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Indian Journal of Natural Products and Resources

Vol. 1 (2), June 2010, pp. 213-215

Antibacterial activity of black pepper (Piper nigrum Linn.) with special reference
to its mode of action on bacteria
Pavithra Vani Karsha and O Bhagya Lakshmi*
Department of Microbiology and Department of Botany
Sarojini Naidu Vanita Maha Vidhyalaya
Exhibition Grounds, M J Road, Nampally, Hyderabad − 500 001, Andhra Pradesh, India
Received 8 June 2009; Accepted 7 December 2009

During present study the antibacterial activity of black pepper (Piper nigrum Linn.) and its mode of action on bacteria
were done. The extracts of black pepper were evaluated for antibacterial activity by disc diffusion method. The minimum
inhibitory concentration (MIC) was determined by tube dilution method and mode of action was studied on membrane
leakage of UV260 and UV280 absorbing material spectrophotometrically.
The diameter of the zone of inhibition against various Gram positive and Gram negative bacteria was measured. The
MIC was found to be 50-500ppm. Black pepper altered the membrane permeability resulting the leakage of the UV260 and
UV280 absorbing material i.e., nucleic acids and proteins into the extra cellular medium. The results indicate excellent
inhibition on the growth of Gram positive bacteria like Staphylococcus aureus, followed by Bacillus cereus and
Streptococcus faecalis. Among the Gram negative bacteria Pseudomonas aeruginosa was more susceptible followed by
Salmonella typhi and Escherichia coli.

Keywords: Antibacterial activity, Black pepper, Piper nigrum, Gram positive bacteria, Gram negative bacteria, Nucleic acids.
IPC code; Int. cl.8  A61K 36/00, A61K 36/67, A61K 131/00, A61P 31/00, A61P 31/04

Introduction In present study the antibacterial activity of black
The plant extracts and secondary metabolites pepper and its mode of action on bacteria by membrane
possess antimicrobial, antifungal or antiviral leakage studies i.e., leakage of UV260 and UV280
activities. The various plant products, that are absorbing materials spectrophotometrically were done.
regularly used for their therapeutic potential, and
plants or plant products that form the part of the food Materials and Methods
Plant material
or as dietary components, have been receiving Black pepper (Piper nigrum) berries were
considerable attention. Though much is known about purchased from local market.
the chemistry and the antimicrobial action of several
phytochemicals, very few reports are available on the Bacterial cultures
possible mechanism of action. For phenols and Gram positive bacteria: Staphylococcus aureus
phenolic compounds, an injury of membrane (NCIM-2079), Bacillus cereus (NCIM-2016) and
functions has been proposed as a mechanism of Streptococcus faecalis (NCIM-2063) and Gram
action1-4. negative bacteria: Escherichia coli (NCIM-2089),
Black pepper (Piper nigrum Linn.) is a flowering Klebsiella pneumoniae (NCIM -2957), Pseudomonas
vine of Piperaceae family. It is native to India and has aeruginosa (NCIM-2200), and Salmonella typhi
been a prized spice since ancient times. The volatile (NCIM-2263) were obtained from National Collection
oil of pepper has been shown to have antimicrobial of Industrial Micro-organisms (NCIM), NCL, Pune.
acitivity5. Black pepper has many medicinal Chemicals
properties like it is used to treat vertigo, asthma, Acetone and Dichloromethane (DCM) as solvents
chronic indigestion, colon toxins, obesity, sinusitis, for the extraction of black pepper (Berry), nutrient
congestion, fever, paralytic, arthritic disorders and agar and nutrient broth for bacterial cultivation,
also advised in diarrhoea and cholera3, 6. standard antibiotic like Ampicillin were purchased
______________ from HI Media laboratories India, Ltd and Piperine
*Correspondent author: E-mail: from M/s Sigma Chemicals.

the test compound was Streptococcus faecalis 500 125 250 1.5 to 125 ppm for control. Streptococcus faecalis 18 15 13 Pseudomonas aeruginosa 15 14 24 Mode of action of pepper extract on bacteria Escherichia coli 10 NI 18 The method of Heipieper10 was followed to Klebsiella pneumoniae 10 12 20 determine the leakage of UV260 and UV280 absorbing Salmonella typhi 14 NI 26 material. At regular intervals of 15 Pseudomonas aeruginosa 62. Ampicillin. After incubation for Staphylococcus aureus 125 125 250 0.98 min aliquots of the samples were drawn. The diameter The DCM extract of black pepper showed good of zone of growth inhibition was recorded.5.625 .5 250 125 30 min at room temperature.214 INDIAN J NAT PROD RESOUR.5 125 250 0. respectively. Disc diffusion method The acetone extract of black pepper displayed The in vitro antibacterial activity of the acetone and excellent inhibition on the growth of Gram positive DCM extracts of pepper was carried out by disc bacteria. coli. The culture tubes were Acetone DCM extract Ampicillin incubated at 37°C for 24 hours. The pepper powder (50g) was then A small portion of the dry extract was used for extracted with two solvents. Two fold serial dilution of the test compound was Table 1Antibacterial activity of Black pepper extracts determined by disc diffusion method carried out in the nutrient broth. The active principle MIC was determined by serial dilution method9. The extracts obtained were concentrated Results and Discussion to dryness by evaporating the solvent under reduced The zone of inhibition was measured for both pressure7. Gram positive bacteria and 125 to 250 ppm for Gram Minimum inhibitory concentration (MIC) negative bacteria (Table 2). acetone and DCM phytochemical screening test11. cultures and incubated for 24h at 37°C. The various extracts (5µl or Among the Gram negative bacteria Pseudomonas was 5mg) were loaded onto different filter paper discs more susceptible to black pepper followed by E. NI-No inhibition aureus cells were grown overnight with continuous Table 2Determination of minimum inhibitory concentration of Black pepper extracts in ppm shaking in nutrient broth at 37°C. harvested.9 and UV260 and UV280 absorbing material in the Klebsiella pneumoniae 125 125 250 1. prepared from Whatman No: 1 filter paper. JUNE 2010 The plant material was ground to a fine powder Phytochemical screening before extraction. Actively growing log phase followed by Bacillus and Streptococcus. S.9 added to suspension at MIC. 250 and 500 ppm. To each test tube 105 CFU/ml of actively growing bacterial cultures in log Bacteria Zone of inhibition (mm) phase was inoculated. bacteria but less efficient than that of standard antibiotic. 12.0 at A450. Staphylococcus was more susceptible diffusion method8.98 Bacillus cereus 250 62. It was found that Gram positive concentration of the extract would be 1g/ml of bacteria were more susceptible than Gram negative DMSO. viz. 125 and 250 ppm. washed with 10mm EDTA and then twice in distilled water by Bacteria MIC in ppm centrifugation each time at 6000 rpm for 15 min at Acetone DCM Piperine Ampicillin 4°C and resuspended such that the absorbance of the extract extract final suspension was 2. centrifuged Escherichia coli 125 125 250 1. After the incubation extract (5µl) (5µl) (10 µg/disc) the tubes are checked for the growth of bacteria and Staphylococcus aureus 20 14 22 MIC of that extract was determined and expressed in Bacillus cereus 15 12 19 ppm. 1% agar) and plated. using soxhlet apparatus by continuous heat extraction for 24 hours. The minimum inhibitory Ampicillin (10µg/disc) and DMSO alone served as concentration ranged between 62. Salmonella typhi 250 250 250 15. The concentration thus obtained was acetone and DCM extract of pepper and the results dissolved in DMSO in such a way that the final depicted in Table 1. The MIC cultures were mixed in soft agar (Nutrient broth with values are 125. The effects activity and inhibited both Gram positive and Gram were compared with that of the standard antibiotic negative bacteria. The bacterial suspension was prepared.9 suspension was measured. The discs Klebsiella and Salmonella (62. were then placed on the agar medium containing the respectively).

Ashok K and Bheema 260nm (nucleic acid material). negative bacteria tested.3 new antibiotics.). 1992.3 2 Mitscher LA. 81-83. 2000. J Food Prot.1 0. 30 0. 1019-1024. Present study shows that both the extracts of black 12 Harborne JB. The material from mechanism of antibacterial action appears to be loss of Staphylococcus aureus spectrophotometrically control over cell membrane permeability. 1978. material (mainly nucleic acid material and protein) 6 Ravindran PN.2 0. membrane permeability resulting the leakage of the Antimicrobial activity of essential oils from plants against UV260 and UV280 absorbing material.). (Table 3). 1978. 1981. KARSHA & LAKSHMI: ANTIBACTERIAL ACTIVITY BLACK PEPPER 215 Table 3Determination of leakage of UV260 and UV280 absorbing bacteria are more susceptible to the extracts. Ph. Time in minutes Absorbance at UV260 nm UV280 nm References 0 0 0 1 Aizenman BE. 11 Trease GE and Evans WC. 89. Center for absorbance at 280nm (protein) was increased in Medicinal Plants Research. Studies on bioactive natural Compounds for 75 0. Joshi SD. submitted to Osmania University. J Chromatogr Libr.3 their antimicrobial and antioxidant Properties.Industrial profiles. 15. at 250 ppm against all the Gram positive and Gram 623-632.3 (Chem Abstr. 348- 383. London. 1973. 44. The results suggest that pepper alters the 8 Elgayyar M. 60 0. 20297t. Plant derived antibiotics. India. which took 75min Chari. volatile oil. Appl Environ Microbiol. 2005. The and Aromatic plants. 1983. 5 Dorman HJ and Deans SG.3 3 Sashidhar NS. Leakage of selected pathogenic and saprophytic microorganisms. Gram positive 1st Edn. Diefenbach R and Kewelol H. The phytochemical analysis showed the presence 10 Heipieper HJ. London. Phytochemical Methods: A Guide to Modern pepper have good antibacterial activity. Kerala. 64 (7). Conversion of of alkaloids. 271. seems to be one of the modes of bactericidal action. The alkaloids like piperine. Hyderabad. 208. 12th Edn.1 0. 35 0. 463-477. p. 90 0. Aravind MB.1 0. 233-241. Antimicrobial agents from plants: antibacterial activity of plant volatile oils. 2001. a possible mechanism for and resins. Antibacterial and Antifungal activity of Thespesia populnea (Linn. Draughon FA. D Thesis. J Appl Microbiol.31 2002. 1975. Edward Arnold Ltd. 57-59. Higher plants as source for the preparation of 15 0. 40. Antimicrobial activity of non- piperine alone showed excellent bactericidal activity halogenated phenolic compounds.34 0. compared to Gram negative bacteria. antibacterial activity. which Prot. London. 58. 9 Stokes EJ. J Food intracellular materials might cause cell death. 308-316. Study on leakage of UV260 and UV280 absorbing 2000. Black pepper: Piper nigrum series: Medicinal was monitored over a period of 90 minutes. Mikrobial Zh (Kiev). but when Techniques of Plant Analysis. piperidine. 4 Davidson PM and Branen AL. Conclusion Balliere & Tindall. the protection of phenol degrading Pseudomonas putida P8 volatile oil and resins might be responsible for the from substrate toxicity. Clinical Bacteriology. 42(2). p. mono.and polysaccharides cis unsaturated fatty acids to trans. Indian Drugs.08 0. Golden DA and Mount JR. . Text book of Pharmacognosy. 88(2). pp. India. 15min only when compared to the absorbance at 7 Shastry CS. 1847-1852. Chapman and Hall.