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Der Pharmacia Lettre, 2015, 7 (4):250-253
(http://scholarsresearchlibrary.com/archive.html)
ISSN 0975-5071
USA CODEN: DPLEB4
Soxhlet extraction of leaf extracts of Andrographis paniculata and its
antibacterial efficacy against few pathogenic bacterial strains
1 * 1 2
B. K. Nayak , S. Pavithera and Anima Nanda
1Department of Botany, K. M. Centre for P. G. Studies (Autonomous), Lawspet, Pondicherry, India
2Department of Biomedical Engineering, Sathyabama University, Rajiv Gandhi Salai, Chennai, India
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ABSTRACT
Andrographis paniculata Burm. F (Acanthaceae) is a potent medicinal plant in the Indian systems of medicine.
Traditionally, the leaves are used as/in influenza, bronchitis, gonorrohea, cholera, fertility and antibacterial,
anticancer, antidiabetic, antiinflammatory and antisnake venom etc. In the present study, the hydroalcohol extracts
of the leaves of Andrographis paniculata was carried out by soxhlet method and their antibacterial activity study
was made by disc diffusion method at different concentrations (5µl,10µl and 20µl) against different gram-positive
(Staphylococcus aureus, Pseudomonas aeruginosa and Bacillus subtilis) and gram negative (Escherichia coli,
Serratia marcescens and Proteus vulgaris). The antibiosis was also compared with standard antibiotics, Rifamycin
and Amoxyclav. It was recorded with a remarkable inhibition ability of the leaf extract against the tested organisms
and was also found better than amoxyclav at low dilutions.
Keywords: Andrographis paniculata, Antibacterial activity, Disc diffusion method, Soxhlet extraction method.
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INTRODUCTION
Infectious diseases are considered as the number one among all causes of death, accounting approximately one-half
all deaths throughout the world. About 50-75% of hospital deaths are reported due to infectious diseases [1]. The
treatment of these infections is mainly based on the use of antibiotics. In recent years, a number of antibiotics have
lost their effectiveness due to the development of resistant strains. Since then, there is a need to develop alternative
antibacterial drugs for the treatment of infectious diseases from various sources such as medicinal plants [2].
Andrographis paniculata is a herbaceous plant belongs to family Acanthaceae, native to India and Sri Lanka. Mostly
the leaves and roots were used for medicinal purposes. Andrographis paniculata is used in traditional Siddha and
Ayurvedic systems of medicine as well as in tribal medicine in India, China, Thailand and other Asian countries
[3,4]. It is present in 26 different polyherbal formulations in the Ayurvedic traditional health system. Andrographis
paniculata plant extract is known to possess a variety of pharmacological activities. Andrographolide, the major
constituent of the extract, which is implicated in its pharmacological activity. A study has been conducted on the
cellular processes and targets modulated by andrographolide treatment in human cancer and immune cells [5].
Andrographolide treatment inhibited the in vitro proliferation of different tumor cell lines, representing various types
of cancers. The compound exerts direct anticancer activity on cancer cells by cell cycle arrest at G0/G1 phase
through induction of cell cycle inhibitory protein p27 and decreased expression of cyclin dependent kinase 4
(CDK4). Immuno stimulatory activity of andrographolide is evidenced by increased proliferation of lymphocytes
and production of interleukin-2[5]. Andrographolide also enhanced the tumor necrosis factor α production and CD
marker expression, resulting in increased cytotoxic activity of lymphocytes against cancer cells, which may
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contribute for its indirect anticancer activity [5]. Medicinal plants are the richest bio-resource of drugs of traditional
systems of medicine, modern medicines, nutraceuticals, food supplements, folk medicines, pharmaceutical
intermediates and chemical entities for synthetic drugs [6]. The aim of the study was to assess the antibacterial
activity of leaf extract of A. paniculata prepared by Soxhlet extractor against disease causing bacteria. This study is
to foster the multi dimensional use of the plant by integrating its antimicrobial efficacy against pathogenic
microorganisms.
MATERIALS AND METHODS
Selection of plant
A. paniculata is an erect annual herb that grows 30 to 110 cm in height and is native to India. The square stem has
wings on the angles of new growth and is enlarged at the nodes, while small white flowers with rose-purple spots are
borne on a spreading panicle. The plant produces yellowish-brown seeds and all parts have an extremely bitter taste.
Collection of leaf material
The healthy and fresh leaf of the plant Andrographis paniculata was collected from Puducherry region. The
collected leaves were washed in tap water for 2-3 times. The excess water was removed.
Preparation of leaf extract
The leaf material was shade dried for 10 days. The dried leaf material was crushed directly by grinder without
adding any solvent [7]. The powder of the leaf materials were initially defatted with petroleum ether followed by
100 ml of hydro-alcohol by using a soxhlet extractor for 24 hours and dried in desiccators [8]. Once the process was
finished, the hydro-alcohol was evaporated using a rotary evaporator leaving a small yield of extracted plant
material in the glass bottom flask [9,10]. The hydro-alcoholic extract yielded dark greenish residues. The extracts
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were then kept in sterile bottles and put in refrigerator at 2-4 c until further use [1].
Antibacterial activity
Three sets of dilution (5µl, 10µl, 20µl) of Andrographis paniculata leaf extract were prepared in milli-Q water.
Mueller Hinton Agar (MHA) was used for antibacterial bioassay. MHA was prepared by dissolving 38g in 1000ml
of distilled water and brought to boil to completely dissolve. Sterilization was achieved by autoclaving at 121 oC for
15 minutes [8]. Extract were screened for their antibacterial activity against the gram-positive (Staphylococcus
aureus, Pseudomonas aeruginosa and Bacillus subtilis) and gram negative (Escherichia coli, Serratia marcescens
and Proteus vulgaris). MHA plates were prepared and bacterial strains were inoculated by cotton swab. The disc
which was prepared with three dilutions (5µl,10µl,20µl) were placed equidistantly over the plates with the
o
antibiotics disc of Rifamycin, Amoxyclav. The plates were incubated at 37 c for 24 hours and it was followed by the
calculation of the zone of inhibition (mm) [1].
RESULTS AND DISCUSSION
The results of the study indicated that the crude extract prepared from the leaves of A. paniculata showed good
inhibitory activity against six bacterial strains (Fig 1 and Table 1) and moreover, 5 µl dilution was found suitable to
control the bacterial growth in comparison to other dilutions. Staphylococcus aureus did not show any zone
formation in lower concentrations i.e., 5µl and 10µl.The antibacterial activity of the leaf extract was compared with
standard drugs and found more or less effective with Rifamycin. It was comparatively showed inhibition on bacterial
strains. Proteus vulgaris, Pseudomonas aeruginosa and Bacillus subtilis were more sensitive than Serratia
marcescens, Staphylococcus aureus and Escherichia coli (Fig 2).The result showed that the extract of Andrographis
paniculata where found to be more effective against all the bacterial species and found suitable in comparison to
amoxyclav antibiotics. According to the findings of Biswas et al [11] about the antibacterial assay, the methanol and
ethanol extracts of the guava leaves showed inhibitory activity against gram-positive bacteria, whereas the gram-
negative bacteria were resistant to all the solvent extracts. The methanol extract had an antibacterial activity with
mean zones of inhibition of 8.27 and 12.3 mm, and the ethanol extract had a mean zone of inhibition of 6.11 and
11.0mm against B. cereus and S. aureus respectively, was agreed with our hydro-alcoholic extract. On the basis of
their finding, guava leaf-extract might be a good candidate in the search for a natural antimicrobial agent [11].
Equivalent to our work, Redfern et al [10] extracted antimicrobial compound successfully by soxhlet method, they
tested the compound against E. coli and S. aureus and found the result, 9 mm diameter when tested against E. coli
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and 16 mm diameter when tested against S. aureus. The results made by them were found similar, more or less to
our results.
Fig 1: Antibacterial potency of leaf extracts and antibiotics against bacterial pathogens.
A: 5µl, B: 10µl, C: 20µl of leaf extract, D: Rifamycin, E: Amoxyclav
Table1: Antibacterial efficacy of Andrographis paniculata leaf extract against pathogenic bacteria.
Andrographis paniculata Soxhlet leaf extracts’ Antibiotics
Bacterial strains Dilutions Rif: Rifamycin, Amc: Amoxyclav
5 µl 10 µl 20 µl Rif Amc
Bacillus subtilis 11 11 10 16 7
Pseudomonas aeruginosa 11 9 10 10 0
Staphylococcus aureus 0 0 6 24 11
Proteus vulgaris 11 12 12 16 7
Escherichia coli 8 7 7 18 16
Serratia marcescens 10 9 9 11 0
Figure 1: Susceptibility/resistnace of bacterial strains towards the leaf extract and antibiotics studied
together
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B. subtilis P. S. aureus P. vulgaris E. coli S.
aeruginosa marcescens
Pathogenic bacteria
5µl 10µl 20µl RIF AMC
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CONCLUSION
The above results about the leaf extract of Andrographis paniculata showed significant antibacterial activity. The
hydro-alcoholic extract of Andrographis paniculata were found to be active on most of the clinically isolated
microorganisms as compared with the standard drugs. Further studies aiming at the isolation and structural
elucidation of antibacterial active constituents from the plant materials would be initiated to find out a solution to
treat the drug resistance bacteria in future for the human welfare.
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