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Vol 9, Issue 1, 2016 ISSN - 0974-2441
ANTIBACTERIAL ACTIVITY OF TABERNAEMONTANA DIVARICATA
1111,2*
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2
Received: 04 August 2015, Revised and Accepted: 29 August 2015
ABSTRACT
To study the antibacterial activity of Tabernaemontana divaricata (Apocynaceae) secondary metabolites capped silver nanoparticles
(SNPs) and gold nanoparticles (AuNPs).
In the present investigation, SNPs and AuNPs were synthesized using an aqueous extract of T. divaricata leaves. Leaf aqueous extract
was mixed with 1 mM silver nitrate and chloroauric acid for the biosynthesis of nanoparticles, and the same was analyzed using ultraviolet-visible
(UV-Vis) spectrophotometer and particle size analyzer. The antibacterial activity of SNPs and AuNPs was determined against various bacterial cultures
including laboratory isolates using the agar well diffusion method.
The results recorded from UV-Vis spectrum supported the biosynthesis and characterization of SNPs and AuNPs. The SNPs when compared
to AuNPs, showed the highest antibacterial activity against Gram-positive and Gram-negative bacteria.
The present study envisions on the biosynthesis of SNPs from T. divaricata plant which are emerging as antibacterial therapy in modern
medical applications.
Antibacterial activity, Gold nanoparticles, Silver nanoparticles, Tabernaemontana divaricata.
Medicinal plants have numerous applications in the field of medical
sciences because most of the prescribed drugs are obtained from
herbs, trees, and shrubs. Plants are well-known as a potential source of
modern medicine [1]. Tabernaemontana divaricata, commonly known
as pinwheel flower belongs to the family Apocynaceae, is a beautifully
shaped evergreen shrub which blooms in spring but flowers may appear
sporadically throughout the year and distributed throughout Bangladesh
and other parts of the South East Asia. The phytochemistry, alkaloids and
non-alkaloid constituents such as terpenoids, steroids, flavonoids, phenyl
propanoids, phenolic acids and enzymes from the leaves, stems, and
roots have been reported. In folklore practice, it is used to treat fever and
diarrhea. The plant is also used as a tonic to the brain, liver, and spleen.
It is reported that plant extract possesses antinociceptive, antioxidant,
anti-inflammatory, and reversible acetylcholinesterase inhibition
activities [2-4]. The flower juice can be mixed with oil and used as eye
drops. The milky juice of the leaves along with oil is applied over the
forehead for pain in the eyes. The roots are ground along with water and
given internally for intestinal worms. Nanobiotechnology is an emerging
field that applies the nanoscale principle and techniques to understand
and transform bio systems (living and non-living). Nanoparticles have
expressed significant advances owing to a wide range of applications in the
field of biomedical, sensors, and antimicrobials. Synthesis of nanoparticles
from plant extract is advantageous because they are of low cost, fast,
efficient and generally lead to the formation of crystalline nanoparticles
with a variety of shapes and sizes. In view of the medicinal applications of
this plant, the present study was designed to investigate the antimicrobial
activity of silver nanoparticles (SNPs) and gold nanoparticles (AuNPs)
synthesized from T. divaricata to examine the pharmacological basis of the
use of the plant in folk medicine for the treatment of infectious diseases.
Flowers of T. divaricata were washed thoroughly with autoclaved
distilled water and dried in shade for a week and ground using a mixer
to coarse powder. The powder was used for preparing the aqueous
extract. 1 g of leaf powder was boiled in 10 ml of deionized water for
10 minutes. It was cooled and filtered through Whatman No. 1 filter
paper, and the filtrate was stored at 4°C until further use.
Silver nitrate (AgNO3) of analytical grade (AR) was purchased from
Merck (India). To synthesize SNPs, 1 ml of the aqueous extract of
T. divaricata flower was added to 100 ml of 1 mM AgNO3 solution in
150 ml glass beaker. Then the beaker was incubated for 24 hrs at room
temperature on a magnetic stirrer in the dark place for the reduction
of SNPs. The color change from light yellow to dark orange indicates
the formation of SNPs. An initial setup was also maintained as flower
extract without the addition of AgNO3.
Chloroauric acid was purchased from Sigma–Aldrich Chemicals. To
synthesize AuNPs, 1 ml of the aqueous extract of T. divaricata flower
was added to 100 ml of 1 mM gold chloride solution in 150 ml glass
beaker. This mixture was kept for 24 hrs at room temperature on
a magnetic stirrer at 100 rpm. The color change from light yellow to
purple indicates the formation of AuNPs. An initial setup was also
maintained as flower extract without the addition of gold chloride.
The particle sizes were determined using particle size analyzer (Malvern
Zetasizer nanosizer). Particle sizes were calculated based on measuring the
time dependent fluctuation of scattering of laser light by the nanoparticles.
Bacillus subtilis (MTCC 441), Enterococcus faecalis (ATCC 29212),
Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis
(MTCC 3615), Escherichia coli (ATCC 25922), Proteus vulgaris
(MTCC 1771), Vibrio cholera (ATCC 14035), Bacillus licheniformis strain
018, Bacillus tequilensis strain ARMATI, and B. subtilis strain AK were
used for antibacterial assay.
Research Article
Asian J Pharm Clin Res, Vol 9, Issue 1, 2016, 63-65
Purushothaman et al.
64
Antibacterial activity was carried out using the well diffusion method
on Mueller Hinton Agar (MHA) plates. The test cultures were swabbed
on the top of the solidified media and allowed to dry for 10 minutes.
The aqueous extract of T. divaricata flowers, synthesized T. divaricata
SNPs and AuNps were used at two different concentrations (100 µl
and 150 µl per well). The wells were loaded and left for 30 minutes at
room temperature for compound diffusion. AgNO3 and gold chloride
solutions were used as control. The plates were incubated for 24 hrs
at 37°C, and the zone of inhibition was measured in millimeters (mm).
MIC of SNPs and AuNPs were determined against B. tequilensis strain
ARMATI, B. subtilis strain AK, B. subtilis, S. aureus, B. licheniformis strain
018, and V. cholera. The bacterial suspension was prepared, and 100 µl
of MHA broth was added to the microtitre plate and incorporated with
different concentration (500, 250, 125, 62.5, 31.25, 15.625, 7.81, 3.90,
1.95, 0.976, 0.48, 0.24 µl) of SNP and AuNPs. The microtitre plate was
incubated at 37°C for 24 hrs.
Nanotechnology has become a very active and vital research topic
which is rapidly developing in industrial sectors and spreading
to almost every field of science and engineering. Nanostructured
inorganic, organic, and biological materials may have existed in
nature since the evolution of life started on earth. Rapid synthesis
and excellent reducing capping of SNPs and AuNPs through the plant
extract mediated process has a time-saving advantage over microbial
synthesis and the laborious and lengthy procedures involved with
the mare the environmental friendly. SNPs and AuNPs biosynthesis
may open new process in pharmaceutical chemistry, microbial
biotechnology, biomedical and material science, nanotechnology,
and biotechnology fields. In the present study, the green synthesis
of SNPs through plant extracts were carried out. The appearances
of yellowish-orange in the reaction vessels suggest the formation of
SNPs (Fig. 1). It is well-known that SNPs exhibit yellowish-brown in
aqueous solution due to excitation of surface plasmon vibrations in
SNPs [5]. AgNO3 is used as reducing agent as silver has distinctive
properties such as good conductivity, catalytic and chemical
stability. The aqueous silver ions, when exposed to herbal extracts,
were reduced in solution, there by leading to the formation of silver
hydrosol. The time duration of the change in color varies from plant to
plant. In the present study, T. divaricata synthesized SNPs after 24 hrs
of reaction. The extracellular synthesis of AuNPs occurred during the
exposure of T. divaricata flower extract to 1 mM gold chloride solution
(Fig. 2). The complete reduction of gold ions was observed within 1 hr.
The color change in the reaction mixture was observed during the
incubation period because the formation of AuNPs is able to produce
the particular color in the reaction mixtures due to their specific
properties. The appearance of dark purple is a clear indication of the
formation of AuNPs.
The antibacterial effects of biologically synthesized SNPs and AuNPs
have been investigated against Gram-positive and Gram-negative
strains; E. faecalis, S. aureus, Staphylococcus epidermis, E. coli,
B. tequilensis strain ARMATI, B. subtilis strain AK, B. subtilis, P. vulgaris,
V. cholera and B. licheniformis strain 018. A clear zone of growth
inhibition was observed against strain ARMATI, B. subtilis, S. aureus,
V. cholera and strain 018. It confirms the antibacterial activity of
biologically synthesized nanoparticles. The highest zone of inhibition
was observed in the well loaded with SNPs, and less zone of inhibition
was observed in the well loaded with AuNPs. On the other hand, there
01 S. epidermis - 6 - - -
02 E. coli - - - - -
03 B. tequilensis strain ARMATI - 5 5 - -
04 P. vulgaris - - - - -
05 B. subtilis strain AK - 5 - - -
06 B. subtilis - 5 4 - -
07 E. faecalis - - - - -
08 S. aureus - 5 5 - -
09 V. cholera - 3 3 - 3
10 B. licheniformis strain 018 - 5 5 - 6
V. cholera: Vibrio cholera, S. epidermis: Staphylococcus epidermis¸ E. coli: Escherichia coli, B. tequilensis: Bacillus tequilensis, P. vulgaris: Proteus vulgaris,
S. aureus: Staphylococcus aureus, B. subtilis: Bacillus subtilis, E. faecalis: Enterococcus faecalis, B. licheniformis: Bacillus licheniformis, SNPs: Silver nanoparticles,
AgNO3: Silver nitrate
Asian J Pharm Clin Res, Vol 9, Issue 1, 2016, 63-65
Purushothaman et al.
65
was no zone of inhibition observed in the well loaded with flower
extract (Table 1).
In the present investigation, nanoparticles show higher inhibition
against the Gram-positive S. aureus (5 mm) compared to other Gram-
negative strain V. cholera (3 mm), employed in this antibacterial assay.
Savithramma and Rao [5] demonstrated the antibacterial effect of SNPs
and the growth of Pseudomonas and Rhizopus species were inhibited
maximum by the SNPs synthesized from leaf extract of Svensonia
hyderobadensis, indicating that the SNPs may have an important
advantage over conventional antibiotics. AuNPs also have potential
activity against microbial pathogens, and it mainly depends on the size
and shape of the particles. Consequently, the interaction between Gram-
positive bacteria and SNPs were certainly stronger than that of Gram-
negative bacteria. The cell wall of gram-negative bacteria consists of an
outer membrane composed of lipid, protein, and lipopolysaccharides
which act as a barrier and provide effective protection against the
antibacterial agent. However, the cell wall of the Gram-positive bacteria
lacks an outer membrane [6].
The synthesized SNPs and AuNPs were effective in inhibiting the
bacterial growth. The MIC was checked against Gram-positive
(B. tequilensis strain ARMATI, B. subtilis strain AK, B. subtilis, B.
licheniformis strain 018, and S. aureus) and Gram-negative (V. cholera)
1. SNPs B. tequilensis strain ARMATI 62.5
2. B. subtilis strain AK 31.25
3. B. subtilis 15.63
4. S. aureus 7.81
5. V. cholera 7.81
6. B. licheniformis strain 018 3.9
7. Gold nanoparticles V. cholera 250
8. B. licheniformis strain 018 125
SNPs: Silver nanoparticles, MIC: Minimum inhibitory concentration,
V. cholera: Vibrio cholera, B. tequilensis: Bacillus tequilensis,
S. aureus: Staphylococcus aureus, B. licheniformis: Bacillus licheniformis
bacteria. The SNPs and AuNPs were used in different concentration
such as 500, 250, 125, 62.5, 31.25, 15.63, 7.8, 3.9, 1.95, 0.976, 0.48,
0.24 µl in order to determine the MIC. The SNPs showed MIC value of
62.5 µl for strain ARMATI, 31.25 µl for strain AK, 15.63 µl for B. subtilis,
7.81 µl for V. cholera, 3.9 µl for strain 018, and 7.81 µl for S. aureus. The
AuNPs showed MIC value of 250 µl for V. cholera and 125 µl for strain
018 (Table 2).
Freely water soluble terpenoids, steroids, alkaloids, and proteins
of T. divaricata are responsible for the biosynthesis and stability of
spherical shaped nanoparticles in an aqueous medium. The SNPs
showed the highest antibacterial activity against both Gram-positive
and Gram-negative bacteria. The AuNPs were not found to be a
potential bactericidal agent. The SNPs showed the MIC concentration
ranging from 62.5 to 3.9 µg against bacteria. The AuNPs showed the
MIC concentration of 250 µg and 125 µg against V. cholera and B.
licheniformis strain 018, respectively.
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