146

Chapter 5

Green synthesis and characterization

of Silver nanoparticles using some

Zingiberaceae plants and their

antimicrobial and catalytic

activities

147

Chapter 5

Green synthesis and characterization of Silver nanoparticles usingsome Zingiberaceae plants and their antimicrobial and catalyticactivities

5.1. Introduction

The field of nanotechnology is one of the most active areas of research in

modern material sciences. The application of nanoscale materials and structures,

usually ranging from 1 to 100 nanometers (nm), is an emerging area of

nanoscience and nanotechnology. Nanoparticles exhibit completely new or

improved properties based on specific characteristics such as size, distribution

and morphology, if compared with larger particles of the bulk material they are

made of. Specific surface area is relevant for catalytic reactivity and other

related properties such as antimicrobial activity in silver nanoparticles. As

specific surface area of nanoparticles is increased, their biological effectiveness

can increase due to the increase in surface area.1

The biosynthesis of nanoparticles by green technique which represents a

connection between biotechnology and nanotechnology, has received increasing

consideration due to the growing need to develop environmentally benign

technologies for material syntheses. The ‘‘green synthesis’’ of metal

nanoparticles receives greater attention due to their unusual optical, chemical,

photo-chemical, electronic properties and antimicrobial activities.1,2 Metal

nanoparticles, especially the noble metals, have a surface plasmon resonances

absorption in the UV-Vis region. The surface plasmon band arises from the

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collective excitation of the free electron gas.3

Among noble metal nanoparticles, silver nanoparticles (AgNPs) have

gained much attention as they have a large number of applications, such as, in

the fields of dentistry, clothing, mirrors, photography, electronics, non-linear

optics, spectrally selective coatings for solar energy absorption, bio-labelling,

intercalation materials for electrical batteries as optical receptors, catalysts in

chemical reactions, food industries etc.4 The AgNPs show efficient

antimicrobial property compared to other salts due to their extremely large

surface area, which provides better contact with micro-organisms.2

Numerous chemical and physical methods are used to reduce Ag+ ion.5-14

However, some chemical methods cannot avoid the use of toxic chemicals in the

synthesis protocol. There is a growing need to develop environmentally friendly

processes of nanoparticles synthesis that do not use toxic chemicals. The use of

plant extracts containing secondary metabolites for the synthesis of

nanoparticles is termed as 'green synthesis', as it is a simple, convenient, less

energy intensive and eco-friendly method. The method is suitable for nanoscale

metal synthesis due to the absence of any requirement to maintain an aseptic

environment.15 The possibility of using plant materials for the synthesis of

nanoscale metals was reported initially by Gardea-Torresdey et al.16,17

The green synthesis of AgNPs involves three main steps, which must be

evaluated based on green chemistry perspectives, including selection of solvent

medium, reducing agent, and nontoxic stabilizers for AgNPs.18 Thus a green

synthesis method has gained much attention and become a major field of

149

interest. Recently synthesis and application of silver nanoparticles were done

previously by many researchers for variety of purpose.19-30

Rhizomes of some medicinal plants of Zingiberaceae family such as

Curcuma aromatica Salisb.31-34, Curcuma caesia Roxb.35,36, Curcuma

leucorrhiza Roxb.37-40 Hedychium coccineum Buch.-Ham. ex Sm41,42 and

Kaempferia galanga Linn.43,44 are used in the preparation of AgNPs using silver

nitrate as silver precursor and these plants as reducing agents and stabilizers.

These plants are used in traditional medicine and cultivated in tropical and

subtropical regions of Asia. From the rhizomes of these plants, many types of

sesquiterpenes, such as carabrane, germacrane, guaiane, elemane and

curcuminoids have been isolated. Terpenoids and water soluble fractions

comprised of complex polyols are believed to play an important role in silver

nanoparticles biosynthesis through the reduction of silver ion. Satishkumar and

his group successfully utilized C. longa tuber powder and extract in the

synthesis of AgNPs and this synthesized AgNPs showed potent activity against

E.Coli.45

Various characterization techniques such as UV–Vis spectroscopy, Fourier

transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), Electron

paramagnetic resonance (EPR), Dispersive X-ray (EDX) spectroscopy, Scanning

electron microscopy (SEM), Transmission Electron Microscopy (TEM) and

selected area electron diffraction pattern (SAED) were used to confirm the

reduction of Ag+ and the formation of AgNPs.

Herein, the use of naturally available some medicinal plants of

150

Zingiberaceae family which have not been investigated so far for the biosynthesis

of AgNPs are used in this study. Further bioactive compounds isolated from

these plants are also used for the synthesis of silver nanoparticles. The

synthesized AgNPs show the antibacterial activity against Proteus mirabilis,

Klebsiella pneumoniae, Escherichia coli, Salmonella paratyphi and Pseudomonas

aeruginosa. The catalytic activity of the synthesized AgNPs is established in the

reduction of DPPH by BHT + AgNPs. The UV–VIS spectra have been recorded

in methanol at regular intervals of time.

5.2. Results and discussion

5.2.1. UV–vis spectrum of silver nanoparticles

In this study, the formation of silver nanoparticles by the rhizomes

extracts were investigated. The control AgNO3 solution (without plant extract)

showed no change of colour and there was no absorption peak in the UV–vis

spectrum. Reduction of the silver ion to silver nanoparticles during exposure to

the plant extracts could be followed by change of colour and thus the aqueous

silver nitrate solution appeared turbid soon after adding the rhizome extract of

C.leucorrhiza (CL). After the solution was kept at 55°C, the intensity of the

colour increased gradually from pale yellow to reddish brown at the end of the

experiment within 3 min. The diluted rhizome extract C. caesia (CC) had a pale

dark in colour and the colour of the solution was intensified as the reaction

progressed after its addition to aqueous AgNO3 solution and turned dark brown

in colour. The colour changes arise from the excitation of surface plasmon

151

vibrations (SPV) with the AgNPs.46,47 The appearance of surface plasmon

resonance (SPR) peak at 431nm and 441nm provides a convenient spectroscopic

signature for the formation of respective AgCL and AgCC NPs, respectively.29

Increase in absorbance with respect to time in case of CL and CC indicates the

increase in productivity of AgNPs [Fig.1]. The increase in the absorbance values

with increasing CL and CC dosage demonstrates the higher production of silver

nanoparticles. This behaviour may be due to the excitation of the surface

plasmon resonance (SPR) effect and to the reduction of silver ions into AgNPs.

The intensity of the brown colour increased in direct proportion to the

incubation period.21 AgCA and AgKG NPs exhibit surface plasmon resonance at

436nm and 422nm respectively.

152

200 300 400 500 600 7000.0

0.1

0.2

0.3

0.4

0.5AgKG422nm

KG

Abs

orba

nce

Wavelength(nm)

Figure 1. UV–Vis spectra of synthesized AgNPs

5.2.2. EPR spectrum of silver nanoparticles

The EPR spectra of the synthesized AgNPs [Fig 2.] were found to be

confined in a single line which showed the presence of an unpaired electron

indicating of the silver in neutral 4d105s1 (Ag0) at room temperature. The EPR

splitting factor of AgCL, AgCC, AgCA, AgHC, AgKG and AgZ were caliberated

at g = 2.00358, 2.00361, 2.00362, 2.00341, 2.0023 and 2.00318 respectively,

which confirmed the reduction of Ag+ ion, i.e. formation of AgNPs.

320 330 340 350

-1000

0

1000

g=2.00358

Inte

nsity

Magnetic Field (mT)

AgCL

320 325 330 335 340 345 350 355

-1000

-500

0

500

1000

g=2.00361

Inte

nsity

Magnetic Field (mT)

AgCC

153

320 325 330 335 340 345 350 355-2000

-1500

-1000

-500

0

500

1000

1500

2000 AgCA

g=2.00362

Inte

nsity

Magnetic Field (mT)320 325 330 335 340 345 350 355

-1500

-1000

-500

0

500

1000

1500

g=2.00341

Inte

nsity

Magnetic Field (mT)

AgHC

330 340 350-2000

-1500

-1000

-500

0

500

1000

1500

2000

g=2.0023

Inte

nsity

Magnetic Field (mT)

AgKG

300 350

g=2.00318

Inte

nsity

Magnetic Field (mT)

AgZ

Figure 2. EPR spectra of AgNPs at room temperature5.2.3. FT-IR spectrum of silver nanoparticles

FT-IR measurements were performed to identify the possible functional

groups present in the rhizomes extract of CL, CC, CA, HC and KG respectively,

that were responsible for the reduction of the metal precursors as well as for the

stabilization of the AgNPs. In the present study, the rhizomes extracts acted as

both the reductant and the capping agent; thus, no extra surfactant or reductant

was added. The representative IR spectra of the rhizomes extract before and

after the reaction are shown in Fig.3. The FT-IR spectra display a number of

absorption peaks, reflecting its complex nature and show significant change in

their respective vibrational spectra. The IR spectrum of the rhizomes extract

before the reaction shows a number of vibration bands for v(O– H) (3420 and

1387cm-1). In the synthesized AgCL NPs, the bands appeared at 1622, 1381,

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1311 and 1102 cm-1 could be assigned to C=O, C–N, C–O and C–O–C

stretchings, respectively. The IR spectrum of the rhizomes extract before the

reaction (CC) shows vibration band for v(O– H) (3420 cm-1). In the synthesized

AgCC NPs, the bands appeared at 1597, 1393, 1381, 1318 and 1114 cm−1

could be assigned to C=C, C–N, –C–O and C–O–C stretching respectively. FT-

IR study indicates that the carboxyl (C=O), hydroxyl (O-H), and amine (N-H)

groups in the rhizome extracts are mainly involved in reduction of Ag+ ions to

Ag0 nanoparticles. The other AgNPs - AgCA, AgHC and AgKG show similar

absorption bands. By comparing the crude extracts, the absorption peaks of

AgNPs are attributed to the interaction between the O-H, C=C, C=O and the

NPs, thus suggesting that the O-H, C=C, C=O groups also act as capping ligands

for the NPs. To a significant extent, functional groups such as O-H, C=O and

C=C can be derived from the water soluble heterocyclic compounds present in

the respective rhizomes extract of CL, CC, CA, HC and KG. The FT-IR

spectroscopic study also confirms that the biomolecules present in rhizomes

extract act as a reducing agents and stabilizers for the AgNPs and prevents

agglomeration. Thus, the biomolecules have a strong binding ability with metal,

suggesting the formation of a layer covering the metal nanoparticles (i.e.

capping of AgNPs) to prevent agglomeration and thereby stabilizing the

medium. Therefore, the rhizome extracts act as an environmentally benign

reductants and stabilizers.

155

4000 3500 3000 2500 2000 1500 1000 500

CL

AgCL

Wavelength(cm-1)

1381 11

02.48

3295

.60

2923

.96

2347

.05

1622

.13

785.9

3

1311

.64

4000 3500 3000 2500 2000 1500 1000 500

CC

AgCC

Wavelength(cm-1)

3177

.84

1597

.28 1393

.0913

18.55

1114

.36 984.7

276

1.30

2322

.20

156

4000 3500 3000 2500 2000 1500 1000 500

709

1375

.29 1153

.47

1546

.961666

.55

1006

.8811

51.54

1531

.53

1662

.69

3307

CA

AgCA

Wavelength(cm-1) 3500 3000 2500 2000 1500 1000 500

40

50

60

70

80

90

100

1006

.88

831.

35

1151

.54

1327

.0714

46.6

615

29.6

016

64.6

2

3282

.95

2908

.75

3130

.43

528.

51

773.

4897

6.01

1076

.32

1313

.57

1384

.94

1600

.97

Wavelength (cm-1)

HC AgHC

4000 3500 3000 2500 2000 1500 1000 500

89932

75

13911595

619

80635

43

113813

25

1678

KG

AgKG

Wavenumber(cm-1)

1679

1015

773

131335

43

3208

Figure 3. FT-IR spectra of AgNPs

5.2.4. EDX spectrum of silver nanoparticles

EDX observation gains further insight into the features of the silver

nanoparticles. Figure 4 shows the strong silver signal confirming the presence

of silver along with a weak oxygen and carbon peak, which might be originated

from the biomolecules that were bound to the surface of the NPs.

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AgCL AgCC

AgCA AgHC

AgKG AgZFigure 4. EDX spectra of AgNPs

5.2.5. X-ray diffraction analysis of silver nanoparticles

The XRD technique was used to determine and confirm the crystal

structure of the Ag NPs. Figure 5 shows a representative XRD pattern of the Ag

NPs. This figure showed the intense XRD peaks corresponding to (111), (200),

(220), (311) at angles, 38.18°, 44.25°, 64.72°, 77.40° respectively. From the

158

broadening of XRD peaks, it could be confirmed that nano sized particles are

getting formed. Some additonal peaks may be due to the interaction of the NPs

and starches of the respective rhizome extract of CL, CC, CA, HC and KG. It

was reported that ginger and turmeric rhizomes had approximately 45 and 40%

of starch.48-50 The sharp intense XRD peaks of AgZ is due to thin film made in

acetone.

20 30 40 50 60 70 800

50

100

150

200

(111) (200 )

(220) (311)

Inte

nsity

220 30 40 50 60 70 80

30

45

60

75

90

(111)

(220) (311)

Inte

nsity

AgCL AgCC

159

30 40 50 60 70 800

50

100

150

200

250

300In

tens

ity

2

AgCA

20 30 40 50 60 70 800

100

200

300

400

500

Inte

nsity

2

AgHC

AgCA AgHC

20 30 40 50 60 70 80

50

100

150

200

250

300

350

Inte

nsity

2

AgKG

30 40 50 60 70 800

200

400

600

(311)(220)

(200)

(111)

2

AgKG AgZ (in acetone)

Figure 5. XRD spectra of AgNPs

5.2.6. Electron microscopy analysis of silver nanoparticles

Scanning electron microscopy (SEM) images are shown in Fig. 6 that

relatively spherical nanoparticles were formed. The morphology and crystalline

phase of the NPs are further confirmed by transmission electron microscopy

(TEM) images, selected area electron diffraction (SAED).

160

AgCL AgCC

AgCA AgHC

AgKG

Figure 6. SEM images of AgNPs

TEM micrographs of the synthesized Ag nanoparticles are presented in

Fig.7 It was observed that most of the Ag NPs were spherical in shape with a

moderate variation in particle sizes. The silver particles were poly crystalline, and

161

as could be seen from the selected area electron diffraction (SAED), all the Ag

NPs have single orientation form a cluster of silver particles. In the selected area

electron diffraction (SAED) pattern recorded from the silver nanoparticles

(Fig.7), the ring-like diffraction pattern indicates that the particles are crystalline.

The diffraction rings could be indexed on the basis of the fcc structure of silver.

Four rings arise due to reflections from (111), (200), (220), and (311) lattice

planes of fcc silver, respectively.

a b

c

0-4 4-8 8-12 12-16 16-200

10

20

30

40

50

60

% o

f Par

ticle

s

Particle size (nm)d

TEM images of AgCL- a (the inset is the SAED pattern), b, c (HR TEM) and d (particle sizedistribution)

162

e f

g

0-4 4-8 8-12 12-16 16-20 20-240

5

10

15

20

25

30

35

40%

of P

artic

les

Particle size (nm)h

TEM images of AgCC - e (the inset is the SAED pattern), f, g (HR TEM) and h (particle sizedistribution)

i jTEM images of AgCA - i (the insets are the SAED pattern and HR TEM) and j (the inset is

particle size distribution)

163

k l

TEM images of AgHC - k (the inset is the SAED pattern) and l (the inset is particle sizedistribution)

m n

TEM images of AgKG - m (the insets are the SAED pattern and HR TEM) and n (the inset isparticle size distribution)

164

o p

q r

TEM images of AgZ - o (the insets are the SAED pattern), p, q (the inset is HR TEM) and r

Figure 7. TEM images of AgNPs

According to the particle size distribution, most of the nanoparticles

ranges from 2 to 20nm. Average size range estimated from our studies is found

to be 4–8nm, 4-12nm, 3-6nm, 2-4nm and 2-4 for AgCL, AgCC, AgCA, AgHC

and AgKG NPs respectively.

165

5.2.7. Antimicrobial activity of silver nanoparticles

The prepared AgNPs were tested against five human pathogens gram

negative bacteria. The antibacterial activity test results of synthesized AgNPs

were shown in Table 1. The synthesized AgCA and AgHC NPs show the highest

level of zone of inhibition against P. aeruginosa, while AgCL, AgCC and AgCA

NPs exhibit the moderate level of zone of inhibition against Salmonella

paratyphi. However, very few research articles have mentioned about mechanism

for antimicrobial activity of AgNPs.9 In earlier report the synthesised AgNPs

from the rhizome extract of C. longa were found to have potent activity against

E.Coli.45 Antimicrobial activitities of the as prepared AgNPs also correlate to

earlier studies.19,51-58 The minimum inhibitory concentrations (MICs) of the

sample were determined by serial dilution against the micro-organisms. The

minimum concentrations at which no visible growth were observed were defined

as the MICs, which were expressed in mg/ml. The antimicrobial tests were

calculated as a mean of three replicates. Table 2 showed the minimal inhibitory

concentrations (MICs) of synthesized AgNPs against five human pathogenic

bacteria.

5.2.8.Catalytic activitity of silver nanoparticles

The catalytic activity of the synthesized AgNPs is established in the

reduction of DPPH by BHT + AgNPs. The UV–VIS spectra have been recorded

at regular intervals of time (Fig.8). AgCA NPs showed the highest catalytic

activity, followed by AgCL, AgHC and AgZ NPS. AgCC presented the least

catalytic activity, followed by AgKG NPs.

166

400 500 600 700 800 9000.0

0.2

0.4

0.6AgCA

Increasing time

Abso

rban

ce

Wavelength(nm)

0min 5min 10min 15min 20min 25min 30min 35min 40min

400 500 600 700 800 9000.0

0.2

0.4

0.6

AgCL

Increasing time

Abso

rban

ce

Wavelength(nm)

0min 5min 10min 15min 20min 25min 30min 35min 40min 45min

400 500 600 700 800 900

0.0

0.2

0.4

0.6

AgZ

Increasing time

Abso

rban

ce

Wavelength(nm)

0min 5min 10min 15min 20min 25min 30min 35min 40min 45min

400 500 600 700 800 9000.0

0.2

0.4

0.6AgHC

Increasing time

Abso

rban

ce

Wavelength(nm)

0min 5min 10min 15min 20min 25min 30min 35min 40min 45min

400 500 600 700 800 9000.0

0.2

0.4

0.6AgKg

Increasing time115min

0min

Abso

rban

ce

Wavelength (nm) 400 500 600 700 800 9000.0

0.1

0.2

0.3

0.4

0.5

0.6 AgCC

Increasing time120min

0min

Abso

rban

ce

Wavelength (nm)

Figure 8. Catalytic activity of AgNPs

The silver nitrate (1mM) can act as catalyst as shown in scheme 1.

O

O

O

CH3

O

O

OH

CH3

O

O

O

CH3

Ag+NaBH4

MeOH 72%

+ e-Ag+ Ag0

Z ZNH Z

Scheme 1. Proposed mechanism for the formation of AgZ NPs

167

5.3. Experimental

5.3.1. Collection of plant materials

The rhizomes of Curcuma leucorrhiza Roxb (CL). Curcuma caesia Roxb.

(CC), Curcuma aromatica Salisb.(CA), Hedychium coccineum Buch.-Ham. ex

Sm (HC) and Kaempferia galanga Linn. (KG) were collected from Thoubal,

Bishnupur and Senapati districts of Manipur, India. AgNO3 (99.98%, Merck) was

used as a silver precursor. All glassware used in experimental procedures were

cleaned in a fresh solution of HNO3/HCl (3:1, v/v), washed thoroughly with

double distilled water, and dried before use.

5.3.2. Preparation of extracts

The rhizomes of these plants were washed to remove the adhering mud

particles and possible impurities. Later, they were dried in the shade for a week to

completely remove the moisture. The rhizomes of each plant were cut into small

pieces, powdered in a mixer, and then sieved using a 20-mesh sieve to get a

uniform size range. For the production of the extract, 1.25 g of each plant

rhizome powder was added to a 100-mL Erlenmeyer flask with 50 mL sterile

distilled water and then heated at 55°C for 5 min. The each extract obtained was

centrifuged at 10,000 rpm for 10 min to remove any undesired impurities. This

extract was used for further experiments. This solution was considered as 100%

extract.

5.3.3. Synthesis of silver nanoparticles (AgNPs)

Forty milliliters of AgNO3 (1mM) were added to 1.0 mL, 5.0 mL and 10.0

mL of C. leucorrhiza (CL)extract and stirred at various temperatures ranging

168

from 25°C - 60°C. A colour change was observed from light yellow to purple to

reddish brown within 3 min. The colloidal sollution was stirred for an additional 5

min and cooled at room temperature. AgCL NPs exhibited surface plasmon

resonance at 431 nm. The obtained colloidal suspensions of AgCL were then

centrifuged at 12,000 rpm for 10 min and washed four times to remove silver ion

residue. The precipitate nanoparticles were then dried overnight at 30°C under

vacuum overnight to obtain the AgCLNPs. The other AgNPs were also

synthesised using the above procedure. AgCC and AgKG NPs exhibited surface

plasmon resonance at 443nm and 422nm respectively.

The AgZ NPs were rapidly synthesized by treating silver ions through a

simple and biosynthetic route using zederone (Z) isolated from the rhizomes of C.

aromatica Salisb.(CA) which acted simultaneously as a reductant and stabilizer.

Twenty milliliters of of AgNO3 (1mM) was added to zederone(Z) (0.0439, 0.3

mmol) dissolved in ethanol (5mL). The formation of Ag-NPs was evidenced by

the appearance of the signatory brown colour of the solution.

5.3.4. Characterization of AgNPs

The prepared AgNPs were characterized by UV–Vis spectroscopy. UV–

Vis spectra were recorded over the 300–800 nm range with a UV-Vis spectro-

photometer (Shimadzu) with samples in quartz cuvette. Deionized water was used

as blank. The spectra recorded were then replotted using Origin 6.1 version. The

FT-IR spectra were performed to the extract which was exposed before and after

addition to AgNO3 solution. The samples were mixed with KBr to make a pellet

and it was placed into the sample holder. The spectrum was recorded over the

169

range of 400–4,000 cm-1 using Shimazdu IR-408 spectrometer and then, re-

plotted using Origin 6.1 version. Electron paramagnetic resonance (EPR) was

recorded for free radical analysis on JEOL JES-FA200 ESR spectrometer with X-

band microwave unit. The XRD pattern was recorded by X’Pert Pro

(PANanlytical) operating X-ray tube at a voltage 40 kV and a current 30 mA. The

radiation used was Cu- Kα(k = 1.5406 A°). The scanning was conducted in the 2θ

range of 20–80°, 2θ with step size [°2Th.] of 0.0500. The grain size and shape of

the particles were given. SEM images were recorded on FEI-QUANTA-250

electron microscope. The compound was adsorbed on a carbon sheet and loaded

on the microscope. The EDX analysis was carried out on EDAX Energy

Dispersion X-ray spectrometer (FIE). The morphological analysis of the particle

was done with transmission electron microscopy (TEM). The drop of aqueous

silver nanoparticles sample was loaded on carbon-coated copper grid and

recorded on JEOL JEM-2100 equipped with selected area electron diffraction

pattern (SAED). The size of particles were measured with the software IMAGE J.

5.3.5. Antimicrobial activity

Five species of bacteria -Proteus mirabilis, Klebsiella pneumoniae,

Escherichia coli, Salmonella paratyphi and Pseudomonas aeruginosa were

employed. Antimicrobial activity was carried out using agar-diffusion method.

Petri plates (100 mm) were prepared with 20 mL of sterile nutrient agar (NA)

(Hi-Media) and Potato-Dextrose Agar (PDA) (Hi-Media) and SDA (Hi-Media)

for testing the bacterial and filamentous fungal and yeast activity. The test

cultures were swabbed on the top of the solidified media and allowed to dry for

170

10 min. Stock solutions of each chemical were diluted. The dilutions were

deposited (20 μL per well) which were subsequently placed on the inoculated

Petri plates and left for 10 min at room temperature for compound diffusion.

Negative control was prepared using DMSO. Ciprofloxacin (Hi-Media) for

bacteria were served as positive control. The plates with bacteria were incubated

at 37ºC for 24 hr and for fungal cultures at 30ºC for 48-72 hr. The experiment

was repeated thrice and the average results were recorded. The antimicrobial

activity was determined by measuring the diameter of the inhibition zone around

the well.

Table 1. Inhibitory action of synthesized AgNPs against five human pathogenic

bacteria

AgNPs Bacteria Inhibition zone, (mm)(mg/well)

Ciprofloxacin(100µg/ml)

5 2.5 1.25 0.625

AgCC Proteus mirabilis 26 24 20 16 16Klebsiella pneumoniae 22 20 18 16 17

Escherichia coli 18 16 14 12 19Salmonella paratyphi 28 26 22 20 19

Pseudomonas aeruginosa 28 24 20 18 20AgCL Proteus mirabilis 28 26 24 18 16

Klebsiella pneumoniae 20 18 16 14 17Escherichia coli 20 18 16 14 19

Salmonella paratyphi 28 24 22 20 19Pseudomonas aeruginosa 32 28 26 24 20

AgCA Proteus mirabilis 26 20 18 16 16Klebsiella pneumoniae 20 18 16 14 17

Escherichia coli 20 18 16 12 19Salmonella paratyphi 30 28 24 20 19

Pseudomonas aeruginosa 36 34 30 28 20AgHC Proteus mirabilis 24 22 20 16 16

Klebsiella pneumoniae 20 18 16 14 17Escherichia coli 20 18 16 12 19

Salmonella paratyphi 20 18 16 14 19Pseudomonas aeruginosa 38 36 32 28 20

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Table 2. The Minimal inhibitory concentrations (MICs) of synthesized AgNPs

against five human pathogenic bacteria

AgNPs Bacteria MIC

Compound (mg/ml)AgCC Proteus mirabilis <0.01953125

Klebsiella pneumoniae 0.01953125Escherichia coli 0.078125

Salmonella paratyphi 0.0048828125Pseudomonas aeruginosa 0.009765625

AgCL Proteus mirabilis <0.009765625Klebsiella pneumoniae 0.01953125

Escherichia coli 0.01953125Salmonella paratyphi 0.0048828125

Pseudomonas aeruginosa <0.00122070312AgCA Proteus mirabilis <0.01953125

Klebsiella pneumoniae >0.01953125Escherichia coli 0.078125

Salmonella paratyphi 0.004828125Pseudomonas aeruginosa 0.00061035156

AgHC Proteus mirabilis 0.009765625Klebsiellapneumoniae 0.01953125

Escherichia coli 0.0.78125Salmonella paratyphi 0.01953125

Pseudomonas aeruginosa <0.00061035156

5.3.6. Preparation of DPPH for catalytic study

The catalytic activity of the as prepared silver nanoparticles is studied

on the reduction of 2,2-Diphenyl-1-Picrylhydrazyl (DPPH, sigma) by

Butylated hydroxyl toluene (BHT, Sigma). 0.1mL of BHT (125µg/mL) and 0.1

mL AgNPs are mixed with 2.8mL of DPPH (45µg/mL, Sigma). Then the UV–

Vis spectra have been recorded in methanol at regular intervals of time.

172

Conclusion

Silver nanoparticles were successfully prepared using C. leucorrhiza, C.

caesia, C. aromatica, H. coccineum, and K. galanga respectively as the starting

raw materials. The secondary metabolites from the rhizomes extracts act as an

environmentally benign reductants and stabilizers. Terpenoids and water soluble

fractions comprised of complex polyols in the biomass were believed to play

major role in the reduction of silver ions. UV/Vis spectroscopy revealed the

surface plasmon property at 431 nm and 441 nm for AgCL and AgCC NPs

respectivly. AgCA and AgKG NPs exhibited surface plasmon resonance at

436nm and 422nm respectively. TEM and SAED images confirmed the surface

morphology, shape and size of the silver nanoparticles. It was observed that

most of the Ag NPs were spherical in shape with a moderate variation in particle

sizes. The silver nanoparticles were polycrystalline. According to the particle

size distribution, average size range estimated from our studies was found to be

4–8nm, 4-12nm, 3-6nm, 2-4nm and 2-4nm for AgCL, AgCC, AgCA, AgHC and

AgKG NPs respectively. The synthesized AgNPs showed the antibacterial

activity against five human pathogenic bacteria. AgCA showed the highest

catalytic activity, followed by AgCL, AgHC and AgZ NPs. AgCC presented the

least catalytic activity, followed by AgKG NPs.

173

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