British Journal of Pharmacology and Toxicology 1(1): 40-44, 2010
ISSN: 2044-2467
© M axwell Scientific Organization, 2010
Submitted Date: May 18, 2010
Accepted Date: May 29, 2010
Published Date: June 20, 2010
Antimicrobial Activity of Catharanthus roseus – A Detailed Study
Prajakta J. Patil and Jai S. Ghosh
Departm ent of Microbiolog y, Shivaji University , Kolhapur 41 600 4. M S. India
Abstract: Catharanthus roseus (periwinkle) is an important medicinal plant for novel pharmaceuticals since
most of the bacterial pathog ens are developing resistance against many of the currently available anti microbial
drugs. Plants have proved to be significant natural resources for effective chemotherapeutic agents and offering
a broad spectrum of activity with greater emphasis on preventive action. This study aims to investigate some
of the anti microbial properties of this plant. The an ticancer prope rties of Catharanthus roseus has been the
major interest in all investigations. The antimicrobial activity has been checked against microorganisms like
Pseudomonas aeruginosa NC IM 2036, Salmonella typhimurium NC IM 2501, Staphylococcus aureus NC IM
5021. The findings show that the extracts from the leaves of this plant can be used as prophylactic agent in
man y of the diseases, which some time are of the m agnitude of an epidemic.
Key w ords: Catharanthus, leafextracts, periwinkle, prophylaxis, Pseudomonas
INTRODUCTION
Medicinal plant products could prove useful in
m i n i m i z in g t h e a d v e r s e e f f e c ts o f v a ri o us
chemotherapeutic agents as well as in prolonging
longe vity and attaining positive general health
(Kaushik et al., 2002). The increasing global interest in
the medicinal potential of plants during the last few
deca des is therefore quite log ical.
Catharanthus roseus is an important medicinal plant
of family Apocynace ae. It is cultivated mainly for its
alkaloids, which are having antican cer activities
(Jaleel et al., 2009). M uhammad et al. (2009) reported
that the antibacterial potential in crude extracts of
different parts (viz., leaves, stem, root and flower) of
C. roseus against clinically significant bacterial strains.
Emerging and reemerging infections and spread of deadly,
drug-resistant strains of organisms pose a challenge to the
global public health for their treatment. Bacterial
resistance to antibiotics is a major therapeutic problem
and the rate at which new antibiotics are being produced
is slowing, (Russell et al., 2002). Thus, the search for
novel antimicrobial agents is of the utmost importance
(Gootz et al., 1990). Global attention has been shifted
towards finding new chemicals, specifically herbals, for
the development of new drugs. These natural products can
provide unique elements of molecular diversity and
biological functionality, which is indispensable for novel
drug discovery (Nisbet and M oore, 1997).
Catharanthus roseus possesses known antibacterial,
antifungal, antidiabetic, anticancer and antiviral activities.
The extracts have dem onstrated significant anticancer
activity against numerous cell types (EL-Sayed and
Corde ll, 1981).
The plant sh ows the presence of various alkaloids,
viz.
1) Vincristine, which binds to tubulin dimers,
inhibiting assem bly of m icrotub ule structures. Disruption
of the microtubules arrests mitosis in metaphase . The
vinca alkaloids therefore affect all rapidly dividing cell
types including cancer cells, but also intestinal epithelium
and bone m arrow (G raf et al., 1996). 2) Vinblastine is an
antimicrotub ule drug used to treat certain kinds of cancer
(Starling, 1976). 3) Yohimbine (Procomil) is an alkaloid
with stimulant and aphrodisiac effects found naturally in
Pausinystalia yohimbine (Millon et al., 2002). C. roseus
also shows the presence of this compound along with
another flavonoide hirsutidin (Piovan and Fillipini, 2007).
The different parts of C. roseus (leaf, stem, flower
and root) were used and extracts were subjected to
antibacterial assay. The extracts of C. roseus did not
exhibit antibacterial activity ag ainst Staphylococcus
aureus. Moreover, leaf, stem and flowe r extracts were
also ineffective against Pseudom onas aeruginosa. The
leave extrac t did not exhibit activity against
Corynebacterium diphtheriae; similarly, the crude extract
of stem d id not sh own activity against Shige lla boydii.
The most effective was the root extract, which exhibited
broad-spectrum antibacterial activity aga inst Salmonella
typhimurium and S. boy dii. The flower extract showed
activity against C. diphtheriae (Perez et al., 1990).
The present investigation is focused on screening of
leaf extracts of the plant for its antibacterial potential
adopting the routine antibacterial assay techniques.
MATERIALS AND METHODS
This work was carried out between June 2009 and
March 2010. The plant being a perennial one, there was
no difficulty in getting the material for the investigation.
Corresponding Author: Dr. Jai S. Ghosh, Department of Microbiology, Shivaji University, Kolhapur 416004, MS, India
Tel: +91 9850515620
40
Br. J. Pharm. Toxicol., 1(1): 40-44, 2010
Table 1: Composition
S. No.
1
2
3
4
of nutrient agar medium
Com ponen ts
Peptone
Yeast extract
NaCl
Agar
Table 2: Composition of mineral base medium
S. No.
Composition
1
So dium nitrate
2
Dip otass ium hyd rog en p hos pha te
3
Kcl
4
Glucose
5
Yeast extract
6
Agar
Tab le 3: Zone of inhibition on nutrient agar usin g 1: 1 0 dilu ted e xtrac ts
Organisms
Methanol Ethanol Acetone
Pseudomonas aeruginosa NCIM 2036 2mm
_
_
S a lm o ne ll a t yp h im u ri um NCIM 2501
_
_
_
Staphyloco ccus aureu s NCIM 5021
_
_
_
P erc en ta ge (% )
1.0
1.0
0.5
2.5
Tab le 4: S olid c onte nt of the e xtrac ts
Ex tracts
Acetone extract
Ethanol extract
Methanol extract
P erc en ta ge (% )
0.20
0.10
0.05
1.00
0.02
2.50
S olid re sid ue (% )
0.16
0.26
0.36
Tab le 5: Growth in presence of respective extracts- concentrated 1:20
using mineral based medium with glucose
Organisms
Methanol
Acetone
Pseudomonas aeruginosa NCIM 2036
_
_
S a lm o ne ll a t yp h im u ri um NCIM 2501
_
_
Staphyloco ccus aureu s NCIM 5021
_
_
_: denotes no growth o f microorganism
Preparation of extract: The crushed leaves were taken
in mortar and pestle and to which different solvents w ere
added individually and mixed well. The solvents used
were ethanol, acetone, methanol. This was then
centrifuged at 3220xg for 10 min and the supernatants
were collected se parately for further study.
Tab le 6: Gro wth in presence of respective extracts- concentrated 1:40
using mineral based medium with glucose
Organisms
Methanol
Acetone
Pseudomonas aeruginosa NCIM 2036
+
+
S a lm o ne ll a t yp h im u ri um NCIM 2501
+
+
Staphyloco ccus aureu s NCIM 5021
+
+
Bacillus subtilis NCIM 2010
+
+
+: denotes grow th of microorgan ism
Cultures used for antimicrobial activity: The
microorganisms used were as follows, Staphylococcus
aureus NC IM 5021, Pseudomonas a erug inosa NC IM
2036, Bacillus subtilis NC IM 2010 and Salm onella
typhimurium NCIM 2501.
Tab le 7: Growth in presence of respective extracts- concentrated 1:60
using mineral based medium with glucose
Organisms
Methanol
Acetone
Pseudomonas aeruginosa NCIM 2036
+
+
S a lm o ne ll a t yp h im u ri um NCIM 2501
+
+
Staphyloco ccus aureu s NCIM 5021
+
+
Bacillus subtilis NCIM 2010
_
_
+: denotes growth of microorganism
Culture medium: Nutrient agar medium and a mineral
based medium were used in all further studies. The
com positions are as shown in Table 1 and 2,
respectiovely.
Table 8: The nutrient agar containing extracts- concentrated 1:20
Methanol
Ethanol
(extracts )
-----------------------------------O r ga ni sm s
0 .1 m l
1ml
0 .1 m l
1 ml
Salmonella typhimurium
+
+
+
+
Staphylococcus aureus
+
+
+
+
Staphylococcus aureus
+
+
+
+
Bac illus sub tilis
+
+
+
+
Pseudom onas aeruginosa
+
+
+
+
+: denotes growth of microorganism
Antimicrobial testing: Sterile molten nutrient agar at
around 40ºC , was taken into which different cultures was
added and p oured in sterile Petri plate and allowed to be
solidified. After solidification 4 mm wells were prepared.
In these wells solvent extracts of the seed coat we re
added. The plate w as incu bated overn ight at 37 ºC. After
incub ation the zones of inhibition were measured and
recorded. Respective solvent controls were also run
simultaneously.
The above procedure was repeated using mineral
based medium with added yeast extract at 0.02%.
Acetone
--------------0 .1 m l 1 m l
+
+
+
+
+
+
+
+
+
+
glucose (1%), yea st extract (0.1%). The organ isms w ere
inoculated respectively and incubated at 37ºC for
overnight on shaker.
Determination of percentage of solid conten t: This was
done gravimetrically using solutions in different solvents.
Each exp eriment 5 time s to get consistent results.
Detection of phytochemicals by GCMS: The acetone
extract was centrifuged at 3220xg for 10 min and the
supernatant was analyzed by GCM S and on MALDI-TOF
MS.
Take 5 ml of extract evaporate all the so lvents
Then weight of test tube – solid aside = Y gm
RESULTS AND DISCUSSION
Determination of minimum inhibitory concentration
of crude extracts: Differe nt con centration of crude
extract as 1:20, 1:40, 1:60, 1:70, 1:80, 1:90 and 1:100 was
added respectively into mineral based medium containing
The results of bioassay conducted on Nutrient agar
using solvent extract are rep orted in Table 3. Th e results
of bioassay conducted on nutrient agar using concentrated
solvent extract are rep orted in Table 4. The results of
41
Br. J. Pharm. Toxicol., 1(1): 40-44, 2010
Tab le 9: G row th on aga r con tainin g ex tracts a nd w ith an d w ithou t yeas t extra cts
Medium with yeast extract (0.1%)
Organisms
M ediu m w ith leaf extra ct (1 m l)
and leaf e xtrac t (1 m l)
Pseudomonas aeruginosa NCIM 2036
_
+
S a lm o ne ll a t yp h im u ri um NCIM 2501
_
+
Staphyloco ccus aureu s NCIM 5021
_
+
Bacillus subtilis NCIM 2010
_
+
_: Deno tes no grow th of microorgan ism, +: Deno tes growth of m icroorganism
Medium with yeast extract (0.1%)
and leaf e xtrac t(10m l)
_
_
_
_
Fig. 1: GCMS analysis of the acetone extract of the leaves showing the presence of 3, 10 dinitrodiftalone
Fig. 2: GCMS analysis of the acetone extract of the leaves showing the presence of desmethylnomifensine
Bacillus subtilis shows the pure culture and Salm onella
typhimurium shows the mixed culture with Gram positive
bioassay conducted on mineral based agar containing
glucose using solvent extract are reported in Tab le 5. The
results of bioassay conducted on Mineral based agar
without glucose using solvent extract are reported in
Table 6.
The results of spot inoculation conducted on mineral
based agar using solvent extract are reported in Table 7.
It was carried out to seed if over a longer period of time
the organism could grow using some of the components
of the leaf extract. The results of spot inoculation
conducted on nutrient agar using solven t extract are
reported in Table 8. The result of spot inoculation after
Gram staining was as shown in Table 9.
W hen all these organism were Gram stained
Pseudomonas aeruginosa, Staphylococcus aureus and
Table 10: Observation table for minimum inhibitory concentration
Dilutions
Control
Extract
1:10
_
_
1:20
+
+
1:30
+
+
1:40
+
+
1:50
+
+
1:50
+
+
1:60
+
+
1:70
+
+
1:80
+
+
1:90
+
+
1:100
+
+
_: Denotes no growth of microorganism,
+: Deno tes growth of m icroorganism
42
Br. J. Pharm. Toxicol., 1(1): 40-44, 2010
Fig. 3: Analysis of Catharanthus roseus by MALDI-TOF showing the presence of catharanthine (m.w. 336.43), perivine (m.w. 341),
Aparicine (m.w. 318.31) etc
rods with Gram negative rod. Table 10 that the minimum
inhibitory concentration was 1:20 or 0.05 ppm in mineral
based m edium b ut not in nutrient agar.
extracts. These extracts may no t find a the rapeu tic use in
immediate future but definitely it can be used as a
prophylactic agent in regions where certain diseases can
occur as en demic if not in pandem ic scale. It would go a
long way to remove the stress on specific vaccine
production to protect such population (which is mostly in
developing nations) from the pathogen, which changes its
invasive strategies by vary ing it antigenic nature.
It can be seen from the results above that the leaf
extract contained many indole alkaloids, and some
phenolic compounds. The phenolic compounds are known
for their antimicrobial properties. The significance of
these com pounds are that the se can sub stitute long term
antibiotic therapy like in case of chronic kidney infection,
bacterial endoca rditis, carrier conditions of typh oid
(where the organism s resides in gall bladde r). These
compounds having minimum side effect can be easily
substituted for a ntibiotics.
Actually the indole alkaloids do no t show direct an ti
microbial actions but strengthens the immune system and
it is this system that takes care of the pathogens.
Similarly, the strong immune system also w ill take care of
many initiation steps in certain onco genesis. H owever, if
the immune system is too weak o r the organism is too
G C MS analysis of the extracts of the leaf of
Catharanthus roseus: The leaf extracts pre pared in
ethanol and m ethan ol, wh en an alyzed using GCM S did
not show the presence of any significant compounds.
How ever, the extract prepared in acetone showed the
presence of following compounds as shown in Fig. 1 and
2, respectively.
Similarly when the sam e extract was subjected
MALDI-TOF MS analysis (using cinnamic acid matrix)
did show certain com pounds to be present. The results are
as shown in Fig. 3.
DISCUSSION
As can be seen from the literature survey that this
plant has been mostly studied with respect to its an ti
cancer properties and its anti diabetic properties. Till date,
very little studies have been done on the anti microbial
properties of the plant extracts. Therefore, this study
focuses on the anti microbial properties of the leaf
43
Br. J. Pharm. Toxicol., 1(1): 40-44, 2010
virulent then certain other medication will have to be
given along with these compounds to show significant
antimicrobial activity.
Millon, M .J., T.A. Newman, V. Audinot, D. Cussac,
F. Lejeune, J.P. Nicolas, F. Cogé, J.P. Galizzi and
J.A. Boutin, 2002. Agonist and antagonist actions of
Yohimbine as compared to fluparoxan at alpha (2)adrenergic receptors (AR). Synapse, 35: 79-95.
Muham mad, L.R., N. Muhammad, A . Tanveer and
S.N. Baqir, 2009. Antimicrobial activity of different
extracts of Catharanthas roseus. Clin. Exp. Med. J.,
3: 81-8 5.
Nisbet, L.J. and M. M oore, 1997. Will natural products
rema in an important source of drug research for the
future. Curr. Opin. Biotechnol., 8: 706-712.
Perez, C., M. Pauli and P. Ba zerqu e, 199 0. An antibiotic
assay by the agar-well diffusion me thod. A cta Biol.
Med. Exp., 2: 708-712.
Piovan, A. an d R. Fillipini, 2007. A nthoc yanins in
Catharanthus roseus in vivo and in vitro: A review.
Phytochem. Rev., 6: 235-242.
Russell, A.D ., 2002. Antibiotic and biocide resistance in
bacteria: Introduction. J. Appl. Microbial. Symp.
Supply, 2: 176-181.
Starling, D., 1976. Two ultra structurally distinct tubulin
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PTSD (1995). J. Cell Sci., 20: 79-89.
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