Research Article

Antimicrobial activity of Premna serratifolia

Antimicrobial activity of crude extracts and fractions of

Premna serratifolia Lin. root
Rekha Rajendran 1 and N. Saleem Basha2
1Department of Pharmacognosy
2 Pharmaceutical Biotechnology,
Mohamed Sathak A.J. College of Pharmacy, Medavakkam, Chennai - 600 119, India

ABSTRACT
Premna serratifolia Lin., belonging to the family Verbenaceae was screened to evaluate in vitro antimicrobial
activity against the selected human pathogenic organisms. In the present investigation, different extracts (nhexane, chloroform, ethylacetate, ethanol and ethanol fractions (No. I to VII) of the roots of the plant were
obtained. Preliminary phytochemical reports of the extracts and fractions showed the presence of phytoconstituents
like flavonoids, steroids, alkaloids, glycosides and phenolic compounds. Findings showed potential antimicrobial
properties of the extracts (133.33mg/ml) and fractions (33.33mg/ml) of root of Premna serratifolia Lin., against
the different bacterial organisms and fungal tested and the zone of inhibition of the extracts and fraction were
comparable with that of standard antibiotics. The study suggests that the plant is promising for development of
phytomedicine for antimicrobial properties and also indicates the potential usefulness of Premna serratifolia Lin.,
in the treatment of various pathogenic disease which in future can be developed as a potential antimicrobial agent
with reduced toxicity and adverse effects when compared with the synthetic chemotherapeutic agents. [Medicinal
Plants 2010; 2(1) : 00-00].
Keywords : Premna serratifolia Linn, root extracts, fractions, antimicrobial activity, minimum inhibitory
concentration, zone of inhibition.

INTRODUCTION
Nature has provided an excellent storehouse of remedies
to cure all the ailments of mankind. In ancient days,
almost all the medicine used was from natural sources,
particularly from plants and plants continue to be an
important source of new drugs even now. The
importance of botanical, chemical and pharmacological
evaluation of plant derived agents used in the treatment
of human ailments has been increasingly recognized in
the last decades.
Many drugs, which are commonly used in modern
day medicines, have been derived either directly or
indirectly from herbal source. People take medicinal
herbs for several reasons such as they may be

Corresponding author : Rekha Rajendran

E-mail : rekhacognosy24@yahoo.com

Medicinal Plants, 2(1) January 2010

dissatisf ied with allopathic medicines or they may

believe in efficacy of herbal medicines and a strong
belief that there are no side effects for herbal medicines.
Several thousand medicinal plants are discussed and
used to cure various health disorders in India and abroad.
In India, almost 45,000 plant species are growing
naturally or being cultivated. There are so many popular
Indian herbs used in traditional practices to cure various
disorders of human beings. Premna serratifolia Lin., is
an important plant belonging to the family Verbenaceae,
is one of the most widespread large shrub in the forests
of India, usually occurring in deciduous forests. The
whole plant possesses medicinal properties useful in
the treatment of cardiovascular disease, skin diseases,
inflammatory diseases, arthritis, gonnorrhoea,
rheumatism, anorexia and jaundice. Its an important
Ayurvedic medicinal herb and its synonym is Premna
integrifolia Lin. Root forms an ingredient in well known
Ayurvedic formulation Dasamula for variety of

Rajendran and Basha

affections (Anonymous, 1972). It is widespread

throughout Micronesia and much of the tropical Pacific
and tropical Asia. It is common all along the Indian
and Andaman coasts. Infusion of the leaves is
administered with pepper in cold and fever. Leaves are
used to cure weakness of limbs and the leaves and
leaf sap were used to alleviate headache (Kapoor, 2001).
Premna serratifolia Lin., has cardiotonic (Rekha et al.,
2008), anti-coagulant (Gopal and Purushothaman,
1984), anti-arthritic (Rathore et al., 1977), and antihyperglycaemic properties (Dash et al., 2005). Most of
the plant parts of Premna serratifolia Lin., have been
used in the traditional system of medicine in India to
treat various infectious disease.
The literature survey revealed that no systematic
approach has been made to study the antimicrobial
acitivty of the plant Premna serratifolia Lin. Hence
the present study was focused to screen for in vitro
antimicrobial activity of root extracts and fractions of
Premna serratifolia Lin.
MATERIALS AND METHODS
Collection of Plant Material
Fresh root of Premna serratifolia Lin., was collected
from the Indian Medical Practitioners Co-operative
Pharmacy and Stores garden, Chennai, Tamil Nadu,
India. Plant material was identified (Henry et al., 1987).
Plant tissues were washed with water and dried in the
shade until a constant weight was obtained. The
voucher specimen of the plant was kept in the
Department of Pharmacognosy, Mohamed Sathak A.J.
College of Pharmacy, for future reference.
Microorganisms used
The following microorganisms were selected for the
study.
Eleven gram +ve and gram -ve bacterial and 4 fungal
organisms viz., Staphylococcus aureus ATCC-25923,
Coagulase Negative Staphylococca, Escherichia coli
ATCC - 25922, Klebsiella pneumoniae ATCC-25922,
Pseudomonas aeuroginosa ATCC-25921, Salmonella
typhi, Salmonella typhi A ATCC-25924, Salmonella
typhi B ATCC-25925, Vibrio chlorea ATCC-25926 and
Entero cocci ATCC- ATCC-25927 and fungal strains
such as Candida albicans ATCC-25928, Aspergillus
niger ATCC-2091, Pencillium chrysogenum ATCC-2021,
Microsporum
gypseum
ATCC-2952
and
Epidermatophyton flocossum ATCC-2950 were used in

the present study. Standard antimicrobials such as

Ciprofloxacin and Ketoconazole were used as controls.
These organisms were obtained from standard
laboratory maintained in the Institute of Microbiology,
Madras Medical College, Chennai, India. The purity of
the cultures prior to their use was checked by
conventional cultural, morphological and biochemical
methods. The bacterial and fungal cultures were
maintained and stored in nutrient and Sabourauds agar
medium at 4C.
Preparation of Plant Extracts
The freshly collected root was chopped, shaded dried
and coarsely powdered. The powder was defatted with
petroleum ether (60-80 oC) and 500g of the powder was
extracted successively with hexane, chloroform, ethyl
acetate and ethanol (1.5 litres) in an aspirator bottle at
room temperature. The powder was soaked in the solvent
for 72 h and nearly 80% of the solvent was removed by
distillation on a water bath at atmospheric pressure and
last traces were removed under reduced pressure using
rotary evaporator. The residues were suitably diluted
with dimethyl formamide (DMF) so as to get the final
concentration of each extract 1000mg/ml and used for
the study.
Column chromatography (Linskens and Jackson,
1987)
As many chemical constituents shown their presence in
the ethanol extract and this extract have been subjected
to column chromatography for the isolation and
separation of the phytoconstituents. A suitable column
of 2.5cm diameter and 60cm in length was selected and
thoroughly washed with water, dried and rinsed with
acetone and then dried completely. Cotton was placed
in to the column with the help of a big glass rod up
to the neck to avoid leakage of smaller particles of the
adsorbent. Piece of suitable size of filter paper was
placed over the cotton. The column was packed with
silica gel 100 - 200 mesh up to 1/3 rd of its length by
carefully pouring through the funnel to prevent
formation of air bubbles. The sides of the column was
tapped slowly in order to facilitate the packing of the
adsorbent. The prepared column is thoroughly washed
with n-Hexane and the liquid level was always kept
above the surface of the column to prevent the cracking
of the column. The filter paper of suitable size was also
placed above the packed column.
Packing of extract in the column
About 2g of the concentrated ethanol extract was

Medicinal Plants, 2(1) January 2010

Antimicrobial activity of Premna serratifolia

weighed and mixed with suitable quantity of silica gel

(100 - 200 mesh) to ensure the free flow of the extract,
along with the adsorbent it was packed in the column
through the funnel then n-Hexane was added through
the column and kept aside without disturbance for
overnight to settle the extract. The column was eluted
with different organic solvents in the order of the
increasing polarity.
The fractions of 100ml of each of the eluate from
the column were collected into series of 500ml of glass
beakers. The fractions collected were subjected to
qualitative chemical test. The fractions were
concentrated by evaporating the excess of the solvent
and the residues were identified by using different TLC
plates.
Phytochemical screening
The extracts and the fractions were then subjected to
preliminary phytochemical screening for the detection
of various plant constituents as per standardized
procedure (Kokate, 2004). The extracts gave positive
tests for various phytoconstituents like alkaloids,
steroids, flavonoids, phenolic compounds and
glycosides specifically iridoid glycosides whereas the
fractions showed the presence for alkaloids.
Antimicrobial activity (Collee et al., 1989;
Anonymous, 1996; Bauer et al., 1966, Agrawal, 1974;
Bailey et al., 2001; UTZ et al., 1977)
Antibacterial activity
Preparation of Mueller Hinton Agar

Minimum inhibitory concentration

determined and tabulated in Table 1.

(MIC)

was

Zone of Inhibition
The discs of 6 mm diameter were prepared from
Whatman Filter Paper No.41 and sterilized in hot air
oven at 160 o C for one hour. The discs were then
impregnated with the MIC of the extracts and fractions,
standard Ciprofloxacin (5mg/disc) and the solvent
DME.
The MH agar plates were inoculated with the
bacterial culture by the standard procedure. The plates
were allowed to dry. The standard, extract, fractions and
DMF discs were placed on the agar plates and then the
plates were kept at 4oC for 30 min to allow prediffusion
of the standard, extracts, fractions and DMF. The plates
were then incubated at 37 oC for 24 h and the zones of
inhibition were recorded (Table 2).
ANTIFUNGAL ACTIVITY (Utz and Shadomy, 1977)
Preparation of Sabourauds Dextrose Agar
Media used for the growth of fungi was Sabourauds
Dextrose Agar. It contains Mycological peptone - 10g/
L, Dextrose - 40g/L, Agar - 15g/L and distilled water
- 1000ml. 65g of Sabourauds Dextrose Agar was
weighed and dissolved in 1000ml of distilled water.
The pH was adjusted to 5.6 0.2 and sterilized by
autoclaving and used for the antifungal screening.
Determination of Minimum Inhibitory Concentration

Media used for the growth of bacteria was Mueller

Hinton agar (MH). It contains, Beef Infusion - 300g/L,
Casein acid hydrolysate - 17.5g/L, Starch -1.5g/L, Agar
- 17g/L and distilled water - 1000ml. 38g of Mueller
Hinton Agar was weighed and dissolved in 1000ml of
distilled water and adjusted to pH 7.3 0.2, sterilized
by autoclaving at 121C at 15lb pressure for 15 minutes
and used for the antibacterial screening.

The plates were prepared by using Sabourauds dextrose

agar media and different extracts of various dilutions
(33.3, 66.6, 133.3 and 200mg/ml), allowed to solidify
and dry. Then a loopful of the fungal cultures was
inoculated at the labeled spot and the plates were
incubated at 37oC for 24 h. The results were read by the
presence or absence of growth of organism and the
Minimum inhibitory concentration (MIC) was
determined.

Determination of Minimum Inhibitory Concentration

Zone of Inhibition

The plates were prepared by using Mueller Hinton agar

(MH) media and different extracts of various dilutions
(33.3, 66.6, 133.3 and 200mg/ml), allowed to solidify
and dry. Then a loopful of the bacterial cultures was
inoculated at the labeled spot and the plates were
incubated at 37 oC for 24 h. The results were read by the
presence or absence of growth of organism and the

The discs of 6 mm diameter were prepared from

Whatman Filter Paper No.41 and sterilized in hot air
oven at 160 o C for one hour. The discs were then
impregnated with the MIC of the extracts and fractions,
standard Ketoconazole (5mg/disc) and the solvent
DME.

Medicinal Plants, 2(1) January 2010

The Sabourauds dextrose agar plates were

Rajendran and Basha

Table 1. Antimicrobial activity of different fractions of root extracts of Premna serratifolia Lin.
Minimum inhibitory
concentration (g/ml)
Micro-Organisms

Fractions

F1

F2

F3

F4

F5

F6

F7

Staphylococcus aureus

133

133

133

133

33

33

33

33

33

33

33

Coagulase Negative Staphylococca

133

133

133

133

33

33

33

33

33

33

33

Escherichia coli

133

133

133

133

33

33

33

33

33

33

33

Klebsiella pneumoniae

133

133

133

133

33

33

33

33

33

33

33

Pseudomonas aeuroginosa

133

133

133

133

33

33

33

33

33

33

33

Salmonella typhi

133

133

133

133

33

33

33

33

33

33

33

Salmonella typhi typhi A.

133

133

133

133

33

33

33

33

33

33

33

Salmonella typhi B

133

133

133

133

33

33

33

33

33

33

33

Vibrio chlorea

133

133

133

133

33

33

33

33

33

33

33

Entero cocci

133

133

133

133

33

33

33

33

33

33

33

Candida albicans

133

133

133

133

33

33

33

33

33

33

33

Ciprofloxcain

133

133

133

133

33

33

33

33

33

33

33

Aspergillus flavus

133

133

133

133

33

33

33

33

33

33

33

Epidermatophyton flocossum

133

133

133

133

33

33

33

33

33

33

33

Pencillium chrysogenum

133

133

133

133

33

33

33

33

33

33

33

Microsporum gypseum

133

133

133

133

33

33

33

33

33

33

33

Ketoconazole

133

133

133

133

33

33

33

33

33

33

33

Bacteria

Fungus

1. n-Hexane; 2. Chloroform; 3. Ethylacetate; 4. Ethanol; F 1-F 7. Fractions of ethanol extract (Zone of inhibition is the average
of triplicate experiments)

inoculated with the fungal culture by the standard

procedure. The plates were allowed to dry. The standard,
extract, fractions and DMF discs were placed on the
agar plates and then the plates were kept at 4oC for 30
min to allow prediffusion of the standard, extracts,
fractions and DMF. The plates were then incubated at
37oC for 24 h and the zones of inhibition were recorded
(Table 2).
RESULTS
The extracts gave positive tests for various
phytoconstituents like alkaloids, steroids, flavonoids,
phenolic compounds and glycosides specifically iridoid
glycosides whereas the fractions showed the presence

for alkaloids.
The ethanol fractions 1 to 7 may be an alkaloid
which is conf irmed by chemical test, thin layer
chromatography and further studies to identify,
characterize and elucidate the structures of each fraction
was in progress.
All the extracts showed inhibitory response against
all the organisms examined. The MIC of all extracts
was 133.33mg/ml against the bacteria studied. But the
ethanol fractions F 1-7 were found to be 33.33mg/ml
against the test organisms. The zones of inhibition
against the bacteria produced by ethanol fractions were
comparable with that of standard antibiotic

Medicinal Plants, 2(1) January 2010

Antimicrobial activity of Premna serratifolia

Table 2. Antimicrobial activity of root of Premna serratifolia Lin.

Diameter of zone of
inhibition (mm)
Micro-Organisms

Fractions

F1

F2

F3

F4

F5

F6

F7

Staphylococcus aureus

22

14

19

19

15

21

20

20

23

16

13

Coagulase Negative Staphylococca

20

10

20

18

16

19

21

20

20

11

15

Escherichia coli

19

12

18

20

15

19

19

19

22

12

12

Klebsiella pneumoniae

17

13

18

22

13

21

24

18

23

18

13

Pseudomonas aeuroginosa

17

14

19

20

14

18

21

17

23

14

14

Salmonella typhi

20

10

17

19

10

20

18

18

20

15

10

Salmonella typhi typhi A.

17

13

19

19

16

19

17

19

23

18

13

Salmonella typhi B

18

11

18

20

15

21

19

20

21

19

18

Vibrio chlorea

12

12

17

21

12

22

22

21

22

17

12

Entero cocci

19

10

20

19

15

19

19

19

21

19

16

Candida albicans

20

10

18

20

18

20

20

19

20

18

10

Ciprofloxcain

22

14

20

21

18

21

22

20

24

19

17

Aspergillus flavus

10

15

20

22

19

17

18

22

19

17

11

Epidermatophyton flocossum

13

13

15

19

18

20

19

19

21

13

14

Pencillium chrysogenum

10

12

19

20

16

19

18

20

21

15

15

Microsporum gypseum

11

15

20

23

17

18

19

22

20

14

15

Ketoconazole

13

16

22

25

19

21

20

26

23

18

16

Bacteria

Fungus

1. n-Hexane; 2. Chloroform; 3. Ethylacetate; 4. Ethanol; F 1-F 7. Fractions of ethanol extract (Zone of inhibition is the average
of triplicate experiments)

Ciprofloxacin. It showed that the extracts possess

significant antibacterial action due to the presence of
phytoconstituents like alkaloids, steroids, flavonoids,
phenolic compounds and glycosides specifically iridoid
glycosides whereas the ethanol fractions possess
powerful antibacterial action against the bacterial strains
which is due to the presence of alkaloid.
In case of antifungal properties, the extracts and the
fractions were effective in concentration of 33.3mg/ml
against the fungi genera studied. The zones of inhibition
of extracts and the ethanol fraction against the fungal
strains were compared with the standard antibiotic
Ketoconazole. Hence it confirmed that all the extracts
and the fractions possessed signif icant antifungal

Medicinal Plants, 2(1) January 2010

activity.
DISCUSSION
Zones of inhibition produced by the root extracts and
ethanol fractions were comparable with that of standard
antibiotic ciprofloxacin and Ketoconazole (Table 2).
These results support the antimicrobial activity of the
extracts and the fractions against the bacterial and
fungal organisms investigated and the activity is
comparable with the standard ciprofloxacin and
Ketoconazole.
Phytochemical screening of all the extracts revealed
presence of flavonoids, steroids, alkaloids, glycosides

Rajendran and Basha

and phenolic compounds which possibly might have

contributed to the antimicrobial activity. A detailed
investigation into the relationship between the
phytoconstituents and the antimicrobial properties may
bring to light the actual chemical constituents of the
extracts and fractions that have antimicrobial activity.
The present study indicates that the plant possess a
signif icant broad spectrum of antimicrobial activity
and may be of use for development of phytomedicine
for the therapy of infectious disease. Our f indings,
confirm that the traditional therapeutic claims for this
plant, in near future surely be able to replace the
conventional anti-microbial agents to which there is
increased incidence of drug interactions.
Future study of in vivo anti-microbial activity and
the ethanol fractions were further required for the
structural elucidation in near future for the exact
mechanism of action for the observed broad spectrum
of anti-microbial activity.
ACKNOWLEDGEMENT
Authors would like to thank the Head, Department of
Microbiology, Madras Medical College, Chennai, for
providing the microbial cultures for the completion of
this study.
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Medicinal Plants, 2(1) January 2010