International Journal of Pure and Applied Mathematics ... · of Cassia fistula were found to be not...

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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.

[email protected]

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].

International Journal of Pure and Applied MathematicsVolume 119 No. 12 2018, 1319-1330ISSN: 1314-3395 (on-line version)url: http://www.ijpam.euSpecial Issue ijpam.eu

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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

International Journal of Pure and Applied Mathematics Special Issue

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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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