Therapeutic - Hindawi Publishing Corporationdownloads.hindawi.com/journals/bca/2014/135824.pdf · Structural Features and In Vivo and In Vitro Therapeutic Effects against Helicobacter

Research ArticleGreen Synthesis of Silver NanoparticlesStructural Features and In Vivo and In Vitro TherapeuticEffects against Helicobacter pylori Induced Gastritis

Muhammad Amin1 Sadaf Hameed1 Asghar Ali1 Farooq Anwar12 Shaukat Ali Shahid3

Imran Shakir4 Aqdas Yaqoob1 Sara Hasan1 Safyan Akram Khan5 and Sajjad-ur-Rahman6

1 Department of Chemistry University of Sargodha Sargodha 40100 Pakistan2 College of Pharmacy Salman bin Abdulaziz University Al-Kharj 11942 Saudi Arabia3 Department of Physics University of Agriculture Faisalabad 38040 Pakistan4Deanship of Scientific Research College of Engineering King Saud University PO Box 800 Riyadh Saudi Arabia5 Center of Excellence in Nanotechnology Research Institute King Fahd University of Petroleum and MineralsDhahran 31261 Saudi Arabia

6 Institute of Microbiology Faculty of Veterinary Sciences University of Agriculture Faisalabad 38040 Pakistan

Correspondence should be addressed to Muhammad Amin muhammadameen20002000yahoocom

Received 20 April 2014 Revised 16 June 2014 Accepted 16 June 2014 Published 6 August 2014

Academic Editor Imre Sovago

Copyright copy 2014 Muhammad Amin et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

This study evaluates in vivo and in vitro anti-Helicobacter pylori (H pylori) efficacy of silver nanoparticles (Ag-NPs) prepared viaa cost-effective green chemistry route wherein Peganum harmala L seeds extract was used as a reducing and capping agent Thestructural features as elucidated by surface plasmon resonance spectrophotometry transmission electron microscopy and powderX-ray diffraction spectroscopy revealed the Ag-NPs synthesized to be polydispersed in nature and spherical in shape with 5ndash40 nm size A typical Ag-NPs suspension (S

5) with size being 15 nm when tested in vitro against forty-two local isolates and two

reference strains showed a considerable anti-H pylori activity In case of in vivo trial against H pylori induced gastritis after oraladministration of 16mgkg bodyweight of S

5for seven days a complete clearance was recorded inmale albino rates In comparative

time-killing kinetics S5exhibited dose- and time-dependent anti-H pylori activity that was almost similar to tetracycline and

clarithromycin less than amoxicillin but higher than metronidazole Furthermore S5was found to be an equally effective anti-H

pylori agent at low (le4) and high pH with no drug resistance observed even up to 10 repeated exposures while a significant drugresistance was recorded for most of the standard drugs employed The present results revealed the potential of the synthesizedAg-NPs as safer bactericidal agents for the treatment of H pylori induced gastritis

1 Introduction

Nanotechnology has become a rapidly growing field ofresearch because of the unique properties and broad rangeof applications of nanobioactive materials in different areasof industrial and biochemical sciences [1 2] Of the nano-materials the silver nanoparticles (Ag-NPs) have attracted agreat deal of scientific interest due to their vast applicationsin the field of medicine [3] catalysis [4] and electronics [5ndash7] So far many methods including physical chemical andbiochemical methods are being employed for the preparation

of Ag-NPs In this regard chemical methods are discouraged[8] as they involve the use of noxious reducing agents andsolvents [9] With the growing awareness about the safe andclean environment the use of green approaches in syntheticprocesses is gainingmuch importance [10]Therefore severalmethods have been developed to avoid the use of syn-theticchemical reducing and stabilizing agents [2] To facil-itate the medicinal applications of Ag-NPs it is fascinatingthat thematerials used as aids in synthesis should be biocom-patible [11] Therefore plant based reducing and stabilizingagents are being preferred over the chemical methods [12]

Hindawi Publishing CorporationBioinorganic Chemistry and ApplicationsVolume 2014 Article ID 135824 11 pageshttpdxdoiorg1011552014135824

2 Bioinorganic Chemistry and Applications

It has been established that H pylori is the major causeof functional dyspepsia chronic mucosal inflammation inthe stomach and duodenum peptic ulcer and many othergastroduodenal abnormalities [13] It is a small curvedGram-positive and microaerophilic bacterium which is fastidiousto grow under ordinary growth medium [13] Triple therapyhas been recommended for the complete eradication of Hpylori [14] Presently the major concern around the worldin medical science is that H pylori has developed resistanceagainst the standard antibiotics being used in the clinicalpractices [15] Therefore there is a need for the developmentof novel antimicrobial agents possessing superior effective-ness against H pylori with reduced toxicity for human cellsSo far metals including zinc [16] bismuth [17] and silver-NPs [11] have been searched for their in vitro antibacterialactivity against H pylori A zinc-based mucosal protectiveagent polaprezinc [18] and various salts of bismuth havebeen recognized as effective antiulcer and anti-H pyloridrugsand indexed in various pharmacopeias [19] Because of well-defined antimicrobial activities and potential role in woundhealing [20] Ag-NPs can be explored as a possible treatmentfor gastrointestinal related H pylori infections

Peganum harmala L commonly known as wild rue isa medicinally important plant from the family NitrariaceaeIn the Soon Valley Punjab Pakistan it is locally knownas ldquoharmalardquo The seeds of Peganum harmala (P harmala)contain several alkaloids [21] and other phytochemicals thatattribute various medicinal properties to this multipurposeplant [21]The seeds of this plant possess antibacterial activityagainst drug-resistant bacteria [22] smoke from the seedskills algae bacteria intestinal parasites and molds and theroots are used to kill lice and insects [21] Cytotoxic activityagainst the tissues of liver and kidney at very high doseof 150 gkg of body weight in rats has been reported [22]However at a dose range of 75ndash100 gkg body weight inrats moderate liver and kidney toxicity was observed [22] Pharmala extract and powdered seeds have been used in folkmedicine of different parts of the world to treat colic in manand animals due to their antispasmodic effect by blockingdifferent types of intestinal calcium channels [23]

Our earlier study showed [11] that Ag-NPs had broad andselective in vitro antimicrobial activity against the antibiotic-resistant and antibiotic-susceptible strains ofH pylori In thepresent study we prepared highly stable Ag-NPs via a cost-effective green chemistry route using P harmala seeds extractas a reducing and capping agent The main objective of thisstudy was to investigate the in vitro and in vivo anti-H pyloriactivity of the synthesized Ag-NPs in a systemic infectionmodel to fill the gap between in vitro characterization andclinical trials This is perhaps the first time that we haveevaluated the in vivo efficacy of Ag-NPs against H pyloriinduced ulcer using an animal model

2 Materials and Methods

21 Preparation of Green Reducing Agent P harmala seedswere collected from Kanhati Garden Soon Valley PunjabPakistan during the months of September-October 2010

Table 1 Inputs of the reagents for the synthesis of silver nanoparti-cles

Sample code PH-sus () AgNO3 (10mM mL) Water ()S1 20 900 80S2 40 900 60S3 60 900 40S4 80 900 20S5 10 900 90S6 10 90lowast 90S7 10 80lowast 90S8 10 70lowast 90S9 10 90dagger 90S10 10 80dagger 90S11 10 70dagger 90lowastrepresents 20mM AgNO3 solution

daggerrepresents 30mM AgNO3 solution

and were identified and authenticated by Dr Amin ShahDepartment of Biological Sciences University of SargodhaSargodha Pakistan A specimen of the seeds was also keptat the Herbarium of the University of Sargodha SargodhaPakistan Dried seeds (100 g) were ground by a conventionalcoffee grinder and extracted with methanol for 48 h usinga Soxhlet extractor Afterwards the residue was reextractedwith the same fresh solvent both of the extracts were pooledand concentrated to dryness with a rotary evaporator (45∘C)The crude concentrated P harmala suspension (PH-sus) waspreserved at minus4∘C and used for further experiments

22 Synthesis of Ag-NPs under Optimized Conditions andStructural Feature To an appropriate amount of PH-sus pre-pared above AgNO

3(90mL 10mM) was added dropwise

under vigorous stirring by the use of a magnetic stirrer-cum-hotplate at ambient to 100∘C The inputs of the reagents usedin the synthesis are given in Table 1

The experiment was repeated by using 20 and 30mMAgNO

3separately in order to study the effect of concentration

of the silver salt on Ag-NPs characteristicsThe effect of reac-tion time (10ndash180min) medium-pH (4ndash10) and temperature(ambient to 100∘C) on the synthesis was studied by surfaceplasmon resonance (SPR) spectral measurements The Ag-NPs suspension was washed several times with Nanopurewater in order to free the NPs from any unreacted silver salt

23 Surface Plasmon Resonance (SPR) Spectroscopic Measure-ments Ag-NPs suspension was appropriately diluted withwater in order to obtain the SPR spectrum within 200ndash700 nm range by using Pharma Spec UV-1700 (ShimadzuTokyo Japan) UV-Vis spectrophotometer by taking extractblank as a reference Average size of the NPs was calculatedfrom SPR measurements according to the already reportedmethod [24] using the equation

119889 =1

1198712

sdot 119871119899(120582SPR minus 120582

∘

1198711

) (1)

where 120582∘ = 512 1198711= 653 and 119871

2= 00216

Bioinorganic Chemistry and Applications 3

24 X-Ray Diffraction Analysis Powder X-ray diffraction (P-XRD) spectra of silver-NPs were recorded on Bruker D8Discover (Germany) diffractometer using monochromaticCuK120572 radiation (120582= 15406 A) operating at 40 kVand 30mAThe data for the lyophilized NPs were collected over a 10ndash80∘ 2120579 rangeThe size of nano-crystallite was calculated fromthe full width at half maxim (FWHM) of the most intensepeak of the spectrum by the use of Debye-Scherrer equation(119863 = 09120582120573 cos 120579)

25 Transmission Electron Microscopy (TEM) An ultrason-ically dispersed sample of the solution of NPs (one drop)was placed on a carbon grid dried at room temperature inclean environment and TEM images were obtained by usingJEM-1200EX (JEOL Japan) microscope at an acceleratingvoltage of 120 kV The average size of NPs was calculatedmeasuring the diameter of about 140 particles by usingOrigin75 software

26 FT-IR Spectroscopy Infrared spectra of the Ag-NPs wererecorded by reflectance method on Perkin-Elmer Spectrum100 FT-IR

27 In Vitro Anti-Helicobacter pylori Activity Forty-two localisolates [11 16] and two standard strains of H pylori NCTC11637 and NCTC 11638 obtained from the National HealthProtection Agency London were used in this study Invitro anti-H pylori activities of the NPs sample S

5 amoxi-

cillin (AMX) tetracycline (TET) clarithromycin (CLT) andmetronidazole (MNZ) were determined by agar dilutionmethod according to already reported method [11 16] Allthe isolates were transferred and inoculation procedures wereconducted under the BSL-III safety cabinet throughout

28 In Vivo Anti-Helicobacter pylori Activity In vivo anti-Hpylori activity of the S

5was studied in male albinoWistar rats

of 72ndash112 days having average weight of 295 plusmn 41 g For thispurpose the rats were infected by oral administration of Hpylori reference strain and then treated with different doses ofsilver nanoparticles An already reported method with slightmodification was adapted in this study [11]

The rats were maintained in the separate animal housefacilities and acclimatized for one week prior to the exper-iment All the experiments were conducted in accordancewith the ethical protocols and guidelines provided by theEthical Committee for Experimental Animals University ofSargodha Pakistan The rats were kept on fasting for 18 hand offered oral dose with 05mL of brain heart infusionbroth (BHIB) containing a fresh diluted culture of H pyloristrain (36 times 108 CFUanimal NCTC 11637) The controlrats were inoculated with blank medium Inoculation wasrepeated after three days and the H pylori was monitoredin the gastric lavage through microscopic examination afterone week postinoculation After confirmation the rats weresegregated into various groups Different concentrations ofthe S5were prepared as per dosage scheme of the already

reported method [11] and administered orally as suspensionin edible oil The dosage scheme was selected by keeping in

view the in vitro MICs of S5experiment The dosing was

started 7 weeks after H pylori inoculation and continuedtwice a day for 7 days On the 3rd day after final dose the ratswere killed and counted for viable H pylori counts This wasdone by grinding the tissue of the removed stomach betweenthe frosted ends of the glass slides and inoculating on theColumbia agar plates under microaerophilic conditions Thecolonies ofH pylori were counted and the clearance rate wasdetermined by Dunnettrsquos method and Fisherrsquos exact test Pvalues below 005 were considered statistically significant

29 Time-Killing Kinetics of Silver Nanoparticles This studywas done according to the standard guide for assessment ofantimicrobial activity using time-killing kinetics procedure[25] The guidelines provided by CLSI for the determinationof time-killing kinetics were adapted throughout this inves-tigation Cultures of H pylori (NCTC 11637) freshly grownover Colombia agar plates were serially adjusted to initialcell concentration of 1 times 106 CFUmL BHIB broths with25 foetal bovine serum (10mL) containing S

5at concen-

trations of 10 120583gmLminus1 20120583gmLminus1 40 120583gmLminus1 80120583gmLminus1and 16 120583gmLminus1 were inoculated with 10 120583L of the freshbacterial culture and shaken at 37∘C in a microaerophilicatmosphere Aliquots (100 120583L) were removed during shakingat various time points (0 1 2 3 6 9 and 24 h) The aliquotswere 10-fold serially diluted in Brucella broth and a 50120583Lportion of each of them was seeded over the plates of BHIAsupplemented with 7 defibrinated horse blood The plateswere kept under microaerophilic atmosphere at 37∘C for72 h Colonies were counted after 72 hours of incubationin a microaerophilic atmosphere and rates of killing weredetermined in duplicate bymeasuring the reduction in viablebacteria (log

10CFUmL)

210 Effects of pH on Bactericidal Activity of Silver Nanopar-ticles Silver-NPs (S

5) in the concentration range of 20ndash

16 120583gmLminus1 were added to BHIB with 7 defibrinated horseblood The pH was adjusted to 7 6 5 and 3 by using 1N ureasolution as appropriate and the bacterial suspension (NCTC11637 10 120583L) was seeded into it Urea was added to control thelethality of acidic pH to H pylori Cultures were incubatedand aliquots were collected at various time points (0 2 34 6 7 9 and 24 h) The aliquots were serially diluted to 10-fold with saline and 10 120583L of the diluted sample was placedonColombia agar platewith 7defibrinated horse bloodTheplatewas incubated at 37∘Cundermicroaerophilic conditionsfor 72 h and colonies of H pylori were counted

211 Determination of Resistance Development in H pyloriExposed to Silver Nanoparticles H pylori strain NCTC 11637adjusted to a culture density of approximately 106 CFUmL inBrucella broth supplemented with 25 FBS was exposed toserial twofold dilutions of S

5(20ndash16 120583gmL) AMX (00125ndash

10 120583gmL) and TET (80ndash64 120583gmL) After the incubationof the bacteria at 37∘C under microaerophilic conditions for24 h the culture was examined for any visible growth ofbacteria The culture that attained turbidity comparable tothat of the untreated culture in the presence of the highest


Table 2 Size of silver nanoparticles calculated by using different techniques

Sample code SPR measurements P-XRD measurements TEMmeasurements (nm)Peak position (nm) Calculated size (nm) FWHM of fcc (111) Calculated size (nm)

S1 440 30 30 25 15ndash40S2 435 22 25 25 15ndash30S3 427 20 25 20 10ndash25S4 419 15 22 15 07ndash14S5 412 12 20 15 05ndash10

level of the test agent was further exposed to increasingconcentrations of the test agent These procedures wererepeated for three times The fluctuations of the MIC in thecourse of the repeated exposure of the bacteria to the testagent were determined

3 Results and Discussion

31 SPR Measurements The SPR absorption was found tobe extremely dependent upon the size shape interparticledistance and the surrounding media of the Ag-NPs [26]Thus the shape and the size of theNPs can easily be optimizedby the use of SPR spectra

For the synthesis of a typical sample S5 PH-sus (10mL)

suspension was added to silver nitrate solution (90mL 1mMaqueous) and heated at 45∘C After 30min a colour change(yellowish brown) was observed in the reaction mixturewhich showed the formation of silver-NPs [11 12] SPR spec-tra were found to be highly sensitive to the concentrationsof PH-sus (Figure 1(a)) For an illustration an increasingamount of the PH-sus (20ndash10mL) caused a significant shiftin the UV-Vis region of the spectrum (440ndash412 nm) Thissignifies the role of reducing agents (PH-sus) in tailoring thesize and shape of NPs as the spectrum can experience a shiftdependent upon the particle size shape and the surroundingmedium [26] Therefore it was observed that beyond anoptimum concentration of the PH-sus the size of the particlesis so increased that is it ceased to be in nanolimits [27]Such observations can be used in calculating the size of theparticles from SPR measurements [2] The size of silver-NPscalculated by various techniques in this study is presented inFigure 1(b)

The effect of temperature on the synthesis of typicalsample S

5is shown in Figure 1(c) No band appeared in SPR

spectrum of the reaction carried out below 25∘C while abroad peak of very low intensity (not shown in Figure 1(c))was observed at 406 nm for the colloidal suspension obtainedafter heating at 30∘C for 30min However for the yellowishbrown colloidal suspension obtained after 30min of stirringat 45∘C an SPR band at 412 nm suggested the formationof Ag-NPs [26] Further increase in temperature resultedin broader peaks until the peak intensity became constantbeyond 100∘CThe optimum temperature for the synthesis oftypical samplewas found to be 45∘C Beyond this temperaturethe solution becomes more viscous perhaps due to theaggregation and increased size of the nanoparticles [11]

The reaction-stirring-time is an important factor affect-ing the optimization of SPR peak intensity A very clear andintense absorption band (Figure 1(d)) appearing at 412 nmafter 30min of stirring for a brown colloidal solutionindicated the presence of spherical NPs [1] Further stirringup to 180min at the same conditions resulted in a viscousdark brown solution having SPR at 499 nm It was thereforeasserted that an optimum time of 30min was required for thecompletion of reaction

Another important factor is pH that affects the synthesisin vivo stability and anti-H pylori activity of Ag-NPs [28]Therefore it is equally important that the silver-NPs must bestable even at low pH (le4) values as they are desired to beadministered in the stomach In order to obtain SPR at 412 nmthe optimum pH was found to be 4 This is in contrast to theprevious studies on the synthesis of Ag-NPs [29] whereby itwas found that the Ag-NPs were stable mostly at high pH andthat at acidic pH clusters were obtained This is perhaps duemainly to the presence of such phytochemicals in the PH-suswhich make it an efficient reducing and capping agent evenat low pH values

The stability of the colloidal suspension S5was deter-

mined periodically by taking the SPR spectraThe suspensionwas found to be stable for a period of more than oneyear and the peak intensity observed was exactly at 412 nm(Figure 1(e))

In order to investigate the effect of silver nitrate concen-tration on the synthesis of Ag-NPs the experiments were alsoperformed by the use of 20mM and 30mM silver nitratesolution while keeping the PH-suspension constant and sixsamples (S

6ndashS11) were synthesized The SPR spectra of these

colloidal suspensions were observed to be between 470 nmand 495 nm indicating large particle size and aggregates Atypical image of an aggregate (S

6) is shown in Figure 2(c)

Under the aforementioned optimized conditions thesynthesis reactions were assumed to be complete because nopeaks due to the residues of the reducing agents were detectedin the UV-Vis spectroscopy SPR measurements have beenproved to be beneficial in determining the size of the NPs[26] The sharpness and symmetry of SPR peaks are reportedto be indicative of the particle size [2]The dependence of theSPR of NPs on particle size and wavelength was analyzed andthe particle size was calculated from SPR measurements andother techniques as listed in Table 2

32 TEM Analysis The particle sizes calculated by TEMmeasurements of five samples (S

1ndashS5) of silver-NPs are


300 400 500 600 700 800

15

10

05

Abso

rban

ce

20mL40mL60mL

80mL100mL

412nm

419nm

427nm

435nm

440nm

Wavelength (nm)

(a)Pa

rtic

le si

ze

0

10

20

30

40

50

60

70

80

0 5 10 15

PH-sus

(b)

Wav

eleng

th (n

m)

500

400

300

200

100

020 40 60 80 120100

(∘C)

(c)

15

10

05

300 400 500 600 700 800

30min

25min

20min

15min

Abso

rban

ce

Wavelength (nm)

(d)

15

10

05

Abso

rban

ce

300 400 500 600 700 800

Wavelength (nm)

1 month6 months1 year

(e)

Figure 1 (a) Effect of PH-sus on the SPR of typical sample S5 (b) variation of the size of the silver-NPswith PH-sus using different techniques

SPR (Q) P-XRD (◼) and TEM analysis (998771) (c) variation of SPR with temperature (S5) (d) effect of time on the synthesis of sample S

5 and

(e) stability of Ag-NPs versus time


50 nm

50 nm

50 nm

50 nm

10

20

30

0 10 20 30 40

Diameter (nm)

10

20

30

0 10 20 30 40

Diameter (nm)50

30 40 50 60 70 80

Rela

tive i

nten

sity

(111

)

(220

)

(222

)

(311

)

2-theta scale

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

Freq

uenc

y (

)Fr

eque

ncy

()

(111)

(220)

(311)

(200)

Figure 2 (a e) TEM image and size distribution of S5 (b f) TEM image and size distribution of S

4 (c) TEM image of S

3 (d) TEM image of

S2 and (g) P-XRD pattern of S

5and SAED pattern of S

5

presented in Table 1 whereas typical TEM images of thecolloidal suspensions S

5and S

4 and their size distribution

histograms are shown in Figures 2(a) 2(b) 2(e) and 2(f)respectively The elongation of the particles that is the ratioof the long to the short axes was found to be between 105 and

125 thereby suggesting the spherical nature of the particles[30] Figure 2(a) consists of almost uniformly sized sphericalNPsThe sizes of S

5and S4were found to be 15 nm and 18 nm

respectively These results are in accordance with the shapeof the SPR bands as reported by some earlier researchers


[28] The size ranges of S3and S

2 calculated from TEM

measurements were found to be 10ndash25 nm and 15ndash30 nmrespectively (Figures 2(c) and 2(d))

33 Powder XRD Analysis P-XRD patterns and selected areaelectron diffraction (SAED) of the typical sample (S

5) are

shown in Figures 2(f) and 2(h) P-XRD consisted of intensepeaks appearing at 382∘ 441∘ 643∘ and 78∘ in the 2120579range of the spectrum These spectra were indexed as (111)(200) (220) and (311) planes of face centred cubic silverwith the help of the data obtained from the database of JointCommittee on Powder Diffraction Standards file number 04-0783 In the spectrum (111) facet reflections were found tobe most intense as compared to the rest of the peaks Thisfeature attributes special bactericidal properties to the silver-NPs [9] The full width at half-maxim (FWHM) of the (111)facewas calculated and the average size of the samples (S

1ndashS5)

calculated by the use of Debye-Scherer equation is shown inTable 2 On the basis of the smallest size spherical shape andthe aforementioned features the tests of the studies were con-ducted only for the typical sample S

5 The patterns appearing

in SAED were characterized as (111) (200) (300) and (200)face-centered cubic (fcc) plane of the crystal structure

34 FT-IR Spectroscopy of Silver Nanoparticles FT-IR mea-surements were carried out in order to identify the potentialof biomolecules in PH-sus responsible for reduction andcapping of silver nanoparticles The characteristic peaksappearing in the spectrum of PH-sus (Figure 3(a)) at about3600 1763 and 1334 cmminus1 are characteristic of ](OH) the CndashO and C=O stretching modes of the carboxylic acid groupThe bands appearing at 1669 and 1535 cmminus1 were assigned toamide I and amide II bands respectively which may arisedue to carboxyl stretch and NndashH deformation vibrations inthe amide linkages of some proteins present in them [5]The disappearance of ](OH) (Figure 3(b)) in the spectra ofsilver nanoparticles is consistent with the rearrangement anddeprotonation of the OndashH and some other groups in the PH-sus to be involved in the stabilizing of silver nanoparticles[5 6]

35 In Vitro Anti-Helicobacter pylori Activity The growthinhibition activities of S

5against H pylori reference strains

(NCTC-11637 and NCTC-11638) and antibiotic-resistant andantibiotic-susceptible isolates ofH pylori are listed in Table 3It was also found that the anti-H pylori activity of S

5against

antibiotic-resistant isolates was nearly comparable to thoseagainst antibiotic-susceptible isolates

36 In Vivo Anti-H pylori Activity All of the vehicle-treatedand control rats were maintained with gastric H pylori at alevel of approximately 1 times 106 CFUThe application of S

5was

found to be efficacious in curing ulcer by inhibitingH pyloriComplete clearance was obtained at a dose of 16mgkg ofbody weight (Figure 4)

Table 3 In vitro minimum inhibitory concentration of S5 againstreference strains and antibiotic-resistant and antibiotic-susceptiblelocal isolates of H pylori

Organism Strainisolate MICs (120583gmLminus1)of S5

Helicobacter pylori NCTC 11637 40NCTC 11638 80Clinical isolates [11 16]AMX-resistant (119899 = 08) 40ndash80AMX-susceptible (119899 = 32) 20ndash80CLT-resistant (119899 = 05) 10ndash16CLT-susceptible (119899 = 35) 40ndash16TET-resistant (119899 = 09) 20ndash80TET-susceptible (119899 = 31) 20ndash16MNZ-resistant (119899 = 036) 20ndash32MNZ-susceptible (119899 = 04) 40ndash80

5001500250035004500

Tran

smitt

ance

()

(cmminus1)

PHSE

minus500

Ag-NPs (S5)

Figure 3 (a) FT-IR spectra of PH-sus and (b) FT-IR spectra of Ag-NPs (S

5)

12

10

8

6

4

2

0010 210 610 810 1010

Clearedtotal

Log

CFU

gas

tric

wal

l

Detection limit

Figure 4 In vivo therapeutic efficacy of silver nanoparticles

37 Time-Killing Kinetics of Silver Nanoparticles Time- anddose-killing curves of S

5 TET AMX and MNZ against

H pylori strain NCTC 11637 as function of viable bacterialcounts versus incubation time are shown in Figures 5(a)ndash5(d) The curves for the rest of the strainsisolates are notshown S

5exhibited (Figure 5(a)) bactericidal effects at con-

centrations of 40 120583gmLminus1 80 120583gmLminus1 and 16 120583gmLminus1 It wasfound that at 16120583gmLminus1 dose S

5was found to be effective for


0 10 20 30

Incubation time (h)

0

2

4

6

8

10

12

14

Viab

le co

unts

(log

CFU

mL)

(a)

0 10 20 30

Incubation time (h)

0

2

4

6

8

10

12

Viab

le co

unts

(log

CFU

mL)

(b)

0

2

4

6

8

10

12

14

Control12 times MIC1 times MIC

2 times MIC4 times MIC8 times MIC

0 10 20 30

Incubation time (h)

Viab

le co

unts

(log

CFU

mL)

(c)

0

2

4

6

8

10

12

0 10 20 30

Viab

le co

unts

(log

CFU

mL)

Incubation time (h)



(d)

Figure 5 Time- and dose-dependent killing curves for (a) silver nanoparticles (b) tetracycline (c)metronidazole and (d) amoxicillin againstH pylori strain NCTC 11637

eradicatingH pylori strains within time range of 9 h followedby 80120583gmLminus1 dose in 12 h time However after 24 h contactstimeNPs atmost of the concentration range (40ndash16 120583gmLminus1)showed potent bactericidal effect against the tested H pyloristrains At a lower concentration of 20120583gmLminus1 no influenceor a slight decrease in CFUmL was noted S

5generally

exhibited rapid killing effect demonstrating concentration-and time-dependent bactericidal activity At 40120583gmLminus1 theinhibition occurred at 12 h after the addition of H pylorisuspension to the concentrationsThese results at higher con-centrations of Ag-NPs were found to be almost comparable

to the clarithromycin-resistant isolates of H pylori (data notshown)

Amongst the standard antibiotics used in this studyTET (Figure 5(b)) exhibited anti-H pylori activities almostcomparable to those of S

5(effective concentrations 40ndash

16 120583gmLminus1 90ndash24 h) whereas AMX (Figure 5(c)) showed thebest bactericidal activities (effective dose 025ndash40120583gmLminus1)MNZ (Figure 5(d)) was found to be less potent than S

5and

almost no H pylori strain was found to be susceptible toit However at higher concentration (1024120583gmLminus1) a slightdecrease in CFUmL was noted


minus2

0

2

4

6

8

10

0 5 10 15 20 25

Time (h)

Viab

le co

unts

(log

CFU

mL)

pH 7

pH 6

pH 5

pH 4

pH 3

Figure 6 Effect of medium pH on the anti-H pylori activities ofsilver nanoparticles against NCTC 11637

38 Effect of pH on the Anti-H pylori Activity of SilverNanoparticles Theeffect ofmedium-pHon the anti-H pyloriactivities of S

5against H pylori strain NCTC 11637 at MIC

16 120583gmLminus1 is presented in Figure 6 It was found that thebactericidal activity of the S

5was not affected by themedium-

pH (3ndash7) S5(concentration 16 120583gmLminus1) at pH 3 and pH 5

exhibited potent bactericidal effects and the viable bacterialcounts reduced rapidly at 7 h after the NPs contact Howeverthe complete eradication was found to be possible after about12 h time The complete eradication (the time at which viablecounts become zero) at pH 5 was possible after 24 h

39 Development of Resistance in H pylori to the SilverNanoparticles The fluctuations of the MICs in the course ofthe repeated exposure of the bacteria to the silver-NPs (S

5)

and other test drugs are shown in Figure 7 No significantchanges to the MICs of S

5and AMX were found However

a growing drug resistance was observed in the case of TETand MNZ after the fifth repeated exposure

The use of solvents during drug synthesis leads to residualsolvents in the final products and causes negative impact onhealth and the environment [31] Currently it is highly rec-ommended that the drug substances should be synthesized bysolvent and noxious chemical free methods [2] The presentinvestigation revealed that the P harmala seeds extract notonly reduced the silver ions but also efficiently caped thesynthesized NPs at least up to more than two years The roleof capping agents in the synthesis of NPs formulations is ofimmense importance and recently in an in vivo study it wasdemonstrated that the capped silver-NPs possessed enhancedantimicrobial activities than the uncapped ones [31]

Synthesis of Ag-NPs using green chemistry principleswhereby some plant extracts can be used as reducing and

0

5

10

15

20

25

30

35

40

0 5 10

MIC

s (120583

g mLminus

1)

AMXAg-NPs

TETCLT

Number of transfers

Figure 7 Resistance developments in H pylori strain NCTC11637 after repeated exposure to silver nanoparticles and standardantibiotics

capping agents has received special attention due to main-taining an aseptic environment during the environment-friendly process [12] Green synthesis of NPs has novelty andinnovationwith regard to variation in particle size shape andsynthesis conditions

In our previous in vitro study [11] we have found that allof the tested clinical isolates (H pylori) were susceptible tosilver-NPs synthesized by the use of a green method Thecurrent study was aimed at facilitating the in vivo clinicalmanifestations of silver-NPs In the current investigation itwas found that an oral administration of 16mgkg bodyweight of S

5resulted in the complete clearance of gastric

infection induced by 36 times 108 CFUanimal of H pyloriinoculums In vitro time-killing kinetics showed that viablecounts were reduced to zero 12 h after bacterial contact with80 120583gmLminus1 of S

5 However the same bacterial eradication

was achieved in 8 h by administering 16 120583gmLminus1 of S5 This

shows that in vitro susceptibilities are in consistency withthe in vivo findings Some discrepancies have previouslybeen reported between the in vitro antibacterial activities andthe clinical efficacies of several antibacterial agents towardsthe eradication of H pylori related infections [32] Someearlier researchers [33] have reported that an ingestion upto 16mg of silver is well tolerated in humans Furthermoreit has low toxicity and minimal side effects when ingestedsince at most 2ndash4 is retained in tissues after absorptionby the body [34] Development of antimicrobial agents forthe eradication of multidrug-resistant (MDR) microbes is achallenge for the synthetic chemists [11] It has been reportedthat the efficacy of the triple therapy regime is decreasing tounacceptable levels (ie le80) [11 12] due to the antibioticresistance in H pylori [11ndash15] Almost similar resistancepatterns were found in the present research in case of TETand CLA However no drug resistance was found in Hpylori after prolonged exposure to S

5 Metallic silver and


its compounds have been used as antimicrobial agents anddisinfectants because of their mild toxicity to humans [3]However with the development of synthetic antimicrobialsfor the treatment of infectious diseases the use of silver inthe clinical setting had been restricted solely to the topicaluse [3] Due to the emergence of drug-resistant bacteriathere has been a resurgence of the promotion of silver-NPs asalternate antibiotics [8] Therefore silver-NPs find extensiveapplications in the field of medicine as anti-inflammatoryagents [35] in wound healing [3 11] and as antimicrobialagents against various classes of Gram-positive and Gram-negative bacteria [20]

In vivo anti-H pylori activity of silver-NPs may beconferred from the small size preferential penetrability tothe target site and potent wound healing properties [11]One possible explanation for the accordance between in vitroMICs and in vivo efficacy of S

5may be its stability under

acidic conditions Somemetals complexes have been reportedfor possessing bactericidal activities against H pylori [11 16]Among these bismuth compounds like bismuth subsalicylatebismuth subcitrate and ranitidine bismuth citrate have offi-cially been recommended as a part of triple therapy [36] Dueto toxic effects of bismuth on human cells [17 35] some othermetals and their NPs including Ag-NPs can be explored asa possible treatment for treatment of gastrointestinal and Hpylori related infections

4 Conclusions

A green method has been reported for the synthesis of Ag-NPs using P harmala L seeds extract as reducing and cappingagent It was found that the size and the shape of the Ag-NPs could be tailored by optimizing the reaction temperaturetime and pH of the media The optimized extract pH valuetemperature and molar ratio of the reactants improvedthe size and the shape of Ag-NPs The adopted methodis compatible with green chemistry approaches as the Pharmala L seeds extract serves as a matrix for both reductionand stabilization of the synthesized NPs These NPs due tobiocompatibility and bactericidal potency against H pylorimay be exploited as an anti-H pylori agent capable ofreplacing the existing triple and quadruple therapy regimens

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors gratefully acknowledge Dr J P Leeming Bris-tol Royal Infirmary Bristol UK for providing H pyloristrains and Dr V I Enne Centre for Immunology andInfectious Disease Blizard Institute of Cell and MolecularScience Barts and The London School of Medicine andDentistry 4 Newark Street Whitechapel London UK forher assistance inH pylori susceptibility testingMoreover theauthors would like to extend their sincere appreciation to the

Deanship of Scientific Research King Saud University for itsfunding of this research through the Research Group Projectno RGP-VPP-312

References

[1] R Bhattacharya and P Mukherjee ldquoBiological properties ofldquonakedrdquo metal nanoparticlesrdquoAdvanced Drug Delivery Reviewsvol 60 no 11 pp 1289ndash1306 2008

[2] M Amin F Iram M S Iqbal M Z Saeed M Raza andS Alam ldquoArabinoxylan-mediated synthesis of gold and silvernanoparticles having exceptional high stabilityrdquo CarbohydratePolymers vol 92 no 2 pp 1896ndash1900 2013

[3] D J Leaper ldquoSilver dressings their role inwoundmanagementrdquoInternational Wound Journal vol 3 no 4 pp 282ndash294 2006

[4] B Baruah G J Gabriel M J Akbashev and M E BooherldquoFacile synthesis of silver nanoparticles stabilized by cationicpolynorbornenes and their catalytic activity in 4-nitrophenolreductionrdquo Langmuir vol 29 no 13 pp 4225ndash4234 2013

[5] P Daizy ldquoGreen synthesis of gold and silver nanoparticles usingHibiscus rosa sinensisrdquo Physica E Low-Dimensional Systems andNanostructures vol 42 no 5 pp 1417ndash1424 2010

[6] KNThakkar S SMhatre andR Y Parikh ldquoBiological synthe-sis of metallic nanoparticlesrdquo Nanomedicine NanotechnologyBiology and Medicine vol 6 no 2 pp 257ndash262 2010

[7] M Montazer F Alimohammadi A Shamei andM K RahimildquoDurable antibacterial and cross-linking cotton with colloidalsilver nanoparticles and butane tetracarboxylic acid withoutyellowingrdquo Colloids and Surfaces B Biointerfaces vol 89 no 1pp 196ndash202 2012

[8] X Devaux C Laurent and A Rousset ldquoChemical synthesisof metal nanoparticles dispersed in aluminardquo NanostructuredMaterials vol 2 no 4 pp 339ndash346 1993

[9] I Pastoriza-Santos and L M Liz-Marzan ldquoFormation of PVP-protected metal nanoparticles in DMFrdquo Langmuir vol 18 no 7pp 2888ndash2894 2002

[10] MAAlbrecht CW Evans andC L Raston ldquoGreen chemistryand the health implications of nanoparticlesrdquo Green Chemistryvol 8 no 5 pp 417ndash432 2006

[11] M Amin F Anwar M R S A Janjua M A Iqbal andU Rashid ldquoGreen synthesis of silver nanoparticles throughreductionwith Solanum xanthocarpum L berry extract charac-terization antimicrobial and urease inhibitory activities againstHelicobacter pylorirdquo International Journal of Molecular Sciencesvol 13 no 8 pp 9923ndash9941 2012

[12] A J Kora R B Sashidhar and J Arunachalam ldquoGumkondagogu (Cochlospermum gossypium) a template for thegreen synthesis and stabilization of silver nanoparticles withantibacterial applicationrdquo Carbohydrate Polymers vol 82 no 3pp 670ndash679 2010

[13] B E Dunn H Cohen and M J Blaser ldquoHelicobacter pylorirdquoClinical Microbiology Reviews vol 10 no 4 pp 720ndash741 1997

[14] P Kostamo L Veijola A Oksanen S Sarna and H RautelinldquoRecent trends in primary antimicrobial resistance ofHelicobac-ter pylori in Finlandrdquo International Journal of AntimicrobialAgents vol 37 no 1 pp 22ndash25 2011

[15] FMegraud N Lehn T Lind et al ldquoAntimicrobial susceptibilitytesting of Helicobacter pylori in a large multicenter trial theMACH 2 studyrdquo Antimicrobial Agents and Chemotherapy vol43 no 11 pp 2747ndash2752 1999


[16] M Amin M S Iqbal R W Hughes et al ldquoMechanochemicalsynthesis and in vitro anti-Helicobacter pylori and uresaseinhibitory activities of novel zinc(II)famotidine complexrdquo Jour-nal of Enzyme Inhibition and Medicinal Chemistry vol 25 no3 pp 383ndash390 2010

[17] J P Gisbert and X Calvet ldquoReview article Non-bismuthquadruple (concomitant) therapy for eradication ofHelicobaterpylorirdquo Alimentary Pharmacology and Therapeutics vol 34 no6 pp 604ndash617 2011

[18] T Matsukura and H Tanaka ldquoApplicability of zinc complex ofL-carnosine for medical userdquo Biochemistry vol 65 no 7 pp817ndash823 2000

[19] S C Sweetman Martindale The Complete Drug ReferencePharmaceutical Press London UK 35th edition 2007

[20] M C Fung and D L Bowen ldquoSilver products for medicalindications risk-benefit assessmentrdquo Journal of ToxicologyClinical Toxicology vol 34 no 1 pp 119ndash126 1996

[21] M L Harsh and T N Nag ldquoAntimicrobial principles from invitro tissue culture of Peganum harmalardquo Journal of NaturalProducts vol 47 no 2 pp 365ndash367 1984

[22] AH SMohamed SM J AL-Jammali and Z J Naki ldquoEffect ofrepeated administration of Peganum harmala alcoholic extracton the liver and kidney in Albino mice a histo-pathologicalstudyrdquo Journal of Scientific amp Innovative Research vol 2 no 3pp 585ndash597 2013

[23] M Moloudizargari P Mikaili S Aghajanshakeri M HAsghari and J Shayegh ldquoPharmacological and therapeuticeffects of harmala and its main alkaloidsrdquo PharmacognosyReview vol 7 no 14 pp 199ndash212 2013

[24] W Haiss N T K Thanh J Aveyard and D G FernigldquoDetermination of size and concentration of gold nanoparticlesfrom UV-Vis spectrardquo Analytical Chemistry vol 79 no 11 pp4215ndash4221 2007

[25] Laboratory Methodologies for Bacterial Antimicrobial Suscep-tibility Testing OIE Terrestrial Manual 2008

[26] R A De Matos T Da Silva Cordeiro R E Samad N D VieiraJr and L C Courrol ldquoGreen synthesis of gold nanoparticlesof different sizes and shapes using agar-agar water solution andfemtosecond pulse laser irradiationrdquoApplied Physics A vol 109no 3 pp 737ndash741 2012

[27] V K Vidhu S A Aromal and D Philip ldquoGreen synthesisof silver nanoparticles using Macrotyloma uniflorumrdquo Spec-trochimica Acta A Molecular and Biomolecular Spectroscopyvol 83 no 1 pp 392ndash397 2011

[28] D L van Hyning W G Klemperer and C F Zukoski ldquoSilvernanoparticle formation predictions and verification of theaggregative growth modelrdquo Langmuir vol 17 no 11 pp 3128ndash3135 2001

[29] S F Shayesteh S Kolahi and Y A Kalandarragh ldquoEffect ofpH on the structure and optical properties of nanoparticlesembadded in PVA matrixrdquo Indian Journal of Pure and AppliedPhysics vol 51 no 11 pp 780ndash783 2013

[30] T C Prathna N Chandrasekaran A M Raichur and AMukherjee ldquoKinetic evolution studies of silver nanoparticles ina bio-based green synthesis processrdquo Colloids and Surfaces APhysicochemical and Engineering Aspects vol 377 no 1ndash3 pp212ndash216 2011

[31] D P Gnanadhas M BThomas RThomas A M Raichur andDChakravortty ldquoInteraction of silver nanoparticles with serumproteins affects their antimicrobial activity vivordquo AntimicrobialAgents and Chemotherapy vol 57 no 10 pp 4945ndash4955 2013

[32] T Kanamaru Y Nakano Y Toyoda et al ldquoIn vitro and invivo antibacterial activities of TAK-083 an agent for treatmentof Helicobacter pylori infectionrdquo Antimicrobial Agents andChemotherapy vol 45 no 9 pp 2455ndash2459 2001

[33] T Kim M Kim H Park U S Shin M Gong and HKim ldquoSize-dependent cellular toxicity of silver nanoparticlesrdquoJournal of Biomedical Materials Research A vol 100 no 4 pp1033ndash1043 2012

[34] J S Kim K S Song J H Sung et al ldquoGenotoxicity acute oraland dermal toxicity eye anddermal irritation and corrosion andskin sensitisation evaluation of silver nanoparticlesrdquo Nanotoxi-cology vol 7 no 5 pp 953ndash960 2013

[35] D MubarakAli N Thajuddin K Jeganathan and MGunasekaran ldquoPlant extract mediated synthesis of silver andgold nanoparticles and its antibacterial activity against clinicallyisolated pathogensrdquo Colloids and Surfaces B Biointerfaces vol85 no 2 pp 360ndash365 2011

[36] J P Gisbert X Calvet A OConnor F Megraud and C AOMorain ldquoSequential therapy for helicobacter pylori eradica-tion a critical reviewrdquo Journal of Clinical Gastroenterology vol44 no 5 pp 313ndash325 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy


Carbohydrate Chemistry

International Journal of


Journal of

Chemistry


Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of


Chromatography Research International


Applied ChemistryJournal of



Theoretical ChemistryJournal of


Journal of

Spectroscopy

Analytical ChemistryInternational Journal of


Journal of


Quantum Chemistry


Organic Chemistry International

ElectrochemistryInternational Journal of



CatalystsJournal of


It has been established that H pylori is the major causeof functional dyspepsia chronic mucosal inflammation inthe stomach and duodenum peptic ulcer and many othergastroduodenal abnormalities [13] It is a small curvedGram-positive and microaerophilic bacterium which is fastidiousto grow under ordinary growth medium [13] Triple therapyhas been recommended for the complete eradication of Hpylori [14] Presently the major concern around the worldin medical science is that H pylori has developed resistanceagainst the standard antibiotics being used in the clinicalpractices [15] Therefore there is a need for the developmentof novel antimicrobial agents possessing superior effective-ness against H pylori with reduced toxicity for human cellsSo far metals including zinc [16] bismuth [17] and silver-NPs [11] have been searched for their in vitro antibacterialactivity against H pylori A zinc-based mucosal protectiveagent polaprezinc [18] and various salts of bismuth havebeen recognized as effective antiulcer and anti-H pyloridrugsand indexed in various pharmacopeias [19] Because of well-defined antimicrobial activities and potential role in woundhealing [20] Ag-NPs can be explored as a possible treatmentfor gastrointestinal related H pylori infections

Peganum harmala L commonly known as wild rue isa medicinally important plant from the family NitrariaceaeIn the Soon Valley Punjab Pakistan it is locally knownas ldquoharmalardquo The seeds of Peganum harmala (P harmala)contain several alkaloids [21] and other phytochemicals thatattribute various medicinal properties to this multipurposeplant [21]The seeds of this plant possess antibacterial activityagainst drug-resistant bacteria [22] smoke from the seedskills algae bacteria intestinal parasites and molds and theroots are used to kill lice and insects [21] Cytotoxic activityagainst the tissues of liver and kidney at very high doseof 150 gkg of body weight in rats has been reported [22]However at a dose range of 75ndash100 gkg body weight inrats moderate liver and kidney toxicity was observed [22] Pharmala extract and powdered seeds have been used in folkmedicine of different parts of the world to treat colic in manand animals due to their antispasmodic effect by blockingdifferent types of intestinal calcium channels [23]

Our earlier study showed [11] that Ag-NPs had broad andselective in vitro antimicrobial activity against the antibiotic-resistant and antibiotic-susceptible strains ofH pylori In thepresent study we prepared highly stable Ag-NPs via a cost-effective green chemistry route using P harmala seeds extractas a reducing and capping agent The main objective of thisstudy was to investigate the in vitro and in vivo anti-H pyloriactivity of the synthesized Ag-NPs in a systemic infectionmodel to fill the gap between in vitro characterization andclinical trials This is perhaps the first time that we haveevaluated the in vivo efficacy of Ag-NPs against H pyloriinduced ulcer using an animal model

2 Materials and Methods

21 Preparation of Green Reducing Agent P harmala seedswere collected from Kanhati Garden Soon Valley PunjabPakistan during the months of September-October 2010

Table 1 Inputs of the reagents for the synthesis of silver nanoparti-cles

Sample code PH-sus () AgNO3 (10mM mL) Water ()S1 20 900 80S2 40 900 60S3 60 900 40S4 80 900 20S5 10 900 90S6 10 90lowast 90S7 10 80lowast 90S8 10 70lowast 90S9 10 90dagger 90S10 10 80dagger 90S11 10 70dagger 90lowastrepresents 20mM AgNO3 solution

daggerrepresents 30mM AgNO3 solution

and were identified and authenticated by Dr Amin ShahDepartment of Biological Sciences University of SargodhaSargodha Pakistan A specimen of the seeds was also keptat the Herbarium of the University of Sargodha SargodhaPakistan Dried seeds (100 g) were ground by a conventionalcoffee grinder and extracted with methanol for 48 h usinga Soxhlet extractor Afterwards the residue was reextractedwith the same fresh solvent both of the extracts were pooledand concentrated to dryness with a rotary evaporator (45∘C)The crude concentrated P harmala suspension (PH-sus) waspreserved at minus4∘C and used for further experiments

22 Synthesis of Ag-NPs under Optimized Conditions andStructural Feature To an appropriate amount of PH-sus pre-pared above AgNO

3(90mL 10mM) was added dropwise

under vigorous stirring by the use of a magnetic stirrer-cum-hotplate at ambient to 100∘C The inputs of the reagents usedin the synthesis are given in Table 1

The experiment was repeated by using 20 and 30mMAgNO

3separately in order to study the effect of concentration

of the silver salt on Ag-NPs characteristicsThe effect of reac-tion time (10ndash180min) medium-pH (4ndash10) and temperature(ambient to 100∘C) on the synthesis was studied by surfaceplasmon resonance (SPR) spectral measurements The Ag-NPs suspension was washed several times with Nanopurewater in order to free the NPs from any unreacted silver salt

23 Surface Plasmon Resonance (SPR) Spectroscopic Measure-ments Ag-NPs suspension was appropriately diluted withwater in order to obtain the SPR spectrum within 200ndash700 nm range by using Pharma Spec UV-1700 (ShimadzuTokyo Japan) UV-Vis spectrophotometer by taking extractblank as a reference Average size of the NPs was calculatedfrom SPR measurements according to the already reportedmethod [24] using the equation

119889 =1

1198712

sdot 119871119899(120582SPR minus 120582

∘

1198711

) (1)

where 120582∘ = 512 1198711= 653 and 119871

2= 00216






5 amoxi-










5at concen-


10CFUmL)































300 400 500 600 700 800

15

10

05

Abso

rban

ce

20mL40mL60mL

80mL100mL

412nm

419nm

427nm

435nm

440nm

Wavelength (nm)

(a)Pa

rtic

le si

ze

0

10

20

30

40

50

60

70

80

0 5 10 15

PH-sus

(b)

Wav

eleng

th (n

m)

500

400

300

200

100

020 40 60 80 120100

(∘C)

(c)

15

10

05

300 400 500 600 700 800

30min

25min

20min

15min

Abso

rban

ce

Wavelength (nm)

(d)

15

10

05

Abso

rban

ce

300 400 500 600 700 800

Wavelength (nm)


(e)



5 and



50 nm

50 nm

50 nm

50 nm

10

20

30

0 10 20 30 40

Diameter (nm)

10

20

30

0 10 20 30 40

Diameter (nm)50

30 40 50 60 70 80

Rela

tive i

nten

sity

(111

)

(220

)

(222

)

(311

)

2-theta scale

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

Freq

uenc

y (

)Fr

eque

ncy

()

(111)

(220)

(311)

(200)



3 (d) TEM image of



5


5and S











5) are


1ndashS5)








5against



5was





5001500250035004500

Tran

smitt

ance

()

(cmminus1)

PHSE

minus500

Ag-NPs (S5)


5)

12

10

8

6

4

2

0010 210 610 810 1010

Clearedtotal

Log

CFU

gas

tric

wal

l

Detection limit









0 10 20 30

Incubation time (h)

0

2

4

6

8

10

12

14

Viab

le co

unts

(log

CFU

mL)

(a)

0 10 20 30

Incubation time (h)

0

2

4

6

8

10

12

Viab

le co

unts

(log

CFU

mL)

(b)

0

2

4

6

8

10

12

14



0 10 20 30

Incubation time (h)

Viab

le co

unts

(log

CFU

mL)

(c)

0

2

4

6

8

10

12

0 10 20 30

Viab

le co

unts

(log

CFU

mL)

Incubation time (h)



(d)



5generally






5and



minus2

0

2

4

6

8

10

0 5 10 15 20 25

Time (h)

Viab

le co

unts

(log

CFU

mL)

pH 7

pH 6

pH 5

pH 4

pH 3









5)






0

5

10

15

20

25

30

35

40

0 5 10

MIC

s (120583

g mLminus

1)

AMXAg-NPs

TETCLT

Number of transfers















4 Conclusions




Acknowledgments



References















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of






5 amoxi-










5at concen-


10CFUmL)































300 400 500 600 700 800

15

10

05

Abso

rban

ce

20mL40mL60mL

80mL100mL

412nm

419nm

427nm

435nm

440nm

Wavelength (nm)

(a)Pa

rtic

le si

ze

0

10

20

30

40

50

60

70

80

0 5 10 15

PH-sus

(b)

Wav

eleng

th (n

m)

500

400

300

200

100

020 40 60 80 120100

(∘C)

(c)

15

10

05

300 400 500 600 700 800

30min

25min

20min

15min

Abso

rban

ce

Wavelength (nm)

(d)

15

10

05

Abso

rban

ce

300 400 500 600 700 800

Wavelength (nm)


(e)



5 and



50 nm

50 nm

50 nm

50 nm

10

20

30

0 10 20 30 40

Diameter (nm)

10

20

30

0 10 20 30 40

Diameter (nm)50

30 40 50 60 70 80

Rela

tive i

nten

sity

(111

)

(220

)

(222

)

(311

)

2-theta scale

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

Freq

uenc

y (

)Fr

eque

ncy

()

(111)

(220)

(311)

(200)



3 (d) TEM image of



5


5and S











5) are


1ndashS5)








5against



5was





5001500250035004500

Tran

smitt

ance

()

(cmminus1)

PHSE

minus500

Ag-NPs (S5)


5)

12

10

8

6

4

2

0010 210 610 810 1010

Clearedtotal

Log

CFU

gas

tric

wal

l

Detection limit









0 10 20 30

Incubation time (h)

0

2

4

6

8

10

12

14

Viab

le co

unts

(log

CFU

mL)

(a)

0 10 20 30

Incubation time (h)

0

2

4

6

8

10

12

Viab

le co

unts

(log

CFU

mL)

(b)

0

2

4

6

8

10

12

14



0 10 20 30

Incubation time (h)

Viab

le co

unts

(log

CFU

mL)

(c)

0

2

4

6

8

10

12

0 10 20 30

Viab

le co

unts

(log

CFU

mL)

Incubation time (h)



(d)



5generally






5and



minus2

0

2

4

6

8

10

0 5 10 15 20 25

Time (h)

Viab

le co

unts

(log

CFU

mL)

pH 7

pH 6

pH 5

pH 4

pH 3









5)






0

5

10

15

20

25

30

35

40

0 5 10

MIC

s (120583

g mLminus

1)

AMXAg-NPs

TETCLT

Number of transfers















4 Conclusions




Acknowledgments



References















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

























300 400 500 600 700 800

15

10

05

Abso

rban

ce

20mL40mL60mL

80mL100mL

412nm

419nm

427nm

435nm

440nm

Wavelength (nm)

(a)Pa

rtic

le si

ze

0

10

20

30

40

50

60

70

80

0 5 10 15

PH-sus

(b)

Wav

eleng

th (n

m)

500

400

300

200

100

020 40 60 80 120100

(∘C)

(c)

15

10

05

300 400 500 600 700 800

30min

25min

20min

15min

Abso

rban

ce

Wavelength (nm)

(d)

15

10

05

Abso

rban

ce

300 400 500 600 700 800

Wavelength (nm)


(e)



5 and



50 nm

50 nm

50 nm

50 nm

10

20

30

0 10 20 30 40

Diameter (nm)

10

20

30

0 10 20 30 40

Diameter (nm)50

30 40 50 60 70 80

Rela

tive i

nten

sity

(111

)

(220

)

(222

)

(311

)

2-theta scale

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

Freq

uenc

y (

)Fr

eque

ncy

()

(111)

(220)

(311)

(200)



3 (d) TEM image of



5


5and S











5) are


1ndashS5)








5against



5was





5001500250035004500

Tran

smitt

ance

()

(cmminus1)

PHSE

minus500

Ag-NPs (S5)


5)

12

10

8

6

4

2

0010 210 610 810 1010

Clearedtotal

Log

CFU

gas

tric

wal

l

Detection limit









0 10 20 30

Incubation time (h)

0

2

4

6

8

10

12

14

Viab

le co

unts

(log

CFU

mL)

(a)

0 10 20 30

Incubation time (h)

0

2

4

6

8

10

12

Viab

le co

unts

(log

CFU

mL)

(b)

0

2

4

6

8

10

12

14



0 10 20 30

Incubation time (h)

Viab

le co

unts

(log

CFU

mL)

(c)

0

2

4

6

8

10

12

0 10 20 30

Viab

le co

unts

(log

CFU

mL)

Incubation time (h)



(d)



5generally






5and



minus2

0

2

4

6

8

10

0 5 10 15 20 25

Time (h)

Viab

le co

unts

(log

CFU

mL)

pH 7

pH 6

pH 5

pH 4

pH 3









5)






0

5

10

15

20

25

30

35

40

0 5 10

MIC

s (120583

g mLminus

1)

AMXAg-NPs

TETCLT

Number of transfers















4 Conclusions




Acknowledgments



References















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


300 400 500 600 700 800

15

10

05

Abso

rban

ce

20mL40mL60mL

80mL100mL

412nm

419nm

427nm

435nm

440nm

Wavelength (nm)

(a)Pa

rtic

le si

ze

0

10

20

30

40

50

60

70

80

0 5 10 15

PH-sus

(b)

Wav

eleng

th (n

m)

500

400

300

200

100

020 40 60 80 120100

(∘C)

(c)

15

10

05

300 400 500 600 700 800

30min

25min

20min

15min

Abso

rban

ce

Wavelength (nm)

(d)

15

10

05

Abso

rban

ce

300 400 500 600 700 800

Wavelength (nm)


(e)



5 and



50 nm

50 nm

50 nm

50 nm

10

20

30

0 10 20 30 40

Diameter (nm)

10

20

30

0 10 20 30 40

Diameter (nm)50

30 40 50 60 70 80

Rela

tive i

nten

sity

(111

)

(220

)

(222

)

(311

)

2-theta scale

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

Freq

uenc

y (

)Fr

eque

ncy

()

(111)

(220)

(311)

(200)



3 (d) TEM image of



5


5and S











5) are


1ndashS5)








5against



5was





5001500250035004500

Tran

smitt

ance

()

(cmminus1)

PHSE

minus500

Ag-NPs (S5)


5)

12

10

8

6

4

2

0010 210 610 810 1010

Clearedtotal

Log

CFU

gas

tric

wal

l

Detection limit









0 10 20 30

Incubation time (h)

0

2

4

6

8

10

12

14

Viab

le co

unts

(log

CFU

mL)

(a)

0 10 20 30

Incubation time (h)

0

2

4

6

8

10

12

Viab

le co

unts

(log

CFU

mL)

(b)

0

2

4

6

8

10

12

14



0 10 20 30

Incubation time (h)

Viab

le co

unts

(log

CFU

mL)

(c)

0

2

4

6

8

10

12

0 10 20 30

Viab

le co

unts

(log

CFU

mL)

Incubation time (h)



(d)



5generally






5and



minus2

0

2

4

6

8

10

0 5 10 15 20 25

Time (h)

Viab

le co

unts

(log

CFU

mL)

pH 7

pH 6

pH 5

pH 4

pH 3









5)






0

5

10

15

20

25

30

35

40

0 5 10

MIC

s (120583

g mLminus

1)

AMXAg-NPs

TETCLT

Number of transfers















4 Conclusions




Acknowledgments



References















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


50 nm

50 nm

50 nm

50 nm

10

20

30

0 10 20 30 40

Diameter (nm)

10

20

30

0 10 20 30 40

Diameter (nm)50

30 40 50 60 70 80

Rela

tive i

nten

sity

(111

)

(220

)

(222

)

(311

)

2-theta scale

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

Freq

uenc

y (

)Fr

eque

ncy

()

(111)

(220)

(311)

(200)



3 (d) TEM image of



5


5and S











5) are


1ndashS5)








5against



5was





5001500250035004500

Tran

smitt

ance

()

(cmminus1)

PHSE

minus500

Ag-NPs (S5)


5)

12

10

8

6

4

2

0010 210 610 810 1010

Clearedtotal

Log

CFU

gas

tric

wal

l

Detection limit









0 10 20 30

Incubation time (h)

0

2

4

6

8

10

12

14

Viab

le co

unts

(log

CFU

mL)

(a)

0 10 20 30

Incubation time (h)

0

2

4

6

8

10

12

Viab

le co

unts

(log

CFU

mL)

(b)

0

2

4

6

8

10

12

14



0 10 20 30

Incubation time (h)

Viab

le co

unts

(log

CFU

mL)

(c)

0

2

4

6

8

10

12

0 10 20 30

Viab

le co

unts

(log

CFU

mL)

Incubation time (h)



(d)



5generally






5and



minus2

0

2

4

6

8

10

0 5 10 15 20 25

Time (h)

Viab

le co

unts

(log

CFU

mL)

pH 7

pH 6

pH 5

pH 4

pH 3









5)






0

5

10

15

20

25

30

35

40

0 5 10

MIC

s (120583

g mLminus

1)

AMXAg-NPs

TETCLT

Number of transfers















4 Conclusions




Acknowledgments



References















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of






5) are


1ndashS5)








5against



5was





5001500250035004500

Tran

smitt

ance

()

(cmminus1)

PHSE

minus500

Ag-NPs (S5)


5)

12

10

8

6

4

2

0010 210 610 810 1010

Clearedtotal

Log

CFU

gas

tric

wal

l

Detection limit









0 10 20 30

Incubation time (h)

0

2

4

6

8

10

12

14

Viab

le co

unts

(log

CFU

mL)

(a)

0 10 20 30

Incubation time (h)

0

2

4

6

8

10

12

Viab

le co

unts

(log

CFU

mL)

(b)

0

2

4

6

8

10

12

14



0 10 20 30

Incubation time (h)

Viab

le co

unts

(log

CFU

mL)

(c)

0

2

4

6

8

10

12

0 10 20 30

Viab

le co

unts

(log

CFU

mL)

Incubation time (h)



(d)



5generally






5and



minus2

0

2

4

6

8

10

0 5 10 15 20 25

Time (h)

Viab

le co

unts

(log

CFU

mL)

pH 7

pH 6

pH 5

pH 4

pH 3









5)






0

5

10

15

20

25

30

35

40

0 5 10

MIC

s (120583

g mLminus

1)

AMXAg-NPs

TETCLT

Number of transfers















4 Conclusions




Acknowledgments



References















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


0 10 20 30

Incubation time (h)

0

2

4

6

8

10

12

14

Viab

le co

unts

(log

CFU

mL)

(a)

0 10 20 30

Incubation time (h)

0

2

4

6

8

10

12

Viab

le co

unts

(log

CFU

mL)

(b)

0

2

4

6

8

10

12

14



0 10 20 30

Incubation time (h)

Viab

le co

unts

(log

CFU

mL)

(c)

0

2

4

6

8

10

12

0 10 20 30

Viab

le co

unts

(log

CFU

mL)

Incubation time (h)



(d)



5generally






5and



minus2

0

2

4

6

8

10

0 5 10 15 20 25

Time (h)

Viab

le co

unts

(log

CFU

mL)

pH 7

pH 6

pH 5

pH 4

pH 3









5)






0

5

10

15

20

25

30

35

40

0 5 10

MIC

s (120583

g mLminus

1)

AMXAg-NPs

TETCLT

Number of transfers















4 Conclusions




Acknowledgments



References















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


minus2

0

2

4

6

8

10

0 5 10 15 20 25

Time (h)

Viab

le co

unts

(log

CFU

mL)

pH 7

pH 6

pH 5

pH 4

pH 3









5)






0

5

10

15

20

25

30

35

40

0 5 10

MIC

s (120583

g mLminus

1)

AMXAg-NPs

TETCLT

Number of transfers















4 Conclusions




Acknowledgments



References















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of






4 Conclusions




Acknowledgments



References















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of
































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of










Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

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Therapeutic - Hindawi Publishing Corporationdownloads.hindawi.com/journals/bca/2014/135824.pdf · Structural Features and In Vivo and In Vitro Therapeutic Effects against Helicobacter