American Journal of Anal yt ical Chemistry, 2011, 2, 475-483
doi:10.4236/ajac.2011.24057 Published Online August 2011 (
Copyright © 2011 SciRes. AJAC
Microwave-Assisted Rapid Extracellular Biosynthesis of
Silver Nanoparticles Using Carom Seed (Trachyspermum
copticum) Extract and in Vitro Studies
Deshpande Raghunandan1, Prashant Arunkumar Borgaonkar2, Basawaraj Bendegumble1, Mahesh
Dhondojirao Bedre3, Mantripragada Bhagawanraju4, Manjunath Sooganna Yalagatti5, Do Sung Huh6,
Venkataramana Abbaraju3
1H.K.E.Ss College of Pharmacy, Gulbarga, India
2R.M.E.S College of Pharmacy, Gulbarga, India
3Materials Chemistry Laboratory, Department of Material Science, Gulbarga University, Gulbarga, India
4CM College of Pharmacy, Hakimpet, Hyderabad, India
5Institute of Pharmaceutical Science, Siddipeth, India.
6Department of Biomedicinal Chemistry, Inje University, Kimhae, South Korea
Received February 28, 2011; revised March 29, 2011; accepted April 15, 2011
Microwave-assisted rapid extracellular biosynthesis of silver nanoparticles was carried out by using carom
seed (Trachyspermum copticum) extract as the reducing agent. The reaction mixture containing AgNO3 and
carom seed extract when exposed to microwave irradiation resulted in reducing silver ions to
bio-functionalized silver nanoparticles of size 6- 50 nm. The AgNP were characterized by UV-vis spectros-
copy (UV-vis), X-ray diffraction (XRD), energy dispersive X-ray analysis (EDAX), field emission scanning
electron microscopy (FESEM), transmission electron microscopy (TEM) and atomic force microscopy (AFM).
Themogravimetric analysis (TGA) and fourier transform infrared spectroscopy (FTIR) are used to understand
the possible mechanism of biosynthesis. In this study, we have also investigated the antimicrobial and anti-
oxidant activities of bio-functionalized AgNP. The antibacterial activity is investigated by measuring the zone
of inhibition and antioxidant study is done using 1,1-diphenyl-2-picryl hydrazyl (DPPH) method.
Keywords: Microwave, Silver Nanoparticles, Biosynthesis, Carom Seed (Trachyspermum copticum),
Antibacterial Activity, Antioxidant Activity
1. Introduction
The perfection to unfold and view the structure and func-
tion of life at the nano level is leading to rapid develop-
ment in technology [1] and health sciences [2]. With bot-
tom-up synthesis approach, tailoring of metal particles at
nanoscale is expected to open new protective and preven-
tive aspects from life threatening diseases [3]. Metal na-
noparticles, which have a high specific surface area, have
been studied extensively because of their remarkable
physicochemical characteristics like catalytic, optoec-
tronic and magnetic properties [4]. It can be expected that
the high fraction of surface atoms of silver nanoparticles
(AgNP) will lead to an excellent antimicrobial activity
when compared to bulk Ag metal [5]. This can be attrib-
uted to the cohesion between the nanoparticles surface
and the microbial cells, and hence is found to be size de-
pendent. AgNP are currently used as active drug in tar-
geted drug delivery [6], gene delivery [7] and artificial
implants [8] and as a diagnostic agent in imaging and
sensors for detection of different diseases in their early
stages. Owing to their mutation-resistant antimicrobial
activity, they are being used in different pharmaceutical
formulations as antibacterial clothing [9], burn ointments
[10] and as coating for medical devices [11].
Methods of nanoparticle production through different
physical and chemical routes have their own demerits
as they produce enormous environmental contamina-
tions and hazardous byproducts. Thus, there is a need
for “green chemistry” that ensures clean, non-toxic, and
environment-friendly methods. The increasing demand
for functionalized nanoparticles has encouraged devel-
oping new bio-routes. These include employing micro-
organisms, such as: Fusarium oxysporum [12], Fusa-
rium semitactum [13], Cladosporium Sp. [14], and also
different plants like alfalfa [15] neem [16] and clove [17].
In this paper, we report on the microwave-assisted
rapid synthesis of highly stable bio-functionalized AgNP
using carom seed extract as a reducing agent. Carom
seed commonly cultivated in India and adjacent countries
as the local environment suits for their growth. It is used
for many domestic and medicinal purposes like diarrhea,
dyspepsia, cholera, flatulence, and indigestion. Carom
seeds are also extensively used in Ayurvedic medicine
and Unani systems [18]. In literature we have not come
across using this type of seed for formation of stable
AgNP in aqueous system.
The formation of AgNP is understood from the UV-
-vis spectroscopy and X-ray diffraction studies. Trans-
mission electron microscope studies indicate that AgNP
are in the rage of 6 - 50 nm and most of them are nearly
spherical in shape. Interestingly the colloidal suspension
of AgNP is stable for 18 - 20 weeks, much greater than
the stability of nanoparticles synthesized from microor-
ganisms [12-14]. Use of microwave exposed extracellu-
lar carom seed extract carries high level reproducibility.
In recent years, the antimicrobial resistance has
emerged as a major public health problem. The metallic
AgNP show lethal effect on the verity of microorganisms
and do not allow the pathogens to develop resistance
unlike conventional and narrow spectrum antibiotics.
There lies a strong challenge to produce stable and safe
AgNP to prevent bacterial growth significantly. Though
the antibacterial activity of AgNP is being studied exten-
sively, reports on the effect of these bio-functionalized
nanoparticles in particular are rare. Free-radical in-
volvement of AgNP surface in antimicrobial activity is
discussed based on their zone of inhibition. AgNP syn-
thesized by this process can be used as an effective tool
in the control of microorganisms at a very low concen-
tration and as a preventive agent in deleterious infections.
The free-radical effect of AgNP is compared with the
well known antioxidant, butyl hydroxy anisole (BHA)
both at the same concentration which is determined using
DPPH method. Our results support simple and cost-eff-
ective production of stable functionalized AgNP which
are suitable for formulation of new types of bactericidal
2. Materials and Methods.
2.1. Materials Used
AgNO3 and analytical grade C2H5OH and butyl hydroxy
anisole (BHA) is procured from Himedia Laboratories.
Highly pure carom seeds are procured from Agricultural
University, Dharwad. Double distilled water is used
throughout the work. DPPH and the readymade agar me-
dia was procured from Sigma Chemicals, U.S.A.
2.2. Silver Nanoparticles (AgNP) Biosynthesis
Using Carom Seed
Established procedure for the extraction of carom seed
essential oils is adopted to prepare the aqueous ethanolic
extracellular extract (C2H5OH:H2O::1:1) [19]. For pre-
paring the aqueous extracellular solution, 1 g freshly
collected and perfectly dried carom seed coarse powder
was taken and added to a solution mixture containing
50 ml of ethanol and 50 ml double deionized water in a
250 ml wide neck Borosil conical flask. The conical
flask containing the reaction mixture was kept on a
shaker for 4 h. Then the aqueous extracellular filtrate of
carom seeds was obtained by passing it through What-
man filter paper no. 40. The clear filtrate contains only
soluble organic moieties of the seed and the solid residue
is discarded. This aqueous ethanolic filtrate is the ex-
tracellular extract of carom seed used for the reduction
process. Exactly 5 ml of the resultant clear extract was
added to 50 ml carefully weighed 10–3 M AgNO3 aque-
ous solution in a 250 ml Borosil flask and exposed to mi-
crowave unifrequency radiation (DAEWOO, 2.45 GHz)
for the reduction process to take place.
2.3. Analysis
Periodically, upper layer of the reaction mixture is taken
for UV-vis spectroscopy observation which was per-
formed on an ECIL 5704SS UV-visible spectropho-
tometer at a resolution of 1 nm. For crystallinity studies,
X-ray diffraction (XRD) measurement of the biosynthe-
sized solution with a drop coated on glass substrate
bio-film was carried out on a Siemens X-ray diffracto-
meter (Japan) instrument operated at 30 kV and a current
of 20 mA with Cu Kα (l = 1.54 Å) radiation. The mor-
phology of the AgNP was examined using field emission
scanning electron microscopy (FESEM, FEI Nova nano
600, Netherlands), and for this, the images were operated
at 15 kV on a 0° tilt position. Transmission electron mi-
croscopy (TEM) image of the sample was obtained using
Technai-20 Philips transmission electron microscope
operated at 190 keV. Atomic force microscopy (AFM)
images were collected under ambient conditions on a
Veeco Innova scanning probe microscope. Etched Si
nanoprobe tips (RTESPA-M) were used for the same.
For fourier transformed infrared radiation (FTIR) spec-
troscopy measurements AgNP powder sample was pre-
Copyright © 2011 SciRes. AJAC
Copyright © 2011 SciRes. AJAC
pared by centrifuging the synthesized AgNP solution at
10,000 rpm for 15 min. The solid residue layer which
contains AgNP was redispersed and washed in sterile
deionized water for three times to remove the unattached
biological impurities. The pure residue was then dried
perfectly in an oven overnight at 65°C. Thus obtained
powder was subjected to FTIR measurements carried out
on a Perkin-Elmer Spectrum-One instrument at a resolu-
tion of 4 cm1 in KBr pellets.
2.4. In Vitro Activity
2.4.1. Antibacterial Activity.
The culture media is prepared with peptone-10 g, NaCl -
10 g and yeast extract 5 g, agar 20 g in 1000 ml of dis-
tilled water and boiled. Initially, the stock cultures of
Bacillus subtilis, methicillin resistant Staphylococcus
aureus (MRSA), Pseudomonas aerosenosa and Salmo-
nella typhi were revived by inoculating in broth media in
separate test tube and grown at 37°C for 18 h. These mi-
croorganisms were selected on the basis of their patho
genicity, resistance and severity in forming infections.
The required volume of test sample (2.5, 5, 10, 20 μg/mL)
was added and mixed well. The media was poured into
the pre-autoclaved petri dishes. The 104 CFU culture
was inoculated and grown at 37°C for 24 h. The control
plate is prepared with respective bacteria without AgNP
samples for comparative studies.
2.4.2. Free-Radical Scavenging Activity.
Both the functionalized AgNP solution and butylated
hydroxy anisole (BHA) (2.5 μg, 5 μg, 7.5 μg and 10 μg)
were taken in different test tubes. The volume was ad-
justed to 1000 μl by adding methanol. Five milliliters of
a 0.1 mM methanolic solution of 1,1-diphenyl-2-picryl
hydrazyl (DPPH) was added to these tubes and shaken
vigorously. The tubes were allowed to stand at 27oC for
20 min. The control was prepared as above without addi-
tion of AgNP aqueous solution. The absorbance of the
functionalized AgNP was measured at 517 nm with
UV-vis spectroscopy. Free-radical scavenging activity
was calculated using the following formula:
controlfunctionalized silver solution
%radical scavengingactivity100
3. Results and Discussion
3.1. Bio-reduction and Characterization.
The detailed study on microwave-assisted extracellular
biosynthesis of AgNP using ethanolic aqueous carom
seed (T. copticum) extract was carried out with the anti-
bacterial and antioxidant effects. The initial color of the
solution after the addition of carom seed extract to the
aqueous AgNO3 solution was nearly colorless. In the first
phase, the intensity of the reaction mixture on exposure
to microwave radiation increases exponentially with time.
The metal ions reduction occurs very rapidly and more
than 90% of the reduction of Ag+ ions will be completed
in 90 seconds. From 90 to 150 sec the reaction phase is
drastically reduced and the reaction rate changes to linear
phase. After 150 sec, the reaction stops as the intensity of
the reaction shows almost a parallel line with x-axis with
respect to time. The change in color of the reaction mix-
ture is noted at every 10 sec interval and is shown in the
inset of Figure 1. Colorlessness of reaction mixture at
the initial stage and the final deep reddish-brown color
after the completion of the reaction are also shown. The
absorbance intensity, that is, the formation of AgNP will
increase with increased exposure of reaction mixture to
microwave and is shown in Figure 1(a). The microwave
exposed methodology is much faster than the earlier
conventional studies using other plants extracts [12-14]
and microorganisms [15-17]. The time required for the
conventional synthesis of AgNP from other plants was 2
- 4 h and from bacteria was 24 - 120 h and are thus rather
It is well known fact that microwave produces super
heating non-ionizing radiations at ambient pressure [20].
The accelerated rate of reaction is also attributed to the
strong agitation and reorientation of the dipolar water
molecules, electron rich biological moieties and con-
ducting silver ions which tremendously enhance the pos-
sibility of collision between them.
Figure 1(b) shows the UV-vis spectrum of the reac-
tion mixture. The peak at 255 nm is attributed to the ab-
sorption band for the water soluble organic moieties pre-
sent in the extract. This also indicates that the organic
moieties are involved in the reduction process of ions to
nanoparticles. The color developed in the solution, as a
result of AgNP formation was observed with a surface
plasmon resonance (SPR) peak in the UV-vis spectros-
copy. Reddish brown color of the AgNP arises due to
SPR vibrations in the metal nanoparticles. It is seen from
the spectra that the SPR of AgNP band occurs at 465 nm
[21]. The hyperchromic shift of this peak with increased
exposure to microwave is found to be directly depending
(a) (b)
Figure 1. (a) Graph showing change in color intensity of the reaction mixture with respect to time. Inset: I. Initial and the
final color of the reaction mixture containing aqueous 10–3 M AgNO3 solution and aqueous ethanolic carom seed extract on
microwave irradiation. II. Change in the color of the reaction mixture with time in sec. (b) UV-visible spectra of AgNP
biosynthesis, absorbance recorded as a function of time. Projected and enlarged view of UV spectra is highlighted with an
arrow line to show the stoppage of reaction at 90 sec. Inset: UV-vis spectrum of the extracellular carom seed extract.
upon the concentration of AgNP formed. In addition to
465 nm peak, another peak at 480 nm also appears as a
shoulder in the visible region after 60 sec of the reaction.
The 465 nm peak corresponds to the transverse plasmon
vibration, whereas, the peak at 480 nm is due to excita-
tion of longitudinal plasmon vibrations. Wavelengths of
these peaks are different, distinctly separated which in-
dicate that AgNP formed in the solution have different
sizes and shapes and are in aggregates form [22]. The
broadening and splitting of the SPR with the increase in
microwave exposure is probably due to the dampening of
surface plasmon caused by a resonance change [23]
which in turn is due to the change in the refractive index
of the surrounding medium and increase in the size of
AgNP in the colloidal solution. An absorption band at
255 nm is clearly visible and is attributed to electronic
excitation of organic moieties. In order to verify the re-
sults of the UV-vis analysis, the sample of bio-reduced
AgNP was examined by XRD, which gave sharp, crys-
talline peaks of Ag as shown in Figure 2. The different
facet markings will agree with the standard JCPDS re-
port. The appearance of ‘‘Al’’ in figure is because of
the aluminum grid base used for the analysis. The dif-
fraction pattern also suggests that the AgNP formed are
polycrystalline in nature. The study of metallic nature of
these AgNP is further strengthened by EDAX image
shown in the inset of Figure 2.
Figure 3 shows FESEM images of functionalized
AgNP. It can be seen that they are thickly coated with
organic moieties on them with core shell morphology of
size 6 - 50 nm. In Figure 3(a), AgNP seem to be ar-
ranged in an organic matrix making it aqueous suspen-
sion. Higher resolution image at 300 nm (Figure 1)
shows a group of particles in embedded in a organic
moieties making a stable suspension. The particles ap-
pear to be polydispersed in nature and are roughly
spherical in shape. Particles size distribution determined
from the FESEM image, shown in the center as an inset
of Figure 3 represents the histogram of the synthesized
AgNP. It is observed that there is a marginal variation in
the particle size. Almost 90% of the particles are in the
range of 6 - 50 nm, 4% are in 51 - 60 nm and approxi-
mately 6% are in the 1- 5 nm range. The preliminary
studies indicate an encouraging fact that by making vari-
ation in the experimental parameters like pH, concentra-
tion of the carom seed extract, frequency opted for mi-
crowave irradiation, and molar concentration AgNO3
will achieve the monodispersivity and uniformity in
The clear morphology is reconfirmed with drop coated
TEM grids and AFM images shown in Figure 4. Figure
4(a) shows a typical bright-field TEM image of the bio-
synthesized AgNP. The AgNP are nearly spherical in
shape, and are in the range of 6 - 50 nm size indicating
the dispersivity to be in a narrow range. On a careful
observation we can see a sensitive layer adsorbed on the
surface and between thin gaps of two nanoparticles
(shown with arrow mark). We can infer that these are the
organic moieties adsorbed on the surface and are respon-
sible for inter particle binding. The same may also be
responsible for bio-reduction of ions and formation of
nanoparticles. Figure 4(b) shows a typical AFM repre-
sentative image exhibiting the morphology of colloidal
AgNP. Uniformity in the morphology of these nanopar-
ticles may be attributed to the soft adsorbed layer and to
the thick cover of organic moieties on the particles.
AgNP appear to be higher in size (100 - 120 nm) than
that are seen in TEM image. The most probable reason
Copyright © 2011 SciRes. AJAC
Figure 2. XRD pattern of crystalline AgNP synthesized using extracellular aqueous ethanolic carom seed extract. Inset fig.
shows Energy dispersive x-ray spectrum EDAX of metallic biosynthesized AgNP.
(a) (b)
Fiure 3. (a)& (b). FESEM images of synthesized AgNP in colloidal condition on different nanometric scale. Inset at the centre
shows histogram indicating size distribution of AgNP.
(a) (b)
Figure 4. (a) TEM image of biosynthesized AgNP showing they are roughly spherical in shape. (b) Medium scale tapping
mode AFM image of bio-functionalized AgNP adsorbed with organic layer.
Copyright © 2011 SciRes. AJAC
for increased size appearance may be due to the bio-ad-
sorbed layer on the nanoparticles. This magnification can
also be attributed to the convolution of the true particle
size with AFM tip. The AFM data also show that the size
of the particles depends on the deposition conditions.
Figure 5 is the TGA graph, shows three stages of
weight loss. In I stage, from 0oC - 100oC, the weight loss
of 4%, is due to the evaporation of adsorbed water mo-
lecules and free –OH groups on the surface of the func-
tionalized AgNP. Second weight loss of 18% from 100oC
- 450oC is slow and steady and is attributed to the eva-
poration of absorbed water molecules. Third weight loss
of 12.5% from 450oC - 1100oC is due to the loss of
strongly bound organic moieties layer present on the
AgNP surface. The total weight loss of 34.5% from stage
I–III gives confirmative evidence that the metallic core is
thickly covered by bio-moieties shell. The undecom-
posed residue of 65.5% contains pure silver microstruc-
tures. The inset image shows the picture of pure silver
microstructures after cooling the residue to ambient con-
ditions. On further heating up to 1300oC the metal nano-
particles get melted to a liquid state. After cooling, pure
spherical silver particles are formed due to the cohesive
force in the molecules of the metal. These silver spheres
are the bulk microstructures which possess the original
color of the bulk silver.
Figure 6 shows FTIR spectrum of AgNP. FTIR shows
peaks at 1741, 1641, 1569, 1460, 1263, 1099, 1020,
800 cm–1. This indicates secretion of some soluble or-
ganic components of carom seeds which could have con-
tributed for the important role in the reduction and func-
tionalization of the metal nanoparticles. Consequently
the organic moieties adsorbed on the nanoparticles con-
Figure 5. TGA graph showing the weight loss pattern of
functional groups and other biological moieties adsorbed on
the AgNP surface. Inset image shows bulk microstructures
of pure silver as the residue left after cooling.
fer the stability. We presume that the polyphenols like
terpenoids (thymol, which is a major constituent of the
essential oils) of carom seed show characteristic absorp-
tion peaks and the same are responsible for bio-reduction
and capping process [24]. Peak at 1460 cm–1 is due to
C-H deformation of gem-dimethyl groups and 1099 and
1020 cm–1 are of CH3-C-CH3 skeletal vibrations. C=C
stretching vibrations at 1641 cm–1 peak are due to aro-
matic rings. Conjugated C=C bonds at 1569 cm–1 and
bending vibration peak at 800 cm–1 suggest the presence
of thymol adsorbed on the surface of AgNP. Among the
major chemical constituents of carom seed (Thymol,
P-Cymene and γ-Terpinene) [25], thymol is the only
constituent which possesses aromatic ring in its structure.
It appears that the same moiety could be adsorbed on the
metal nanoparticle surface by with π-electrons. The
peaks of phenolic -OH are seen at 3743 cm–1 and -CH3,
-CH2, -CH stretching vibrations are observed at 2923,
2854 and 2690 cm–1 respectively.
3.2. Antibacterial Activity and Free Radical
Scavenging Activity
Two different pathogens were chosen from each group of
gram positive and gram negative segments for our stud-
ies. Pseudomonas aeruginosa can cause chronic oppor-
tunistic nosocomial infections which can’t be treated
with regular antibiotics. P. mirabilis causes maximum
'Proteus' infections. Methicillin-resistant Staphylococcus
aureus (MRSA) causes infections which are diffi-
cult-to-treat. Typhoid is one of the serious infections
developed from the simple strains of S. Typhi and is re-
sponsible for enteric fever.
AgNP synthesized with this green-clean technology, is
exposed to all the strains on MHA plates treated with
different concentrations (from 2.5 μg to 20 μg/mL) for
studying antibacterial application as shown in Figure
7(a). The inhibitory effects of the sample are compared
with the control plate prepared without addition of any
drug. AgNP showed satisfactory growth inhibition effect
against salmonella typhi, and significant growth inhibi-
tion is observed in all other pathogens. The zone of inhi-
bition of AgNP in different microorganisms is different
and is concentration-dependent. Inhibition in S. Typhi, P.
aeruginosa, and B. subtilis is 5, 6 and 7 mm respectively
at the highest concentration of 20 μg/mL. MRSA is inhib-
ited at the same concentration of AgNP but the rate of
inhibition appears to be slow with increasing concentra-
tion compared to B. subtilis and P. aeruginosa. It is ob-
served that the MIC of B. subtilis is higher than the other
pathogens. The mechanism by which the nanoparticles
are able to inhibit bacterial growth is not well understood,
but it can be conceived that the AgNP affect the mem-
Copyright © 2011 SciRes. AJAC
Figure 6. Typical FTIR absorption spectra of the bio-moieties of the macerated extracellular aqueous ethanolic carom seed
extract adsorbed on the AgNP. Functionalized AgNP is shown as an inset.
(a) (b)
Figure 7. A. Antimicrobial activities of bio-functionalized AgNP using carom seed extract. For brevity only 1/4th portion of
the plates are shown. The center potion marked as “C” shows control of respective microorganisms. All the concentrations
are taken in μg/mL. B. Free-radical scavenging activity graph of AgNP and BHA indicating the quenching effect on DPPH
radical at different μg concentration.
brane of both the bacterial strains. It may lead to signifi-
cant increase in the permeability and affect membrane
transport. Also, there is no antimicrobial activity in the
solution devoid of AgNP (produced out of carom seed
extract) in the control plates shown in the center marked
in figure as “C”. Separate test is carried out for the an-
timicrobial analysis for carom seed extract only; even at
the highest concentration, the effect is similar as seen in
case of control plates. This study concludes that the an-
timicrobial effect is only due to synthesized AgNP. The
study also infers that the functionalized AgNP using
carom seed extract have good bactericidal activity at low
concentration in different microbes. Further studies on
the formulation of these nanoparticles, stability studies
and other analysis for comparing the efficacy against
commercial products are on the way.
Figure 7(b) shows very encouraging results. These
functionalized AgNP have DPPH. free radical scaveng-
ing in a concentration dependent manner [26]. It could be
seen from figure that at the same concentration AgNP
Copyright © 2011 SciRes. AJAC
scavenged the DPPH free radical five times more effec-
tively than BHA. Even at concentration as low as 5 μg/mL,
where BHA has less than 10% efficiency, functionalized
AgNP mopped up more than 40% free radicals in-vitro.
Similarly the percentage of quenching effect on DPPH
free radical was 9% with AgNP at a minimum concen-
tration of 2.5 μg, where BHA shows only 3% at same
concentration. AgNP and BHA scavenged 60% and 12%
respectively at a maximum concentration of 10 μg.
4. Conclusions
Carom seeds and silver are generally used as bactericidal
agents; combination of both in the form of functionalized
silver nanoparticles is envisaged. With the help of mi-
crowave-assisted top-down green chemistry approach,
functionalized silver nanoparticles were synthesized in
the range of 6 - 50 nm using aqueous ethanolic carom
seed (Trachyspermum copticum) extract. Usage of AgNP
thus produced are tested for in vitro applications and
found to be more effective as protective and preventive
antibacterial and antioxidant agent. Silver nanoparticles
based on these findings may lead to valuable discoveries
in various fields such as medicine and pharmaceutical
research. As this method of biosynthesis is simple and
handy; can be thought for commercial level of produc-
5. Acknowledgements
Financial supports from BRNS (Grant No. 2009/34/
14/BRNS), DST (Grant No.SR/S1/PC-10/2005) and
UGC Major Research Project (33-307/2007 (SR) are
acknowledged. We also acknowledge the help from
Biogenics, Hubli for antimicrobial studies. We thank
Prof. B. G. Mulimani, Vice-Chancellor, BLDE Univer-
sity, Bijapur for encouragement in the work. Raghunan-
dan Deshpande thanks his father Shri. J. M. Deshpande
for editing work & Dr. Appala Raju, Principal of HKES
college of pharmacy, Gulbarga for encouraging the re-
search program.
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