Post on 28-Oct-2020
1
Antibiotic sensitivity test against medicinal plant
1 Jayalakshmi.T,
2 MG.Amarnath Reddy
Associate Professor, 2 UG Student
Dept. of Genetic Engineering
BIHER, BIST, Bharath University
Chennai- 600073.
jayamaniraaja07@gmail.com
ABSTRACT
The antimicrobial activity was determined in the extracts using agar disc diffusion
method. The antibacterial activities of extracts (5, 25, 50, 100, 250 μg/ml) of Cassia
fistula were tested against two Gram-positive—Staphylococcus aureus, Streptococcus
pyogenes; two Gram-negative—Escherichia coli, Pseudomonas aeruginosa human
pathogenic bacteria. Zone of inhibition of extracts were compared with that of
different standards like ampicillin, ciprofloxacin, norfloxacin, and chloramphenicol
for antibacterial activity. The results showed that the remarkable inhibition of the
bacterial growth was shown against the tested organisms. The phytochemical analyses
of the plants were carried out. The microbial activity of the Cassia fistula was due to
the presence of various secondary metabolites. Hence, these plants can be used to
discover bioactive natural products that may serve as leads in the development of new
pharmaceuticals research activities.
Keywords: Cassia fistula, in vitro antibacterial activity, secondary metabolites
INTRODUCTION
Antibiotics are one of our most important weapons in fighting bacterial infections and
have greatly benefited the health-related quality of human life since their introduction.
However, over the past few decades, these health benefits are under threat as many
commonly used antibiotics have become less and less effective against certain
illnesses not, only because many of them produce toxic reactions, but also due to
emergence of drug-resistant bacteria. It is essential to investigate newer drugs with
lesser resistance. Drugs derived from natural sources play a significant role in the
prevention and treatment of human diseases. In many developing countries, traditional
medicine is one of the primary healthcare systems [1, 2]
Herbs are widely exploited in the traditional medicine and their curative potentials are
well documented [3]. About 61% of new drugs developed between 1981 and 2002
were based on natural products and they have been very successful, especially in the
areas of infectious disease and cancer [4]. Recent trends, however, show that the
discovery rate of active novel chemical entities is declining [5].
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Natural products of higher plants may give a new source of antimicrobial agents with
possibly novel mechanisms of action [6, 7]. The effects of plant extracts on bacteria
have been studied by a very large number of researchers in different parts of the world
[8]. Much work has been done on ethnomedicinal plants in India.
Plants are rich in a wide variety of secondary metabolites such as tannins, terpenoids,
alkaloids, flavonoids, glycosides, etc., which have been found in vitro to have
antimicrobial properties. [10, 11]
Herbal medicines have been known to man for centuries. Therapeutic efficacy of
many indigenous plants for several disorders has been described by practitioners of
traditional medicine. [12]. Antimicrobial properties of medicinal plants are being
increasingly reported from different parts of the world. The World Health
Organization estimates that plant extracts or their active constituents are used as folk
medicine in traditional therapies of 80% of the world's population. The harmful
microorganisms can be controlled with drugs and these results in the emergence of
multiple drug-resistant bacteria and it has created alarming clinical situations in the
treatment of infections [9]. The pharmacological industries have produced a number
of new antibiotics; resistance to these drugs by microorganisms has increased. In
general, bacteria have the genetic ability to transmit and acquire resistance to
synthetic drugs which are utilized as therapeutic agents.
Cassia fistula, known as the golden shower tree and by other names, is a flowering
plant in the family Fabaceae. The species is native to the Indian subcontinent and
adjacent regions of Southeast Asia. It ranges from southern Pakistan eastward
throughout India to Myanmar and Thailand and south to Sri Lanka. In literature, it is
closely associated with the Mullai (forest) region of Sangam landscape. It is the
national tree of Thailand, and its flower is Thailand's national flower. It is also the
state flower of Kerala in India and of immense importance amongst
the Malayali population. It is a popular ornamental plant and is also used in herbal
medicine.
MATERIALS AND METHODS
Collection of Plant Materials
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The fresh and healthy leaves of the plant Cassia fistula were collected between June
and August, 2009 from various areas of Jamnagar district, Gujarat, India. The plant
specimens were identified in department of Pharmacognosy I.P.G.T and R.A.
(Institute of post graduate teaching and research in ayurveda), Jamnagar. Plant parts
were collected on the basis of the information provided in the ethno botanical survey
of India. Each specimen/plant material was labeled, numbered, a noted with the date
of collection, locality, and their medicinal uses were recorded.[10-17]
LEAVES OF CASSIA FISTULA
Preparation of Plant Extract
Extraction
The extraction of the Cassia fistula leaves was carried out using known standard
procedures. The plant materials were dried in shade and powdered in a mechanical
grinder. The powder (25.0 g) of the plant materials were initially defatted with
petroleum ether (60-80°C), followed by 900 ml of hydroalcohol by using a Soxhlet
extractor for 72 hours at a temperature not exceeding the boiling point of the solvent.
The extracts were filtered using Whatman filter paper (No.1) while hot, concentrated
in vacuum under reduced pressure using rotary flask evaporator, and dried in a
desiccator [13]. The hydroalcoholic extract yields a dark greenish solid residue
weighing 5.750 g (23.0% w/w). More yields of extracts were collected by this method
of extractions. The extracts were then kept in sterile bottles, under refrigerated
conditions, until further use [8]. The dry weight of the plant extracts was obtained by
the solvent evaporation and used to determine concentration in mg/ml. The extract
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was preserved at 2- to 4°C. This crude extracts of hydroalcohol was used for further
investigation for potential of antimicrobial properties [14].
LEAF EXTRACT OF CASSIA FISTULA
Preliminary Phytochemical Screening
Plants are a source of large amount of drugs comprising to different groups such as
Kaur, Harleen Kaur antispasmodics, emetics, anti-cancer, antimicrobials etc. A large
number of the plants are claimed to possess the antibiotic properties in the traditional
system and are also used extensively by the tribal people worldwide. It is now
believed that nature has given the cure of every disease in one way or another. Plants
have been known to relieve various diseases in Ayurveda. [18-21]Therefore, the
researchers today are emphasizing on evaluation and characterization of various
plants and plant constituents against a number of diseases based on their traditional
claims of the plants given in Ayurveda. Extraction of the bioactive plant constituents
has always been a challenging task for the researchers. In this present review, an
attempt has been made to give an overview of certain extractants and extraction
processes with their advantages and disadvantages.[22-34]
The extracts were subjected to preliminary phytochemical testing to detect for the
presence of different chemical groups of compounds. Air-dried and powdered plant
materials were screened for the presence of saponins, tannins, alkaloids, flavonoids,
triterpenoids, steroids, glycosides, anthraquinones, coumarin, saponins, gum,
mucilage, carbohydrates, reducing sugars, starch, protein, and amino acids, as
described in literatures.
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Test Microorganisms and Growth Media
A growth medium or culture medium is a liquid or gel designed to support the growth
of microorganisms or cells, or small plants like the moss Physcomitrella patens. There
are different types of media for growing different types of cells. [30-34]
There are two major types of growth media: those used for cell culture, which use
specific cell types derived from plants or animals, and microbiological culture, which
are used for growing microorganisms, such as bacteria or yeast. The most common
growth media for microorganisms are nutrient broths andagar plates; specialized
media are sometimes required for microorganism and cell culture growth.[1]
Some
organisms, termed fastidious organisms, require specialized environments due to
complex nutritional requirements. Viruses, for example, are obligate
intracellular parasites and require a growth medium containing living cells.
The following microorganisms were tested:
Staphylococcus aureus (MTCC 96), Streptococcus pyogenes (MTCC
442), Escherichia coli (MTCC 443), Pseudomonas aeruginosa (MTCC 424) and
fungal strains Aspergillus niger (MTCC 282), Aspergillus clavatus (MTCC
1323), Candida albicans (MTCC 227) were chosen based on their clinical and
pharmacological importance. The bacterial strains obtained from Institute of
Microbial Technology, Chandigarh, were used for evaluating antimicrobial activity.
The bacterial and fungal stock cultures were incubated for 24 hours at 37°C on
nutrient agar and potato dextrose agar (PDA) medium (Microcare laboratory, Surat,
India), respectively, following refrigeration storage at 4°C. The bacterial strains were
grown in Mueller-Hinton agar (MHA) plates at 37°C (the bacteria were grown in the
nutrient broth at 37°C and maintained on nutrient agar slants at 4°C), whereas the
yeasts and molds were grown in Sabourauds dextrose agar and PDA media,
respectively, at 28°C.[35-38] The stock cultures were maintained at 4°C and
antimicrobial activity was determined by zone of inhibition method.
RESULTS
Preliminary phytochemical screening
It was found that hydroalcoholic extracts of Cassia fistula leaves contained tannins,
flavonoids, saponins, triterpenoids, steroids, glycosides, anthraquinones, reducing
sugars, carbohydrates, proteins, and amino acids.
Microbial activity
The antimicrobial activity of the extracts of Cassia fistula were studied in different
concentrations (5, 25, 50, 100, and 250 μg/ml) against four pathogenic bacterial
strains, two Gram-positive (Staphylococcus aureus MTCC 96, Streptococcus
pyogenes MTCC 442) and two Gram-negative (Escherichia coli MTCC
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443,Pseudomonas aeruginosa MTCC 424),. These strains have been selected for the
basis of its application purpose of further formulation study.
ZONE INHIBITION OF P.AERUGINOSA
ZONE INHIBITION IN S.PYOGENES
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S.AUREUS ZONE INHIBITION
Antibacterial potential of extracts were assessed in terms of zone of inhibition of
bacterial growth.[41-44] The results of the antibacterial activities are presented in
Tables Tables11–4.
DISCUSSION
Antimicrobial properties of medicinal plants are being increasingly reported from
different parts of the world. The World Health Organization estimates that plant
extract or their active constituents are used as folk medicine in traditional therapies of
80% of the world's population. In the present work, the extracts obtained from Cassia
fistula show strong activity against most of the tested bacterial and fungal strains. The
results were compared with standard antibiotic drugs. In this screening work, extracts
of Cassia fistula were found to be not inactive against any organism, such as Gram-
positive, Gram-negative, and fungal strains were resistant to all the extracts of Cassia
fistula.
The above results show that the activity of hydroalcohol extracts of Cassia
fistula shows significant antibacterial activities. This study also shows the presence of
different phytochemicals with biological activity that can be of valuable therapeutic
index. The result of phytochemicals in the present investigation showed that the plant
contains more or less same components like saponin, triterpenoids, steroids,
glycosides, anthraquinone, flavonoids, proteins, and amino acids. Results show that
plant rich in tannin and phenolic compounds have been shown to posses antimicrobial
activities against a number of microorganisms.[42-45]
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CONCLUSION
In the current investigation, the hydroalcohol extract in the ratio of 8 : 2 has been
selected after study of such a selected plant with water extracts and methanol extracts,
hydroalcohol extract gave higher yield of chemical constituents expected for this
research work; the originality of this work is that good results have been found with
hydroalcohol ratio, and it will be helpful to carry out other data with MIC and other
formulation study, because in comparison of methanol or water extracts, hydroalcohol
is more suitable for clinical study. The hydro alcoholic extracts of Cassia fistula were
found to be active on most of the clinically isolated microorganism and fungi, as
compare with standard drugs. The present study justified the claimed uses of leaves in
the traditional system of medicine to treat various infectious disease caused by the
microbes. However, further studies are needed to better evaluate the potential
effectiveness of the crude extracts as the antimicrobial agents. The present results will
form the basis for selection of plant species for further investigation in the potential
discovery of new natural bioactive compounds. Further studies which aimed at the
isolation and structure elucidation of antibacterial active constituents from the plant
have been initiated.
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