Biotechnological Approaches for Sustainable Development (2004)

M Sudhakara Reddy and Sunil Khanna (editors)

Allied Publishers Pvt. Ltd., New Delhi
ISBN: 81-7764-669-9
Pages: 196- 200

ANTIBACTERIAL ACTIVITY IN VITRO OF PLUMBAGIN

ISOLATED FROM PLUMBAGO ZEYLANICA

V. P. ZAMBARE, P. S. KOTHARI, M. V. KULKARNI

School of Life Sciences,

North Maharashtra University, Umavi nagar,
Jalgaon, (MH),
INDIA

ABSTRACT

Plumbagin is a naturally occurring naphthoquinone isolated from roots of

Plumbago zeylanica which used as a traditional medicine for the treatment of
several diseases. Crude plumbagin (0.67%) was extracted with ethanol solvent
from plant roots. All eight microorganisms are multiple antibiotics resistant. The
compound exhibited relatively specific activity against bacteria. The minimum
inhibitory concentration test showed the growth inhibition of Pseudomonas sp.,
Proteus sp., Salmonella paratyphi, Klebsiella sp., Salmonella typhi, Shigella sp.
and Staphylococcus sp., E.coli at a concentration of 80µg/ ml and 90µg/ ml of
plumbagin respectively. The results suggest the naphthoquinone plumbagin as a
promising antibacterial agent.

INTRODUCTION

Antimicrobial agents represent the greatest advance in modern curative medicine. Recent evidence
however points to an inexorable increase in the prevalent of microbial drug resistance apparently
paralleling the expansion of the antimicrobial usage in various fields. Clinically most important
resistance to antibiotics is the result plasmid encoded gene (Neu, 1992). Genetic resistance can
also occur through the acquisition of plasmids, which are circular DNA molecules much smaller
than the bacterial chromosome (Davies, 1994). The antibacterial resistance is due to antibiotic
modification (Spratt, 1994), prevention of antibiotic entry (Williams et al. 1996), antibiotic efflux
(Saier et al. 1998, Levy, 1992) and alternation of antibiotic target (Lancini et al. 1995). Antibiotic
as well as metal resistance was also coded at plasmid level in Acinitobacter baumannii (Shakibaie
et al. 1998).
Antimicrobial Activity In Vitro of Plumbagin Isolated from Plumbago zeylanica 197

Root of plant Plumbago zeylanica contains bioactive compound plumbagin, having

potential as a chemotherapeutic agent (Parimala et al. 1993). MIC of isonizid for Mycobacterium
tuberculosis was reduced by this compound (Frank, 2002). Plumbagin has reproductive effect
(Bhargawa, 1986), cardiotonic action (Itigowa et al. 1991) antimicrobial activity (Dama et al.
1998), insecticidal activity (Krishna et al.1996) hypolipidemic and antiatherosclerotic effect by
reducing the level of cholesterol and LDL-cholesterol in rabbit (Sharma et al.1991). The present
work is the study of antibacterial effect of plumbagin isolated from Plumbago zeylanica on the
multiple antibiotic resistant microorganisms.

MATERIALS AND METHODS

Isolation and Identification of Bacterial Species

Eight bacterial species were isolated from patients at the Govt. Medical College Hospital,
Aurangabad, India. Identification and characterization was carried out as per as Bergey”s manual
of Systematic Bacteriology (Krieg etal.1984).

Antibiotic Profile

All eight isolates were checked for the antibiotic response in octa disk (Himedia, Bombay) on
Muller Hinton agar medium at 370C (Baur et al. 1996). The isolates showing zone of clearance
around the octa disc were sensitive while the growth around the disc were resistant against that
antibiotic.

Source and Partial Purification of Plumbagin

The roots of Plumbago zeylanica plant were collected at the campus of Charak Medicinal
Plantation Field, Jalgaon (India). The extraction of plumbagin was carried out in 95% ethanol by
continues solvent extraction process (Dama, et al. 1998). The extract was concentrated on rotary
vacuum evaporator and a dark brown semi solid mass was obtained. The semi solid mass was
separated into phenolic and neutral fractions by treatment with 5% NaOH solution and neutral
fractions by subsequent acidification of aqueous layer with 2M HCl. The phenolic fraction was
again extracted with diethyl ether and concentrated to obtain crude plumbagin.

Minimum Inhibitory Concentrations (MIC)

MIC of partially purified plumbagin was determined to test the extent to which the isolates were
capable to tolerating the plumbagin. MIC was determined by double dilution method taking
appropriate contents like clarity, turbidity and sterility. Solutions of partially purified plumbagin
10-100 µg/ml were taken and inoculated with 0.1 ml of 24 h old culture of respective organism.
The tubes were incubated at 370C for 24h. MIC was reported as the no growth of organism as
compare to control.
198 Biotechnological Approaches for Sustainable Development

RESULTS AND DISCUSSION

Extraction, Purification and Characterization of Plumbagin

Crude plumbagin (0.67 % w/w of root powder) was obtained by ethanol solvent extraction. Partial
characterization of plumbagin was studied by using silica TLC plate (Merck, Germany) with
diethyl ether: hexane: glacial acetic acid (6:14:80) as a solvent system. Plate was showing the same
Rf value for both standard as well as test sample. Spectral analysis (Shimadzu, UV-VIS 1601,
Japan) showing the same absorption spectra and melting point was near to the standard plumbagin
(Sigma, USA). Comparative accounts of all parameters with standard and experimental plumbagin
are shown in Table.1.

Table 1.
Comparative account of standard and experimental plumbagin

Plumbagin
Sr. No. Criteria
Standard Experimental
1 Rf Value (in TLC) 0.52 0.52
2 UV spectra (λ Max) 250,255,415,455 240,255,415,455
3 Melting point (0C) 78 80

Antibiotic Profile

Among the eight isolates Pseudomonas sp, Proteus sp, Salmonella paratyphi and Klebsiella sp.
were found to be resistance to 8, 8, 6 and 5 antibiotics respectively while E.coli, Staphylococcus
sp., Salmonella typhi and Shigella sp. were less resistance to antibiotics. It was reported that the
resistivity pattern was acquired due to the presence of R- plasmid (Shakibaie et al. 1998).

Minimum Inhibitory Concentrations (MIC)

The MIC value for plumbagin was in the range of 80 - 90 µg/ml for all microorganisms (Table 2).

Antibacterial Activity

All isolates were sensitive to the MIC values of plumbagin isolated from P. zeylanica. Table. 3
showing that staphylococcus sp. E. coli and Shigella sp. are more sensitive while Pseudomonas sp.
and Proteus sp. are less sensitive to experimental plumbagin. Like wise P. scandens has been
reported to treat many diseases, some of them caused by bacteria (Duke et al. 2002).
Antimicrobial Activity In Vitro of Plumbagin Isolated from Plumbago zeylanica 199

Table 2.
MIC of plumbagin (experimental)
Isolate Plumbagin
Isolates name
No. MIC (µg /ml) SIC (µg /ml)
1. Pseudomonas sp. 80 70
2. Proteus sp. 80 70
3. Salmonella paratyphi 80 70
4. Staphylococcus sp. 90 80
5. Klebsiella sp. 80 70
6. Escherichia coli 90 80
7. Salmonella typhi 80 70
8. Shigella sp. 80 70

Table 3.
Antibacterial activity of plumbagin (experimental)
Isolate
Zone of clearance (mm)
No.
1. 10
2. 12
3. 15
4. 16
5. 14
6. 20
7. 14
8. 19

CONCLUSION

Plumbagin showed an interesting activity against the gram-negative bacteria such as Pseudomonas
sp., Proteus sp. and Salmonella paratyphi, Proteus sp., Klebsiella sp., E.coli, Salmonella typhi and
Shigella sp. The experimental results indicated the naphthoquinone plumbagin, as a potential
antibiotic to be consider in the moment of spread of diseases like typhoid, dysentery, diarrhoea etc.
200 Biotechnological Approaches for Sustainable Development

REFERENCES

Baur AW, Kirby WMM, Sherris JC, Turck M (1996). Antibiotic susceptibility testing by a
standardized single disc method. American Journal of Clinical Pathology; 45: 493 -496
Bhargawa SK (1986). Effect of testosterone replacement therapy on quantitative spermatogenesis
following plumbagin treatment in immature rats. Acta Europian Fertilization; 17: 217 –
219
Dama LB, Poul BN, Jadhav BV (1998). Antimicrobial activity of naphthoquinonic compound.
Journal of Ecotoxicology and Environmental Monitoring; 8: 213 – 215
Davies J (1994). Inactivation of antibiotic and the dissemination of resistance gene. Science; 264:
375 – 382
Duke JA, Beckstrom-Sternberg SM (2002). Phytochemical database, USDA-ARS-NGRL,
Beltsville agricultural Research Center, Beltsville, Maryland. http://www.ars-
grin.gov/duke/ethnobot.html.
Frank R (2002). Oxidative stress increases suseptible Mycobacterium tuberculosis to isoniazid.
Antimicobial Agent and Chemotherapy; 46: 2765 – 2771
Frederick MA, Roger B, Robert EK, David DM, Seidman JG, Johnn AS, Kevin S (1994).
Minipreap of plasmid DNA. Current Protocols in Molecular Biology, John Wiley and
Sons Inc, USA,; 1: 1.6.1
Itigowa M, Takeya K (1991). Cardiotonic action of plumbagin on guinea pig papillary muscle.
Planta Medica; 57: 317 – 319
Krieg NR, Holt JG (1984). Gram negative rods and cocci. Bergey's manual of Systematic
Bacteriology (I). Williams and Wilkinsons, Baltimore.
Krishna PV, Rao PJ (1996). Plumbagin a natural bioactive principle agent against insect. The
Andhra Agriculture; 43: 1 -5
Lancini G, Parenti F, Gallo GG (1995). Antibiotic - A multidisciplinary approach. Plenum Plub
Comp, New York.
Levy SB (1992). Active efflux mechanisms for antimicrobial resistance. Antimicrobial Agent and
Chemotherapy; 36: 695 – 703
Neu HC (1992. The crisis of antibiotic resistance. Science; 257: 1067 – 1073
Parimala R, Sachdanandan P (1993) Effect of plumbagin on some glucose metabolizing enzymes
studied in rats in experimental hepatoma. Molecular and Cellular Biochemistry; 12: 59 –
63
Saier MHJ, Paulsen IT, Slivinski MK, Pao SS, Skurry RA, Nikaido H (1998). Evolutionary origins
of multidrug and drug specific efflux pumps in bacteria. FASEB Journal; 12: 265 – 274
Shakibaie MR, Dhakephalkar PK, Kapadnis BP, Salajaghe GA, Chopade BA (1998). Plasmid
mediated silver and antibiotic resistance in Acinitobacter baumannii BL54. Iranian Journal
of Medical Science;23: 36
Sharma I, Gussain D, Dixit VP (1991). Hypolidaemic and antiarthrosclerotic effect of plumbagin
in rabbits. Indian Journal of Experimantal Biology; 18: 215 – 218
Spratt BG (1994). New comprehensive Biochemstry. Elsevier; Amsterdam; 27: 517- 534
Williams RAD, Lambert PA, Singleton P (1996). Antimicrobial drug action. Bios Scientific Publ
Ltd Oxford UK.