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| Received by Qing Received on 2012-5-7|
ID No. B330 Revised on
Screening and Evaluation of antimicrobial activity of selected medicinal plants in Eastern Ghats India.
N.Chndrasekharnath1a, Dr.Y.V Mahalaxmi a, P.BrahmaNaidu 1a, B.Venkannaa, D.SubbaRaob, Dr.A.Umaa
Punica Granatum, phyllanthus niruri, Antibacterial activity, Minimum Inhibitory Concentration, Zone Of Inhibition
The present study is that due to increasing concerns about the development of antimicrobial resistance among
Pathogenic bacteria, so alternative strategies are sought that do not use antibiotics to reduce pathogenic bacteria
from foods and patients. Traditional Plants have been in use for thousands of years to conserve food and treat health
diseases. The peels of Punica granatum and whole plant of phyllanthus niruri has been commonly employed as a
crude drug in indian traditional medicine for treatment of diarrhea as well as used for pathogenic bacteria.
Antibacterial properties of Punica granatum peels and whole plant of phyllanthus niruri extracts(Aqueous,
methanolic and ethanolic) were evaluated against klebsiella, Enterococci, E.coli, P.aeruginosa and S.aureus using
agar well diffusion method. Aqueous,methanolic and ethanolic extracts of Punica granatum peels and whole plant
of phyllanthus niruri show an average inhibitory zone diameter of 12,14,16mm respectively which indicates that
ethanolic extract shows best result having ZOI greater than that of the standard antibiotics ampcillin is 14 mm.
Mehanolic extract of Punica Granatum and whole plant of phyllanthus niruri has lowest MIC of 1.45,1.36µg/ml
showing that it is most effective as compared to MICs of other extracts.
Centre for Innovative Research,
Institute of Science and Technology,
Jawaharlal Nehru Technological University Hyderabad,
An antimicrobial is a substance that kills or inhibits the growth of micro-organisms such as bacteria, fungi,
protozoan’s, etc. On the basis of their mode of action, antimicrobials are classified into two broad
categories as Microbicidal that kill microbes without leaving any option for their survival and Microbistatic that
cease all the metabolic activities of microbes that are important for their survival so they are called as growth
inhibitors of microbes.(1,2) The history of antimicrobials begins with the observation of Pasteur and Joubert who
discovered that one type of microbe could prevent the growth of other. That growth inhibition was due to secretion
of a compound that later got called as Antibiotic. Nowadays the term antibiotics is not confined to secretions of
microbes only but also includes all those synthetic drugs(3,4) that help body to get rid of any bacterial infection. The
discovery of antimicrobials like Penicillin and Tetracycline paved way for better health of people in the
world by curing diseases like Gonorrhea, Strep throat and Pneumonia(16). Synthetic drugs causes some adverse
effects to host. Within the recent years, infections have increased to a great extent and antibiotics resistance effects
become an ever-increasing therapeutic problem. Natural products of higher plants may possess a new source of
antimicrobial agents with possibly novel mechanisms of action(18,19). They are effective in the treatment of infectious
diseases while simultaneously mitigating many of the side effects that are often associated with synthetic
antimicrobials(17). Therefore, it is of great interest to carry out a screening of these plants in order to validate their
use in folk medicine and to reveal the active principle by isolation and characterisation of their constituents.
Systematic screening of them may result in the discovery of novel active compounds.
MATERIALS AND METHODS
Fruit peels of pomegranate was collected from a juice shop and whole plant of phyllanthus niruri was collected
from Visakhapatnam hills (Eastern Ghats) andharapradesh. Plant material and peel of fruit were then cut into smaller
pieces and then first washed with tap water followed by washing with distilled water. It was than dried under
sharing sunlight until water droplets got completely evaporated. Peel and plant were then kept in hot air oven for
two days so that it could get dried. Dried pericarp was then taken for grinding by the help of mixer grinder. The
coarse powder of plant sample was then used throughout the study.
Microbial strains of urinary track resistant pathogens Staphylococcus aureus (Gram positive), Klebsiella,
Enterococci Escherichia coli, Pseudomonas aeruginosa (Gram negative) were provided by different diagnostic
centeres They were subcultured and used throughout the studies.
The coarse powder of plant material and peel of fruit was dissolved in different solvents. The solvents used were
non polar as well as polar (methanol, ethanol and water).50 gm of ground peel and 50 gms of whole plant powder
were added 500 ml of water contain soxhlet apparatus so that secondary metabolites got completely extracted. The
extracts were then filterate obtained was evaporated to dryness at 50-650 c in a rotary vacuum evaporator to obtain
dark color molten mass.
Phytochemical Analysis of Extract:
The method described by Harborne with slight modifications were used to screen the bioactive compounds present
in the extracts.(15)
Test for steroids:
10 ml of the plant extract was evaporated to dry mass and dissolved in 0.5 ml of solvent. Acetic anhydride (0.5
ml)and 2ml of concentrated sulphuric acid were added. A green color or blue color or a mixture of these two colors
was indicated as positive for in the presence of steroid compounds (15).
Test for Tannins:
the presence of tannins.
The mixture was boiled for five mintues. Two drops of 5% FeCl3 were then added. Production of greenish
precipitate indicated the presence of tannins (15).
Test for Flavanoids:
A small piece of magnesium ribbon was added to extract of the plant material,this was followed by the drop wise
addition of concentrated hydrochloric acid. Colors varying from orange to red crimson to magneta indicated
Test for Alkaloids:
The extract of plant sample(0.5g) was stirred with 5ml of HCL on a steam bath. The solution obtained filtrate was
treated with two drops of mayer's reagent. The two solutions were mixed and made up to 100 ml with distilled
water. Turbidity of the extract filtrate on addition of Mayer’s reagent was regarded as evidence for the presence of
Test for saponins:
The extract of plant sample (0.5g) was introduced in to a tube containing 5.0 ml of distilled water and shake
Vigorously for 2 minutes formation of froth indicates the presence of saponins(15).
Test for glycosides:
The extract of plant sample 1g added in to separate beakers .to one of the beakers was added 5ml dilute sulphuric
acid while 5ml sulphuricacid is added to other beaker. The two beakers were heated for 3-5 minutes and the contents
Filtered in to labeled test tubes. The filtrate was made alkaline with 5%sodium hydroxide and heated with fehlings
Solution for 3 mins. The presence of reddish precipitate in the acid filtrate and the absence of such precipitate in the
aqueous filtrate were regarded as positive for glycosides (15).
Antibacterial activity was assessed by Agar well diffusion method of Kirby Bauer where in Muller hinton (5) agar
plates were prepared and were spreaded with 30ul of the available pathogenic cultures. Wells of 6mm diameter
were bored using sterile borer. Wells were loaded with antimicrobial, tetracycline as standard and distilled water as
Control and were incubated at 37oC for 24 hours. (6)
Minimum Inhibitory Concentration
MIC of the antimicrobial extracts was also determined using broth serial dilution technique wherein the
Antimicrobial was diluted serially in a series of test tubes containing nutrient broth and they were loaded with the
respective pathogen against which MIC was to be calculated. The tubes were incubated and then growth of the
pathogen was detected using spectrophotometer at 600 nm. Concentration in the tube where growth increased
drastically was stated as Minimum inhibitory concentration.
The development of drug resistance in human pathogens against commonly used antibiotics necessitated a search for
new antimicrobials of mainly plant origin. The Preliminary phytochemical screening and antibacterial screening of
various extracts of Punica granatum and whole plant of Tribulus terrestris showed good results as illustrated in
the Table-1 and 2.
preliminary phytochemical screening of the whole plant extract of phyllanthus and peel of punica granatum showed positive results for the presence of secondry metabolites like flavonoids, alkaloids, tannins, glycosides, steroids, saponins, tannins are less amount in both plants. Bio active compounds like flavonoids,alkaloids, glycosides are rich in methanolic extract.
Nearly 80% of the world populations depends on the traditional medicine for primary health care, mainly including
the use of natural products . Researchers have extensively studied the biological properties of Punica granatum
and whole plant of phyllanthus niruri extracts their results showed that this plant is ethno medically (7)valuable . Punica granatum peel and whole plant of phyllanthus niruri extracts extracts arecurrently used for treatment of UTI diseases and in the preparation of therapeutic formulae. The tannin rich ellagitannins and phenolic acids of Punica granatum have antibacterial, antifungal and antiprotozoal activity(8-10) In the current study the hot aqueous, methanolic and ethanolic extracts of Punica granatum and whole plant of phyllanthus niruri extracts showed Zone ofinhibition of atleast 12mm against P.aeruginosa which was greater than that of ampcillin 14 , 16mm against E.coli which was a little lesser than that of Standard (16mm) and 14mm against E.coli which was greater than thatof standard ampcillin (16mm) respectively. The antibacterial activity of peels of Punica granatum and whole plant of phyllanthus niruri extracts may beindicative of presence of metabolic toxins or broad spectrum antimicrobial compounds that act against bothgram+ve and gram –ve bacteria. Ethanolic extracts exhibited higher degree of antibacterial activity as compared tothat of other extracts tested against bacteria that cause gut infection, stomachache, diarrhea(11). Reported that P.granatum and whole plant of phyllanthus niruri contains large amount of alkaloids (25%) and antibacterial activity may be indicative of presence of secondary metabolites. The ethanolic extract of P. granatum and whole plant of phyllanthus niruri extracts showed some extent of antibacterial activity against E.coli and S. aureus .(12-13).
My special thanks to Dr.Uma Shankar who has given me constant encouragement and support to carry this research work. My sincere thanks to Dr. Uma for constant encouragement and key hypothesis for this work.
United Kingdom. Century Hutchinson Ltd.
Table:1 Preliminary phytochemical screening Methanolic extract peel of Punica granatum and whole plant of phyllanthus niruri
Table:2 antibacterial screening of various extracts peel of Punica granatum and whole plant of phyllanthus niruri
Figure:1 antimicrobial activity of aqueous extracts
Figure:2 antimicrobial activity of ethanol extracts
Figure:3 antimicrobial activity of Methanol extracts
Series1-klebsiella ,series2-enterococci,series3-E.coli, series4-pseudomonas, series5-S.aureus
X-axis-microorganisms, Y-axis-plant extract activity (zone of inhibition in mm)
In the present study an attempt has been made to decipher the preliminary screening of plant extracts and antimicrobial activity of peels of Punica granatum and whole plant of phyllanthus niruri extracts (which are generally treated as wastes). Peels of Punica granatum and whole plant of phyllanthus niruri extracts are reported to have polyphenols, flavonoids, alkaloids, tannins, and anthocyanins (Cyanidins, delphinidins) as bioactive compounds in previous studies. All the three extracts have antibacterial activity against bacterial strains, Klebsiella, Enterococci E. coli, P. aeruginosa, S. aureus). After further purification and characterization of the active metabolites present in Punica granatum and whole plant of phyllanthus niruri extracts followed by detailed study of toxicity and pharmacological effects of the compound, the peel extracts of pomegranate and whole plant of phyllanthus niruri extracts may be used as remedy against various diseases without any side effects.