A Centre for innovative research, Jawaharlal Nehru technological university, Hyderabad. Department of Chemistry, Sri Venkateswara University, Tirupati. Key words




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НазваниеA Centre for innovative research, Jawaharlal Nehru technological university, Hyderabad. Department of Chemistry, Sri Venkateswara University, Tirupati. Key words
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Received by Qing Received on 2012-5-7

ID No. B330 Revised on


东高止山脉所选药用植物的抗菌活性的筛选和评估

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


  1. Centre for innovative research, Jawaharlal Nehru technological university, Hyderabad.

  2. Department of Chemistry, Sri Venkateswara University, Tirupati.



Key words:

Punica Granatum, phyllanthus niruri, Antibacterial activity, Minimum Inhibitory Concentration, Zone Of Inhibition


ABSTRACT


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.


Corresponding Author

N.Chandrasekharnath

Centre for Innovative Research,

Institute of Science and Technology,

Jawaharlal Nehru Technological University Hyderabad,

Kukatpally, Hyderabad-500072.

Telephone: 91-9493871986

Email: chandrasekharnath@gmail.com


Introduction

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


Plant Materials


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.


Test Organisms:


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.


Extraction procedure:


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:

  • 1 cm3 of freshly prepared 10% KOH was added to 1 cm3 of the extract. A dirty white precipitate indicated


the presence of tannins.




  • Powdered coarse powder of test plant (1.0) was weighed into a beaker and 10 ml of distilled water added.


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


flavonones(15).


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


alkaloids (15).


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).


Antimicrobial activity:


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.


Results:


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.


DISCUSSION

Phytochemical analysis:

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.

Antimicrobial activity:

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).

ACKNOWLEDGEMENT:

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.


REFERENCES:


  1. Anonymous, Pharmacopiea of India Indian Pharmacopiea), 3 Edn., Govt. of India, Delhi, Ministry of Health and Family Welfare1996.

  2. Gerhartz, W., Y.S. Yamamota, F.T. Campbell, R. Pfefferkorn and J.F. Rounsaville, Ullmann’s Encyclopedia of Industrial 1985.

  3. Harborne, J.B., Phytochemical methods. In A guide to modern techniques of plant analysis 3rd ed.,pp:40-137. 1998.

  4. Kroschwitz, J.I. and M. Howe-Grant, Kirk- Kandha tribe of Orissa, India. J. Ethnopharmacol. 1992.

  5. Baur AW, Kirby WM, Sherris JC, Turck M. Antibiotic susceptibility testing by a standard single disk method. Am J Clin Path 1966; 45: 493-496.

  6. Prashanth D, Asha M, Amit A Antibacterial activity of Punica granatum. Fitoterapia. 2001-72:171–173.

  7. sahuvinod.k,irchhaiyaraghuveer,shashialok,gurjar himanshu phytochemical investigation and chromatographic evolution of the ethanol extract of whole plant extract. Issue 1, Vol. 1 2010.

  8. Shibumon G and Benny P J A review on the medicinal significance of common fruits. Int. J.Biomed. Res. Analysis. 2010.1(2): 60-64.

  9. Stockwell, C., 1988. Nature’s pharmacy. London United Kingdom. Century Hutchinson Ltd.

  10. Supayang PV, Treechada S, Surasak L, Thanomjit S, Tetsuya I, Takeshi H Inhibitory effect of active compounds from Punica granatum pericarp on verocytotoxin production by enterohaemorrhagic Escherichia coli O 157: H 7: J. Health Science. 2005.51: 590-596.

  11. Taylor, R.S.L., N.P. Manandhar, J.B. Hudson and I. G.H.N. Towers, Screening of selected antifungal medicinal plants of Nepal for antimicrobial activities.J. Ethnopharmacol., 1995. 546: 153-159.

  12. Thomson, W.A.R., Medicines from the Earth. propilis and antimicrobial drugs. Microbiol. Res.,Maidenhead, United Kingdom. McGraw-Hill Book 1978. 158: 353-357.Co.

United Kingdom. Century Hutchinson Ltd.

  1. Vasconcelos LCD, Sampaio MCC, Sampaio FC, Higino JSUse of punicca granatum as an antifungal agent against candidosis associated with denature stomatitis. Mycoses. 2003. 46(5-6): 192-196.

  2. Voravuthikunchai S, Sririrak T, Limsuwan S, Supawita T, Iida T, Honda T Inhibitory effects of active compounds from Punica granatum pericarp on verocytotoxin production by enterohaemorrhagic Escherichia coli O157:H7. J Health Sci. 2005.51:590–596.

  3. Voravuthikunchai S, Lortheeranuwat A, JeejuW, Sririrak T, Phongpaichit S, Supawita T Effective medicinal plants against enterohaemorrhagic Escherichia coli O157:H7. J Ethnopharmacol 2004; 94:49–54.

  4. Mahesh B, Satish S. Antimicrobial activity of some important medicinal plants against plant and human pathogens. World J Agric Sci 2008; 4: 839-843.

  5. Ahmad I, Aqil F. In vitro efficacy of bioactive extracts of 15 medicinal plants against ESbL-producing multidrug-resistant enteric bacteria. Microbiol Res 2007; 162: 264-275.

  6. Barbour EK, Al Sharif M, Sagherian VK, Habre AN, Talhouk RS, Talhouk SN. Screening of selected indigenous plants of Lebanon for antimicrobial activity. J Ethnopharmacol 2004, 93: 1-7.

  7. Iwu MW, Duncan AR, Okunji CO. New antimicrobials of plant origin In: Perspectives on new Crops and new Uses, eds. J. Janick, ASHS Press, Alexandria, VA, 1999; 457-/462.

  8. Tomoko N, Takashi A, Hiromu T, Yuka I, Hiroko M, Munekazu I, Totshiyuki T, Tetsuro I., Fujio A, Iriya I, Tsutomu N, Kazuhito W. Antibacterial activity of extracts prepared from tropical and subtropical plants on methicillin-resistant Staphylococcus aureus. J Health Sci 2002; 48: 273-276.


Tables:


Table:1 Preliminary phytochemical screening Methanolic extract peel of Punica granatum and whole plant of phyllanthus niruri


Methanol extract

Contain chemical compounds

Color test result
Punica granatum

Color test result
phyllanthus niruri

flavonoids

++

++

alkaloids

+++

+++

tanins

+

+

glycosides

++

++

steroids

++

+

saponins



++

+



Table:2 antibacterial screening of various extracts peel of Punica granatum and whole plant of phyllanthus niruri

Test organisms

Aqeous extracts

Methanolic extracts

Ethanolic extracts

ZONE OF INIHIBITIONS

ZONE OF INIHIBITIONS

ZONE OF INIHIBITIONS




Peel of punica granatum

(mm)

Whole plant of phyllanthus niruri (mm)

Ampcillin antibiotic

(mm)

Peel of punica granatum

(mm)

Whole plant of phyllanthus niruri

(mm)

Ampcillin antibiotic

(mm)

Peel of punica granatum

(mm)

Whole plant of phyllanthus niruri

(mm)

Ampcillin antibiotic

(mm)

klebsiella

12

11

11

13

12

12

12

12

10

Enterococci

13

14

10

14

16

10

13

13

11

E.coli

16

12

12

16

13

11

14

14

12

Pseudomonas

14

13

10

12

14

10

16

13

13

S.aureus

12

15

11

13

11

11

12

13

11



Figures:


Figure:1 antimicrobial activity of aqueous extracts


Figure:2 antimicrobial activity of ethanol extracts


Figure:3 antimicrobial activity of Methanol extracts


Abbreviations:

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)


CONCLUSION:

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.

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