible for phenolic tox- microbial activity of A. paniculata.
icity to microorganisms include enzyme inhibition by the
oxidized compounds, possibly through reaction with sulfhy- Acknowledgments AG acknowledges financial assistance from
dryl groups or through more nonspecific interactions with the AICTE, New Delhi, India.
proteins (Geissman 1963). There are reports of phenols being
responsible for the antimicrobial activity in medicinal plants
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