# Green Synthesis of Silver Nanoparticles: Structural Features andIn Vivo and In Vitro Therapeutic Effects against Helicobacter pylori Induced Gastritis

Authors: Muhammad Amin; Sadaf Hameed; Asghar Ali; Farooq Anwar; Shaukat Ali Shahid; Imran Shakir; Aqdas Yaqoob; Sara Hasan; Safyan Akram Khan; Sajjad-ur-Rahman
Journal: Bioinorganic Chemistry and Applications
 (2014)

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## Abstract

This study evaluatesin vivo and in vitro anti-Helicobacter pylori (H. pylori) efficacy of silver nanoparticles (Ag-NPs) prepared via a cost-effective green chemistry route wherein Peganum harmala L. seeds extract was used as a reducing and capping agent. The structural features, as elucidated by surface plasmon resonance spectrophotometry, transmission electron microscopy, and powder X-ray diffraction spectroscopy, revealed the Ag-NPs synthesized to be polydispersed in nature and spherical in shape with 5–40 nm size. A typical Ag-NPs suspension (S5), 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 oral administration of 16 mg/kg body weight of S5 for seven days, a complete clearance was recorded in male albino rates. In comparative time-killing kinetics, S5 exhibited dose- and time-dependent anti-H. pylori activity that was almost similar to tetracycline and clarithromycin, less than amoxicillin, but higher than metronidazole. Furthermore, S5 was found to be an equally effective anti-H. pylori agent at low (≤4) and high pH with no drug resistance observed even up to 10 repeated exposures while a significant drug resistance was recorded for most of the standard drugs employed. The present results revealed the potential of the synthesized Ag-NPs as safer bactericidal agents for the treatment of H. pylori induced gastritis.


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## Body

## 1. Introduction

Nanotechnology has become a rapidly growing field of research because of the unique properties and broad range of applications of nanobioactive materials in different areas of industrial and biochemical sciences [1, 2]. Of the nanomaterials, the silver nanoparticles (Ag-NPs) have attracted a great deal of scientific interest due to their vast applications in the field of medicine [3], catalysis [4], and electronics [5–7]. So far, many methods including physical, chemical, and biochemical 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 and solvents [9]. With the growing awareness about the safe and clean environment the use of green approaches in synthetic processes is gaining much importance [10]. Therefore, several methods have been developed to avoid the use of synthetic/chemical reducing and stabilizing agents [2]. To facilitate the medicinal applications of Ag-NPs, it is fascinating that the materials used as aids in synthesis should be biocompatible [11]. Therefore, plant based reducing and stabilizing agents are being preferred over the chemical methods [12].It has been established thatH. pylori is the major cause of functional dyspepsia, chronic mucosal inflammation in the stomach and duodenum, peptic ulcer, and many other gastroduodenal abnormalities [13]. It is a small curved Gram-positive and microaerophilic bacterium which is fastidious to grow under ordinary growth medium [13]. Triple therapy has been recommended for the complete eradication ofH. pylori [14]. Presently, the major concern around the world in medical science is thatH. pylorihas developed resistance against the standard antibiotics being used in the clinical practices [15]. Therefore, there is a need for the development of novel antimicrobial agents possessing superior effectiveness againstH. pyloriwith reduced toxicity for human cells. So far, metals including zinc [16], bismuth [17], and silver-NPs [11] have been searched for theirin vitro antibacterial activity againstH. pylori. A zinc-based mucosal protective agent, polaprezinc [18], and various salts of bismuth have been recognized as effective antiulcer and anti-H. pylori drugs and indexed in various pharmacopeias [19]. Because of well-defined antimicrobial activities and potential role in wound healing [20] Ag-NPs can be explored as a possible treatment for gastrointestinal relatedH. pylori infections.Peganum harmala L., commonly known as wild rue, is a medicinally important plant from the family Nitrariaceae. In the Soon Valley, Punjab, Pakistan, it is locally known as “harmala.” The seeds ofPeganum harmala(P. harmala) contain several alkaloids [21] and other phytochemicals that attribute various medicinal properties to this multipurpose plant [21]. The seeds of this plant possess antibacterial activity against drug-resistant bacteria [22], smoke from the seeds kills algae, bacteria, intestinal parasites, and molds, and the roots are used to kill lice and insects [21]. Cytotoxic activity against the tissues of liver and kidney at very high dose of 150 g/kg of body weight in rats has been reported [22]. However, at a dose range of 75–100 g/kg body weight in rats, moderate liver and kidney toxicity was observed [22].P. harmala extract and powdered seeds have been used in folk medicine of different parts of the world to treat colic in man and animals due to their antispasmodic effect by blocking different types of intestinal calcium channels [23].Our earlier study showed [11] that Ag-NPs had broad and selectivein vitro antimicrobial activity against the antibiotic-resistant and antibiotic-susceptible strains ofH. pylori. In the present study, we prepared highly stable Ag-NPs via a cost-effective green chemistry route usingP. harmalaseeds extract as a reducing and capping agent. The main objective of this study was to investigate thein vitro andin vivo anti-H. pylori activity of the synthesized Ag-NPs in a systemic infection model to fill the gap betweenin vitro characterization and clinical trials. This is perhaps the first time that we have evaluated thein vivo efficacy of Ag-NPs againstH. pylori induced ulcer using an animal model.


## 2. Materials and Methods

### 2.1. Preparation of Green Reducing Agent

P. harmala seeds were collected from Kanhati Garden, Soon Valley, Punjab, Pakistan, during the months of September-October 2010 and were identified and authenticated by Dr. Amin Shah, Department of Biological Sciences, University of Sargodha, Sargodha, Pakistan. A specimen of the seeds was also kept at the Herbarium of the University of Sargodha, Sargodha, Pakistan. Dried seeds (100 g) were ground by a conventional coffee grinder and extracted with methanol for 48 h using a Soxhlet extractor. Afterwards, the residue was reextracted with the same fresh solvent; both of the extracts were pooled and concentrated to dryness with a rotary evaporator (45°C). The crude concentratedP. harmala suspension (PH-sus) was preserved at −4°C and used for further experiments.


### 2.2. Synthesis of Ag-NPs under Optimized Conditions and Structural Feature

To an appropriate amount of PH-sus, prepared above, AgNO3 (90 mL, 1.0 mM) 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 used in the synthesis are given in Table 1.Table 1
Inputs of the reagents for the synthesis of silver nanoparticles.
Sample code
PH-sus (%)
AgNO3 (1.0 mM; mL)
Water (%)
S1
20
90.0
80
S2
40
90.0
60
S3
60
90.0
40
S4
80
90.0
20
S5
10
90.0
90
S6
10
90*
90
S7
10
80*
90
S8
10
70*
90
S9
10
90†
90
S10
10
80†
90
S11
10
70†
90
*represents 2.0 mM AgNO3 solution; †represents 3.0 mM AgNO3 solution.The experiment was repeated by using 2.0 and 3.0 mM AgNO3 separately in order to study the effect of concentration of the silver salt on Ag-NPs characteristics. The effect of reaction time (10–180 min), medium-pH (4–10), and temperature (ambient to 100°C) on the synthesis was studied by surface plasmon resonance (SPR) spectral measurements. The Ag-NPs suspension was washed several times with Nanopure water in order to free the NPs from any unreacted silver salt.


### 2.3. Surface Plasmon Resonance (SPR) Spectroscopic Measurements

Ag-NPs suspension was appropriately diluted with water in order to obtain the SPR spectrum within 200–700 nm range by using Pharma Spec UV-1700 (Shimadzu, Tokyo, Japan) UV-Vis spectrophotometer by taking extract blank as a reference. Average size of the NPs was calculated from SPR measurements according to the already reported method [24] using the equation
(1)d=1L2·Ln(λSPR-λ°L1),
where λ°=512, L1 = 6.53, and L2 = 0.0216.


### 2.4. X-Ray Diffraction Analysis

Powder X-ray diffraction (P-XRD) spectra of silver-NPs were recorded on Bruker D8 Discover (Germany) diffractometer using monochromatic Cu Kα radiation (λ = 1.5406 Å) operating at 40 kV and 30 mA. The data for the lyophilized NPs were collected over a 10–80° 2θ range. The size of nano-crystallite was calculated from the full width at half maxim (FWHM) of the most intense peak of the spectrum by the use of Debye-Scherrer equation (D=0.9λ/βcos⁡θ).


### 2.5. Transmission Electron Microscopy (TEM)

An ultrasonically dispersed sample of the solution of NPs (one drop) was placed on a carbon grid, dried at room temperature in clean environment, and TEM images were obtained by using JEM-1200EX (JEOL, Japan) microscope at an accelerating voltage of 120 kV. The average size of NPs was calculated measuring the diameter of about 140 particles by using Origin 7.5 software.


### 2.6. FT-IR Spectroscopy

Infrared spectra of the Ag-NPs were recorded by reflectance method on Perkin-Elmer Spectrum 100 FT-IR.


### 2.7.In Vitro Anti-Helicobacter pylori Activity

Forty-two local isolates [11, 16] and two standard strains ofH. pylori,NCTC 11637 and NCTC 11638, obtained from the National Health Protection Agency, London, were used in this study.In vitroanti-H. pylori activities of the NPs sample S5, amoxicillin (AMX), tetracycline (TET), clarithromycin (CLT), and metronidazole (MNZ) were determined by agar dilution method according to already reported method [11, 16]. All the isolates were transferred and inoculation procedures were conducted under the BSL-III safety cabinet throughout.


### 2.8.In Vivo Anti-Helicobacter pylori Activity

In vivo anti-H. pylori activity of the S5 was studied in male albino Wistar rats of 72–112 days having average weight of 295±4.1 g. For this purpose, the rats were infected by oral administration ofH. pylori reference strain and then treated with different doses of silver nanoparticles. An already reported method with slight modification was adapted in this study [11].The rats were maintained in the separate animal house facilities and acclimatized for one week prior to the experiment. All the experiments were conducted in accordance with the ethical protocols and guidelines provided by the Ethical Committee for Experimental Animals, University of Sargodha, Pakistan. The rats were kept on fasting for 18 h and offered oral dose with 0.5 mL of brain heart infusion broth (BHIB) containing a fresh diluted culture ofH. pylori strain (3.6×108 CFU/animal; NCTC 11637). The control rats were inoculated with blank medium. Inoculation was repeated after three days and the H. pylori was monitored in the gastric lavage through microscopic examination after one week postinoculation. After confirmation the rats were segregated into various groups. Different concentrations of the S5 were prepared as per dosage scheme of the already reported method [11] and administered orally as suspension in edible oil. The dosage scheme was selected by keeping in view thein vitroMICs of S5 experiment. The dosing was started 7 weeks afterH. pyloriinoculation and continued twice a day for 7 days. On the 3rd day after final dose, the rats were killed and counted for viableH. pylori counts. This was done by grinding the tissue of the removed stomach between the frosted ends of the glass slides and inoculating on the Columbia agar plates under microaerophilic conditions. The colonies ofH. pyloriwere counted and the clearance rate was determined by Dunnett’s method and Fisher’s exact test.P values below 0.05 were considered statistically significant.


### 2.9. Time-Killing Kinetics of Silver Nanoparticles

This study was done according to the standard guide for assessment of antimicrobial activity using time-killing kinetics procedure [25]. The guidelines provided by CLSI for the determination of time-killing kinetics were adapted throughout this investigation. Cultures ofH. pylori (NCTC 11637) freshly grown over Colombia agar plates were serially adjusted to initial cell concentration of 1×106 CFU/mL. BHIB broths with 2.5% foetal bovine serum (10 mL) containing S5 at concentrations of 1.0 μgmL−1, 2.0 μgmL−1, 4.0 μgmL−1, 8.0 μgmL−1, and 16 μgmL−1 were inoculated with 10 μL of the fresh bacterial culture and shaken at 37°C in a microaerophilic atmosphere. Aliquots (100 μL) were removed during shaking at various time points (0, 1, 2, 3, 6, 9, and 24 h). The aliquots were 10-fold serially diluted in Brucella broth and a 50 μL portion of each of them was seeded over the plates of BHIA supplemented with 7% defibrinated horse blood. The plates were kept under microaerophilic atmosphere at 37°C for 72 h. Colonies were counted after 72 hours of incubation in a microaerophilic atmosphere and rates of killing were determined in duplicate by measuring the reduction in viable bacteria (log⁡10⁡ CFU/mL).


### 2.10. Effects of pH on Bactericidal Activity of Silver Nanoparticles

Silver-NPs (S5) in the concentration range of 2.0–16 μg mL−1 were added to BHIB with 7% defibrinated horse blood. The pH was adjusted to 7, 6, 5, and 3 by using 1N urea solution as appropriate and the bacterial suspension (NCTC 11637; 10 μL) was seeded into it. Urea was added to control the lethality of acidic pH toH. pylori. Cultures were incubated and aliquots were collected at various time points (0, 2, 3, 4, 6, 7, 9, and 24 h). The aliquots were serially diluted to 10-fold with saline and 10 μL of the diluted sample was placed on Colombia agar plate with 7% defibrinated horse blood. The plate was incubated at 37°C under microaerophilic conditions for 72 h and colonies ofH. pyloriwere counted.


### 2.11. Determination of Resistance Development inH. pylori Exposed to Silver Nanoparticles

H. pylori strain NCTC 11637, adjusted to a culture density of approximately 106 CFU/mL in Brucella broth supplemented with 2.5% FBS, was exposed to serial twofold dilutions of S5 (2.0–16 μg/mL), AMX (0.0125–1.0 μg/mL), and TET (8.0–64 μg/mL). After the incubation of the bacteria at 37°C under microaerophilic conditions for 24 h, the culture was examined for any visible growth of bacteria. The culture that attained turbidity comparable to that of the untreated culture in the presence of the highest level of the test agent was further exposed to increasing concentrations of the test agent. These procedures were repeated for three times. The fluctuations of the MIC in the course of the repeated exposure of the bacteria to the test agent were determined.


## 2.1. Preparation of Green Reducing Agent

P. harmala seeds were collected from Kanhati Garden, Soon Valley, Punjab, Pakistan, during the months of September-October 2010 and were identified and authenticated by Dr. Amin Shah, Department of Biological Sciences, University of Sargodha, Sargodha, Pakistan. A specimen of the seeds was also kept at the Herbarium of the University of Sargodha, Sargodha, Pakistan. Dried seeds (100 g) were ground by a conventional coffee grinder and extracted with methanol for 48 h using a Soxhlet extractor. Afterwards, the residue was reextracted with the same fresh solvent; both of the extracts were pooled and concentrated to dryness with a rotary evaporator (45°C). The crude concentratedP. harmala suspension (PH-sus) was preserved at −4°C and used for further experiments.


## 2.2. Synthesis of Ag-NPs under Optimized Conditions and Structural Feature

To an appropriate amount of PH-sus, prepared above, AgNO3 (90 mL, 1.0 mM) 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 used in the synthesis are given in Table 1.Table 1
Inputs of the reagents for the synthesis of silver nanoparticles.
Sample code
PH-sus (%)
AgNO3 (1.0 mM; mL)
Water (%)
S1
20
90.0
80
S2
40
90.0
60
S3
60
90.0
40
S4
80
90.0
20
S5
10
90.0
90
S6
10
90*
90
S7
10
80*
90
S8
10
70*
90
S9
10
90†
90
S10
10
80†
90
S11
10
70†
90
*represents 2.0 mM AgNO3 solution; †represents 3.0 mM AgNO3 solution.The experiment was repeated by using 2.0 and 3.0 mM AgNO3 separately in order to study the effect of concentration of the silver salt on Ag-NPs characteristics. The effect of reaction time (10–180 min), medium-pH (4–10), and temperature (ambient to 100°C) on the synthesis was studied by surface plasmon resonance (SPR) spectral measurements. The Ag-NPs suspension was washed several times with Nanopure water in order to free the NPs from any unreacted silver salt.


## 2.3. Surface Plasmon Resonance (SPR) Spectroscopic Measurements

Ag-NPs suspension was appropriately diluted with water in order to obtain the SPR spectrum within 200–700 nm range by using Pharma Spec UV-1700 (Shimadzu, Tokyo, Japan) UV-Vis spectrophotometer by taking extract blank as a reference. Average size of the NPs was calculated from SPR measurements according to the already reported method [24] using the equation
(1)d=1L2·Ln(λSPR-λ°L1),
where λ°=512, L1 = 6.53, and L2 = 0.0216.


## 2.4. X-Ray Diffraction Analysis

Powder X-ray diffraction (P-XRD) spectra of silver-NPs were recorded on Bruker D8 Discover (Germany) diffractometer using monochromatic Cu Kα radiation (λ = 1.5406 Å) operating at 40 kV and 30 mA. The data for the lyophilized NPs were collected over a 10–80° 2θ range. The size of nano-crystallite was calculated from the full width at half maxim (FWHM) of the most intense peak of the spectrum by the use of Debye-Scherrer equation (D=0.9λ/βcos⁡θ).


## 2.5. Transmission Electron Microscopy (TEM)

An ultrasonically dispersed sample of the solution of NPs (one drop) was placed on a carbon grid, dried at room temperature in clean environment, and TEM images were obtained by using JEM-1200EX (JEOL, Japan) microscope at an accelerating voltage of 120 kV. The average size of NPs was calculated measuring the diameter of about 140 particles by using Origin 7.5 software.


## 2.6. FT-IR Spectroscopy

Infrared spectra of the Ag-NPs were recorded by reflectance method on Perkin-Elmer Spectrum 100 FT-IR.


## 2.7.In Vitro Anti-Helicobacter pylori Activity

Forty-two local isolates [11, 16] and two standard strains ofH. pylori,NCTC 11637 and NCTC 11638, obtained from the National Health Protection Agency, London, were used in this study.In vitroanti-H. pylori activities of the NPs sample S5, amoxicillin (AMX), tetracycline (TET), clarithromycin (CLT), and metronidazole (MNZ) were determined by agar dilution method according to already reported method [11, 16]. All the isolates were transferred and inoculation procedures were conducted under the BSL-III safety cabinet throughout.


## 2.8.In Vivo Anti-Helicobacter pylori Activity

In vivo anti-H. pylori activity of the S5 was studied in male albino Wistar rats of 72–112 days having average weight of 295±4.1 g. For this purpose, the rats were infected by oral administration ofH. pylori reference strain and then treated with different doses of silver nanoparticles. An already reported method with slight modification was adapted in this study [11].The rats were maintained in the separate animal house facilities and acclimatized for one week prior to the experiment. All the experiments were conducted in accordance with the ethical protocols and guidelines provided by the Ethical Committee for Experimental Animals, University of Sargodha, Pakistan. The rats were kept on fasting for 18 h and offered oral dose with 0.5 mL of brain heart infusion broth (BHIB) containing a fresh diluted culture ofH. pylori strain (3.6×108 CFU/animal; NCTC 11637). The control rats were inoculated with blank medium. Inoculation was repeated after three days and the H. pylori was monitored in the gastric lavage through microscopic examination after one week postinoculation. After confirmation the rats were segregated into various groups. Different concentrations of the S5 were prepared as per dosage scheme of the already reported method [11] and administered orally as suspension in edible oil. The dosage scheme was selected by keeping in view thein vitroMICs of S5 experiment. The dosing was started 7 weeks afterH. pyloriinoculation and continued twice a day for 7 days. On the 3rd day after final dose, the rats were killed and counted for viableH. pylori counts. This was done by grinding the tissue of the removed stomach between the frosted ends of the glass slides and inoculating on the Columbia agar plates under microaerophilic conditions. The colonies ofH. pyloriwere counted and the clearance rate was determined by Dunnett’s method and Fisher’s exact test.P values below 0.05 were considered statistically significant.


## 2.9. Time-Killing Kinetics of Silver Nanoparticles

This study was done according to the standard guide for assessment of antimicrobial activity using time-killing kinetics procedure [25]. The guidelines provided by CLSI for the determination of time-killing kinetics were adapted throughout this investigation. Cultures ofH. pylori (NCTC 11637) freshly grown over Colombia agar plates were serially adjusted to initial cell concentration of 1×106 CFU/mL. BHIB broths with 2.5% foetal bovine serum (10 mL) containing S5 at concentrations of 1.0 μgmL−1, 2.0 μgmL−1, 4.0 μgmL−1, 8.0 μgmL−1, and 16 μgmL−1 were inoculated with 10 μL of the fresh bacterial culture and shaken at 37°C in a microaerophilic atmosphere. Aliquots (100 μL) were removed during shaking at various time points (0, 1, 2, 3, 6, 9, and 24 h). The aliquots were 10-fold serially diluted in Brucella broth and a 50 μL portion of each of them was seeded over the plates of BHIA supplemented with 7% defibrinated horse blood. The plates were kept under microaerophilic atmosphere at 37°C for 72 h. Colonies were counted after 72 hours of incubation in a microaerophilic atmosphere and rates of killing were determined in duplicate by measuring the reduction in viable bacteria (log⁡10⁡ CFU/mL).


## 2.10. Effects of pH on Bactericidal Activity of Silver Nanoparticles

Silver-NPs (S5) in the concentration range of 2.0–16 μg mL−1 were added to BHIB with 7% defibrinated horse blood. The pH was adjusted to 7, 6, 5, and 3 by using 1N urea solution as appropriate and the bacterial suspension (NCTC 11637; 10 μL) was seeded into it. Urea was added to control the lethality of acidic pH toH. pylori. Cultures were incubated and aliquots were collected at various time points (0, 2, 3, 4, 6, 7, 9, and 24 h). The aliquots were serially diluted to 10-fold with saline and 10 μL of the diluted sample was placed on Colombia agar plate with 7% defibrinated horse blood. The plate was incubated at 37°C under microaerophilic conditions for 72 h and colonies ofH. pyloriwere counted.


## 2.11. Determination of Resistance Development inH. pylori Exposed to Silver Nanoparticles

H. pylori strain NCTC 11637, adjusted to a culture density of approximately 106 CFU/mL in Brucella broth supplemented with 2.5% FBS, was exposed to serial twofold dilutions of S5 (2.0–16 μg/mL), AMX (0.0125–1.0 μg/mL), and TET (8.0–64 μg/mL). After the incubation of the bacteria at 37°C under microaerophilic conditions for 24 h, the culture was examined for any visible growth of bacteria. The culture that attained turbidity comparable to that of the untreated culture in the presence of the highest level of the test agent was further exposed to increasing concentrations of the test agent. These procedures were repeated for three times. The fluctuations of the MIC in the course of the repeated exposure of the bacteria to the test agent were determined.


## 3. Results and Discussion

### 3.1. SPR Measurements

The SPR absorption was found to be extremely dependent upon the size, shape, interparticle distance, and the surrounding media of the Ag-NPs [26]. Thus, the shape and the size of the NPs can easily be optimized by the use of SPR spectra.For the synthesis of a typical sample S5, PH-sus (10 mL) suspension was added to silver nitrate solution (90 mL; 1 mM aqueous) and heated at 45°C. After 30 min a colour change (yellowish brown) was observed in the reaction mixture which showed the formation of silver-NPs [11, 12]. SPR spectra were found to be highly sensitive to the concentrations of PH-sus (Figure 1(a)). For an illustration, an increasing amount of the PH-sus (2.0–10 mL) caused a significant shift in the UV-Vis region of the spectrum (440–412 nm). This signifies the role of reducing agents (PH-sus) in tailoring the size and shape of NPs as the spectrum can experience a shift dependent upon the particle size, shape, and the surrounding medium [26]. Therefore, it was observed that beyond an optimum concentration of the PH-sus the size of the particles is so increased; that is, it ceased to be in nanolimits [27]. Such observations can be used in calculating the size of the particles from SPR measurements [2]. The size of silver-NPs calculated by various techniques in this study is presented in Figure 1(b).(a) Effect of PH-sus on the SPR of typical sample S5, (b) variation of the size of the silver-NPs with PH-sus using different techniques, SPR (♦), P-XRD (■), and TEM analysis (▲), (c) variation of SPR with temperature (S5), (d) effect of time on the synthesis of sample S5, and (e) stability of Ag-NPs versus time.
(a)
(b)
(c)
(d)
(e)The effect of temperature on the synthesis of typical sample S5 is shown in Figure 1(c). No band appeared in SPR spectrum of the reaction carried out below 25°C, while a broad peak of very low intensity (not shown in Figure 1(c)) was observed at 406 nm for the colloidal suspension obtained after heating at 30°C for 30 min. However, for the yellowish brown colloidal suspension obtained after 30 min of stirring at 45°C, an SPR band at 412 nm suggested the formation of Ag-NPs [26]. Further increase in temperature resulted in broader peaks until the peak intensity became constant beyond 100°C. The optimum temperature for the synthesis of typical sample was found to be 45°C. Beyond this temperature the solution becomes more viscous perhaps due to the aggregation and increased size of the nanoparticles [11].The reaction-stirring-time is an important factor affecting the optimization of SPR peak intensity. A very clear and intense absorption band (Figure1(d)) appearing at 412 nm after 30 min of stirring, for a brown colloidal solution, indicated the presence of spherical NPs [1]. Further stirring up to 180 min, at the same conditions, resulted in a viscous dark brown solution having SPR at 499 nm. It was, therefore, asserted that an optimum time of 30 min was required for the completion of reaction.Another important factor is pH that affects the synthesis,in vivo stability, and anti-H. pyloriactivity of Ag-NPs [28]. Therefore, it is equally important that the silver-NPs must be stable even at low pH (≤4) values as they are desired to be administered in the stomach. In order to obtain SPR at 412 nm the optimum pH was found to be 4. This is in contrast to the previous studies on the synthesis of Ag-NPs [29] whereby it was found that the Ag-NPs were stable mostly at high pH and that at acidic pH clusters were obtained. This is perhaps due mainly to the presence of such phytochemicals in the PH-sus which make it an efficient reducing and capping agent even at low pH values.The stability of the colloidal suspension S5 was determined periodically by taking the SPR spectra. The suspension was found to be stable for a period of more than one year and the peak intensity observed was exactly at 412 nm (Figure 1(e)).In order to investigate the effect of silver nitrate concentration on the synthesis of Ag-NPs, the experiments were also performed by the use of 2.0 mM and 3.0 mM silver nitrate solution while keeping the PH-suspension constant and six samples (S6–S11) were synthesized. The SPR spectra of these colloidal suspensions were observed to be between 470 nm and 495 nm indicating large particle size and aggregates. A typical image of an aggregate (S6) is shown in Figure 2(c).Figure 2
(a, e) TEM image and size distribution of S5, (b, f) TEM image and size distribution of S4, (c) TEM image of S3, (d) TEM image of S2, and (g) P-XRD pattern of S5 and SAED pattern of S5.Under the aforementioned optimized conditions the synthesis reactions were assumed to be complete because no peaks due to the residues of the reducing agents were detected in the UV-Vis spectroscopy. SPR measurements have been proved to be beneficial in determining the size of the NPs [26]. The sharpness and symmetry of SPR peaks are reported to be indicative of the particle size [2]. The dependence of the SPR of NPs on particle size and wavelength was analyzed and the particle size was calculated from SPR measurements and other techniques as listed in Table 2.Table 2
Size of silver nanoparticles calculated by using different techniques.
Sample code
SPR measurements
P-XRD measurements
TEM measurements (nm)
Peak position (nm)
Calculated size (nm)
FWHM offcc (111)
Calculated size (nm)
S1
440
30
30
25
15–40
S2
435
22
25
25
15–30
S3
427
20
25
20
10–25
S4
419
15
22
15
07–14
S5
412
12
20
15
05–10


### 3.2. TEM Analysis

The particle sizes, calculated by TEM measurements, of five samples (S1–S5) of silver-NPs, are presented in Table 1 whereas typical TEM images of the colloidal suspensions, S5 and S4, 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 ratio of the long to the short axes, was found to be between 1.05 and 1.25, thereby suggesting the spherical nature of the particles [30]. Figure 2(a) consists of almost uniformly sized spherical NPs. The sizes of S5 and S4 were found to be 15 nm and 18 nm, respectively. These results are in accordance with the shape of the SPR bands as reported by some earlier researchers [28]. The size ranges of S3 and S2, calculated from TEM measurements, were found to be 10–25 nm and 15–30 nm, respectively (Figures 2(c) and 2(d)).


### 3.3. Powder XRD Analysis

P-XRD patterns and selected area electron diffraction (SAED) of the typical sample (S5) are shown in Figures 2(f) and 2(h). P-XRD consisted of intense peaks appearing at 38.2°, 44.1°, 64.3°, and 78° in the 2θ range of the spectrum. These spectra were indexed as (111), (200), (220), and (311) planes of face centred cubic silver with the help of the data obtained from the database of Joint Committee on Powder Diffraction Standards file number 04-0783. In the spectrum (111) facet reflections were found to be most intense as compared to the rest of the peaks. This feature attributes special bactericidal properties to the silver-NPs [9]. The full width at half-maxim (FWHM) of the (111) face was calculated and the average size of the samples (S1–S5) calculated by the use of Debye-Scherer equation is shown in Table 2. On the basis of the smallest size, spherical shape, and the aforementioned features, the tests of the studies were conducted only for the typical sample S5. The patterns appearing in SAED were characterized as (111), (200), (300), and (200) face-centered cubic (fcc) plane of the crystal structure.


### 3.4. FT-IR Spectroscopy of Silver Nanoparticles

FT-IR measurements were carried out in order to identify the potential of biomolecules in PH-sus responsible for reduction and capping of silver nanoparticles. The characteristic peaks appearing in the spectrum of PH-sus (Figure3(a)) at about 3600, 1763, and 1334 cm−1 are characteristic of ν(OH), the C–O, and C=O stretching modes of the carboxylic acid group. The bands appearing at 1669 and 1535 cm−1 were assigned to amide I and amide II bands, respectively, which may arise due to carboxyl stretch and N–H deformation vibrations in the amide linkages of some proteins present in them [5]. The disappearance of ν(OH) (Figure 3(b)) in the spectra of silver nanoparticles is consistent with the rearrangement and deprotonation of the O–H and some other groups in the PH-sus to be involved in the stabilizing of silver nanoparticles [5, 6].Figure 3
(a) FT-IR spectra of PH-sus and (b) FT-IR spectra of Ag-NPs (S5).


### 3.5.In Vitro Anti-Helicobacter pylori Activity

The growth inhibition activities of S5 againstH. pylori reference strains (NCTC-11637 and NCTC-11638) and antibiotic-resistant and antibiotic-susceptible isolates ofH. pylori are listed in Table 3. It was also found that the anti-H. pyloriactivity of S5 against antibiotic-resistant isolates was nearly comparable to those against antibiotic-susceptible isolates.Table 3
In vitro minimum inhibitory concentration of S5 against reference strains and antibiotic-resistant and antibiotic-susceptible local isolates of H. pylori.
Organism
Strain/isolate
MICs (µgmL−1) of S5
Helicobacter pylori
NCTC 11637
4.0
NCTC 11638
8.0
Clinical isolates [11, 16]
AMX-resistant (n=08)
4.0–8.0
AMX-susceptible (n=32)
2.0–8.0
CLT-resistant (n=05)
1.0–16
CLT-susceptible (n=35)
4.0–16
TET-resistant (n=09)
2.0–8.0
TET-susceptible (n=31)
2.0–16
MNZ-resistant (n=036)
2.0–32
MNZ-susceptible (n=04)
4.0–8.0


### 3.6.In Vivo Anti-H. pylori Activity

All of the vehicle-treated and control rats were maintained with gastricH. pyloriat a level of approximately 1×106 CFU. The application of S5 was found to be efficacious in curing ulcer by inhibitingH. pylori.Complete clearance was obtained at a dose of 16 mg/kg of body weight (Figure 4).Figure 4
In vivo therapeutic efficacy of silver nanoparticles.


### 3.7. Time-Killing Kinetics of Silver Nanoparticles

Time- and dose-killing curves of S5, TET, AMX, and MNZ againstH. pyloristrain NCTC 11637 as function of viable bacterial counts versus incubation time are shown in Figures 5(a)–5(d). The curves for the rest of the strains/isolates are not shown. S5 exhibited (Figure 5(a)) bactericidal effects at concentrations of 4.0 μgmL−1, 8.0 μgmL−1, and 16 μgmL−1. It was found that, at 16 μgmL−1 dose, S5 was found to be effective for eradicatingH. pylori strains within time range of 9 h followed by 8.0 μgmL−1 dose in 12 h time. However, after 24 h contacts time NPs at most of the concentration range (4.0–16 μgmL−1) showed potent bactericidal effect against the testedH. pylori strains. At a lower concentration of 2.0 μgmL−1, no influence or a slight decrease in CFU/mL was noted. S5 generally exhibited rapid killing effect demonstrating concentration- and time-dependent bactericidal activity. At 4.0 μgmL−1 the inhibition occurred at 12 h after the addition ofH. pylori suspension to the concentrations. These results at higher concentrations of Ag-NPs were found to be almost comparable to the clarithromycin-resistant isolates ofH. pylori (data not shown).Time- and dose-dependent killing curves for (a) silver nanoparticles, (b) tetracycline, (c) metronidazole, and (d) amoxicillin, againstH. pylori strain NCTC 11637.
(a)
(b)
(c)
(d)Amongst the standard antibiotics used in this study TET (Figure5(b)) exhibited anti-H. pylori activities almost comparable to those of S5 (effective concentrations 4.0–16 μgmL−1; 9.0–24 h) whereas AMX (Figure 5(c)) showed the best bactericidal activities (effective dose 0.25–4.0 μgmL−1). MNZ (Figure 5(d)) was found to be less potent than S5 and almost noH. pylori strain was found to be susceptible to it. However, at higher concentration (1024 μgmL−1) a slight decrease in CFU/mL was noted.


### 3.8. Effect of pH on the Anti-H. pylori Activity of Silver Nanoparticles

The effect of medium-pH on the anti-H. pylori activities of S5 againstH. pyloristrain NCTC 11637 at MIC 16 μgmL−1 is presented in Figure 6. It was found that the bactericidal activity of the S5 was not affected by the medium-pH (3–7). S5 (concentration 16 μgmL−1) at pH 3 and pH 5 exhibited potent bactericidal effects and the viable bacterial counts reduced rapidly at 7 h after the NPs contact. However, the complete eradication was found to be possible after about 12 h time. The complete eradication (the time at which viable counts become zero) at pH 5 was possible after 24 h.Figure 6
Effect of medium pH on the anti-H. pylori activities of silver nanoparticles against NCTC 11637.


### 3.9. Development of Resistance inH. pylori to the Silver Nanoparticles

The fluctuations of the MICs in the course of the repeated exposure of the bacteria to the silver-NPs (S5) and other test drugs are shown in Figure 7. No significant changes to the MICs of S5 and AMX were found. However, a growing drug resistance was observed in the case of TET and MNZ after the fifth repeated exposure.Figure 7
Resistance developments inH. pylori strain NCTC 11637 after repeated exposure to silver nanoparticles and standard antibiotics.The use of solvents during drug synthesis leads to residual solvents in the final products and causes negative impact on health and the environment [31]. Currently, it is highly recommended that the drug substances should be synthesized by solvent and noxious chemical free methods [2]. The present investigation revealed that theP. harmalaseeds extract not only reduced the silver ions but also efficiently caped the synthesized NPs at least up to more than two years. The role of capping agents in the synthesis of NPs formulations is of immense importance and, recently, in anin vivo study, it was demonstrated that the capped silver-NPs possessed enhanced antimicrobial activities than the uncapped ones [31].Synthesis of Ag-NPs, using green chemistry principles whereby some plant extracts can be used as reducing and capping agents, has received special attention due to maintaining an aseptic environment during the environment-friendly process [12]. Green synthesis of NPs has novelty and innovation with regard to variation in particle size, shape, and synthesis conditions.In our previousin vitro study [11] we have found that all of the tested clinical isolates (H. pylori) were susceptible to silver-NPs synthesized by the use of a green method. The current study was aimed at facilitating thein vivoclinical manifestations of silver-NPs. In the current investigation it was found that an oral administration of 16 mg/kg body weight of S5 resulted in the complete clearance of gastric infection induced by 3.6×108 CFU/animal ofH. pylori inoculums.In vitro time-killing kinetics showed that viable counts were reduced to zero 12 h after bacterial contact with 8.0 μgmL−1 of S5. However, the same bacterial eradication was achieved in 8 h by administering 16 μgmL−1 of S5. This shows thatin vitro susceptibilities are in consistency with thein vivo findings. Some discrepancies have previously been reported between thein vitro antibacterial activities and the clinical efficacies of several antibacterial agents towards the eradication ofH. pylori related infections [32]. Some earlier researchers [33] have reported that an ingestion up to 16 mg of silver is well tolerated in humans. Furthermore, it has low toxicity and minimal side effects when ingested since at most 2–4% is retained in tissues after absorption by the body [34]. Development of antimicrobial agents for the eradication of multidrug-resistant (MDR) microbes is a challenge for the synthetic chemists [11]. It has been reported that the efficacy of the triple therapy regime is decreasing to unacceptable levels (i.e., ≤80%) [11, 12] due to the antibiotic resistance inH. pylori [11–15]. Almost similar resistance patterns were found in the present research in case of TET and CLA. However, no drug resistance was found inH. pylori after prolonged exposure to S5. Metallic silver and its compounds have been used as antimicrobial agents and disinfectants because of their mild toxicity to humans [3]. However, with the development of synthetic antimicrobials for the treatment of infectious diseases, the use of silver in the clinical setting had been restricted solely to the topical use [3]. Due to the emergence of drug-resistant bacteria, there has been a resurgence of the promotion of silver-NPs as alternate antibiotics [8]. Therefore, silver-NPs find extensive applications in the field of medicine as anti-inflammatory agents [35], in wound healing [3, 11], and as antimicrobial agents against various classes of Gram-positive and Gram-negative bacteria [20].In vivoanti-H. pylori activity of silver-NPs may be conferred from the small size, preferential penetrability to the target site, and potent wound healing properties [11]. One possible explanation for the accordance betweenin vitro MICs andin vivo efficacy of S5 may be its stability under acidic conditions. Some metals complexes have been reported for possessing bactericidal activities againstH. pylori[11, 16]. Among these bismuth compounds like bismuth subsalicylate, bismuth subcitrate, and ranitidine bismuth citrate have officially been recommended as a part of triple therapy [36]. Due to toxic effects of bismuth on human cells [17, 35] some other metals and their NPs including Ag-NPs can be explored as a possible treatment for treatment of gastrointestinal andH. pylori related infections.


## 3.1. SPR Measurements

The SPR absorption was found to be extremely dependent upon the size, shape, interparticle distance, and the surrounding media of the Ag-NPs [26]. Thus, the shape and the size of the NPs can easily be optimized by the use of SPR spectra.For the synthesis of a typical sample S5, PH-sus (10 mL) suspension was added to silver nitrate solution (90 mL; 1 mM aqueous) and heated at 45°C. After 30 min a colour change (yellowish brown) was observed in the reaction mixture which showed the formation of silver-NPs [11, 12]. SPR spectra were found to be highly sensitive to the concentrations of PH-sus (Figure 1(a)). For an illustration, an increasing amount of the PH-sus (2.0–10 mL) caused a significant shift in the UV-Vis region of the spectrum (440–412 nm). This signifies the role of reducing agents (PH-sus) in tailoring the size and shape of NPs as the spectrum can experience a shift dependent upon the particle size, shape, and the surrounding medium [26]. Therefore, it was observed that beyond an optimum concentration of the PH-sus the size of the particles is so increased; that is, it ceased to be in nanolimits [27]. Such observations can be used in calculating the size of the particles from SPR measurements [2]. The size of silver-NPs calculated by various techniques in this study is presented in Figure 1(b).(a) Effect of PH-sus on the SPR of typical sample S5, (b) variation of the size of the silver-NPs with PH-sus using different techniques, SPR (♦), P-XRD (■), and TEM analysis (▲), (c) variation of SPR with temperature (S5), (d) effect of time on the synthesis of sample S5, and (e) stability of Ag-NPs versus time.
(a)
(b)
(c)
(d)
(e)The effect of temperature on the synthesis of typical sample S5 is shown in Figure 1(c). No band appeared in SPR spectrum of the reaction carried out below 25°C, while a broad peak of very low intensity (not shown in Figure 1(c)) was observed at 406 nm for the colloidal suspension obtained after heating at 30°C for 30 min. However, for the yellowish brown colloidal suspension obtained after 30 min of stirring at 45°C, an SPR band at 412 nm suggested the formation of Ag-NPs [26]. Further increase in temperature resulted in broader peaks until the peak intensity became constant beyond 100°C. The optimum temperature for the synthesis of typical sample was found to be 45°C. Beyond this temperature the solution becomes more viscous perhaps due to the aggregation and increased size of the nanoparticles [11].The reaction-stirring-time is an important factor affecting the optimization of SPR peak intensity. A very clear and intense absorption band (Figure1(d)) appearing at 412 nm after 30 min of stirring, for a brown colloidal solution, indicated the presence of spherical NPs [1]. Further stirring up to 180 min, at the same conditions, resulted in a viscous dark brown solution having SPR at 499 nm. It was, therefore, asserted that an optimum time of 30 min was required for the completion of reaction.Another important factor is pH that affects the synthesis,in vivo stability, and anti-H. pyloriactivity of Ag-NPs [28]. Therefore, it is equally important that the silver-NPs must be stable even at low pH (≤4) values as they are desired to be administered in the stomach. In order to obtain SPR at 412 nm the optimum pH was found to be 4. This is in contrast to the previous studies on the synthesis of Ag-NPs [29] whereby it was found that the Ag-NPs were stable mostly at high pH and that at acidic pH clusters were obtained. This is perhaps due mainly to the presence of such phytochemicals in the PH-sus which make it an efficient reducing and capping agent even at low pH values.The stability of the colloidal suspension S5 was determined periodically by taking the SPR spectra. The suspension was found to be stable for a period of more than one year and the peak intensity observed was exactly at 412 nm (Figure 1(e)).In order to investigate the effect of silver nitrate concentration on the synthesis of Ag-NPs, the experiments were also performed by the use of 2.0 mM and 3.0 mM silver nitrate solution while keeping the PH-suspension constant and six samples (S6–S11) were synthesized. The SPR spectra of these colloidal suspensions were observed to be between 470 nm and 495 nm indicating large particle size and aggregates. A typical image of an aggregate (S6) is shown in Figure 2(c).Figure 2
(a, e) TEM image and size distribution of S5, (b, f) TEM image and size distribution of S4, (c) TEM image of S3, (d) TEM image of S2, and (g) P-XRD pattern of S5 and SAED pattern of S5.Under the aforementioned optimized conditions the synthesis reactions were assumed to be complete because no peaks due to the residues of the reducing agents were detected in the UV-Vis spectroscopy. SPR measurements have been proved to be beneficial in determining the size of the NPs [26]. The sharpness and symmetry of SPR peaks are reported to be indicative of the particle size [2]. The dependence of the SPR of NPs on particle size and wavelength was analyzed and the particle size was calculated from SPR measurements and other techniques as listed in Table 2.Table 2
Size of silver nanoparticles calculated by using different techniques.
Sample code
SPR measurements
P-XRD measurements
TEM measurements (nm)
Peak position (nm)
Calculated size (nm)
FWHM offcc (111)
Calculated size (nm)
S1
440
30
30
25
15–40
S2
435
22
25
25
15–30
S3
427
20
25
20
10–25
S4
419
15
22
15
07–14
S5
412
12
20
15
05–10


## 3.2. TEM Analysis

The particle sizes, calculated by TEM measurements, of five samples (S1–S5) of silver-NPs, are presented in Table 1 whereas typical TEM images of the colloidal suspensions, S5 and S4, 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 ratio of the long to the short axes, was found to be between 1.05 and 1.25, thereby suggesting the spherical nature of the particles [30]. Figure 2(a) consists of almost uniformly sized spherical NPs. The sizes of S5 and S4 were found to be 15 nm and 18 nm, respectively. These results are in accordance with the shape of the SPR bands as reported by some earlier researchers [28]. The size ranges of S3 and S2, calculated from TEM measurements, were found to be 10–25 nm and 15–30 nm, respectively (Figures 2(c) and 2(d)).


## 3.3. Powder XRD Analysis

P-XRD patterns and selected area electron diffraction (SAED) of the typical sample (S5) are shown in Figures 2(f) and 2(h). P-XRD consisted of intense peaks appearing at 38.2°, 44.1°, 64.3°, and 78° in the 2θ range of the spectrum. These spectra were indexed as (111), (200), (220), and (311) planes of face centred cubic silver with the help of the data obtained from the database of Joint Committee on Powder Diffraction Standards file number 04-0783. In the spectrum (111) facet reflections were found to be most intense as compared to the rest of the peaks. This feature attributes special bactericidal properties to the silver-NPs [9]. The full width at half-maxim (FWHM) of the (111) face was calculated and the average size of the samples (S1–S5) calculated by the use of Debye-Scherer equation is shown in Table 2. On the basis of the smallest size, spherical shape, and the aforementioned features, the tests of the studies were conducted only for the typical sample S5. The patterns appearing in SAED were characterized as (111), (200), (300), and (200) face-centered cubic (fcc) plane of the crystal structure.


## 3.4. FT-IR Spectroscopy of Silver Nanoparticles

FT-IR measurements were carried out in order to identify the potential of biomolecules in PH-sus responsible for reduction and capping of silver nanoparticles. The characteristic peaks appearing in the spectrum of PH-sus (Figure3(a)) at about 3600, 1763, and 1334 cm−1 are characteristic of ν(OH), the C–O, and C=O stretching modes of the carboxylic acid group. The bands appearing at 1669 and 1535 cm−1 were assigned to amide I and amide II bands, respectively, which may arise due to carboxyl stretch and N–H deformation vibrations in the amide linkages of some proteins present in them [5]. The disappearance of ν(OH) (Figure 3(b)) in the spectra of silver nanoparticles is consistent with the rearrangement and deprotonation of the O–H and some other groups in the PH-sus to be involved in the stabilizing of silver nanoparticles [5, 6].Figure 3
(a) FT-IR spectra of PH-sus and (b) FT-IR spectra of Ag-NPs (S5).


## 3.5.In Vitro Anti-Helicobacter pylori Activity

The growth inhibition activities of S5 againstH. pylori reference strains (NCTC-11637 and NCTC-11638) and antibiotic-resistant and antibiotic-susceptible isolates ofH. pylori are listed in Table 3. It was also found that the anti-H. pyloriactivity of S5 against antibiotic-resistant isolates was nearly comparable to those against antibiotic-susceptible isolates.Table 3
In vitro minimum inhibitory concentration of S5 against reference strains and antibiotic-resistant and antibiotic-susceptible local isolates of H. pylori.
Organism
Strain/isolate
MICs (µgmL−1) of S5
Helicobacter pylori
NCTC 11637
4.0
NCTC 11638
8.0
Clinical isolates [11, 16]
AMX-resistant (n=08)
4.0–8.0
AMX-susceptible (n=32)
2.0–8.0
CLT-resistant (n=05)
1.0–16
CLT-susceptible (n=35)
4.0–16
TET-resistant (n=09)
2.0–8.0
TET-susceptible (n=31)
2.0–16
MNZ-resistant (n=036)
2.0–32
MNZ-susceptible (n=04)
4.0–8.0


## 3.6.In Vivo Anti-H. pylori Activity

All of the vehicle-treated and control rats were maintained with gastricH. pyloriat a level of approximately 1×106 CFU. The application of S5 was found to be efficacious in curing ulcer by inhibitingH. pylori.Complete clearance was obtained at a dose of 16 mg/kg of body weight (Figure 4).Figure 4
In vivo therapeutic efficacy of silver nanoparticles.


## 3.7. Time-Killing Kinetics of Silver Nanoparticles

Time- and dose-killing curves of S5, TET, AMX, and MNZ againstH. pyloristrain NCTC 11637 as function of viable bacterial counts versus incubation time are shown in Figures 5(a)–5(d). The curves for the rest of the strains/isolates are not shown. S5 exhibited (Figure 5(a)) bactericidal effects at concentrations of 4.0 μgmL−1, 8.0 μgmL−1, and 16 μgmL−1. It was found that, at 16 μgmL−1 dose, S5 was found to be effective for eradicatingH. pylori strains within time range of 9 h followed by 8.0 μgmL−1 dose in 12 h time. However, after 24 h contacts time NPs at most of the concentration range (4.0–16 μgmL−1) showed potent bactericidal effect against the testedH. pylori strains. At a lower concentration of 2.0 μgmL−1, no influence or a slight decrease in CFU/mL was noted. S5 generally exhibited rapid killing effect demonstrating concentration- and time-dependent bactericidal activity. At 4.0 μgmL−1 the inhibition occurred at 12 h after the addition ofH. pylori suspension to the concentrations. These results at higher concentrations of Ag-NPs were found to be almost comparable to the clarithromycin-resistant isolates ofH. pylori (data not shown).Time- and dose-dependent killing curves for (a) silver nanoparticles, (b) tetracycline, (c) metronidazole, and (d) amoxicillin, againstH. pylori strain NCTC 11637.
(a)
(b)
(c)
(d)Amongst the standard antibiotics used in this study TET (Figure5(b)) exhibited anti-H. pylori activities almost comparable to those of S5 (effective concentrations 4.0–16 μgmL−1; 9.0–24 h) whereas AMX (Figure 5(c)) showed the best bactericidal activities (effective dose 0.25–4.0 μgmL−1). MNZ (Figure 5(d)) was found to be less potent than S5 and almost noH. pylori strain was found to be susceptible to it. However, at higher concentration (1024 μgmL−1) a slight decrease in CFU/mL was noted.


## 3.8. Effect of pH on the Anti-H. pylori Activity of Silver Nanoparticles

The effect of medium-pH on the anti-H. pylori activities of S5 againstH. pyloristrain NCTC 11637 at MIC 16 μgmL−1 is presented in Figure 6. It was found that the bactericidal activity of the S5 was not affected by the medium-pH (3–7). S5 (concentration 16 μgmL−1) at pH 3 and pH 5 exhibited potent bactericidal effects and the viable bacterial counts reduced rapidly at 7 h after the NPs contact. However, the complete eradication was found to be possible after about 12 h time. The complete eradication (the time at which viable counts become zero) at pH 5 was possible after 24 h.Figure 6
Effect of medium pH on the anti-H. pylori activities of silver nanoparticles against NCTC 11637.


## 3.9. Development of Resistance inH. pylori to the Silver Nanoparticles

The fluctuations of the MICs in the course of the repeated exposure of the bacteria to the silver-NPs (S5) and other test drugs are shown in Figure 7. No significant changes to the MICs of S5 and AMX were found. However, a growing drug resistance was observed in the case of TET and MNZ after the fifth repeated exposure.Figure 7
Resistance developments inH. pylori strain NCTC 11637 after repeated exposure to silver nanoparticles and standard antibiotics.The use of solvents during drug synthesis leads to residual solvents in the final products and causes negative impact on health and the environment [31]. Currently, it is highly recommended that the drug substances should be synthesized by solvent and noxious chemical free methods [2]. The present investigation revealed that theP. harmalaseeds extract not only reduced the silver ions but also efficiently caped the synthesized NPs at least up to more than two years. The role of capping agents in the synthesis of NPs formulations is of immense importance and, recently, in anin vivo study, it was demonstrated that the capped silver-NPs possessed enhanced antimicrobial activities than the uncapped ones [31].Synthesis of Ag-NPs, using green chemistry principles whereby some plant extracts can be used as reducing and capping agents, has received special attention due to maintaining an aseptic environment during the environment-friendly process [12]. Green synthesis of NPs has novelty and innovation with regard to variation in particle size, shape, and synthesis conditions.In our previousin vitro study [11] we have found that all of the tested clinical isolates (H. pylori) were susceptible to silver-NPs synthesized by the use of a green method. The current study was aimed at facilitating thein vivoclinical manifestations of silver-NPs. In the current investigation it was found that an oral administration of 16 mg/kg body weight of S5 resulted in the complete clearance of gastric infection induced by 3.6×108 CFU/animal ofH. pylori inoculums.In vitro time-killing kinetics showed that viable counts were reduced to zero 12 h after bacterial contact with 8.0 μgmL−1 of S5. However, the same bacterial eradication was achieved in 8 h by administering 16 μgmL−1 of S5. This shows thatin vitro susceptibilities are in consistency with thein vivo findings. Some discrepancies have previously been reported between thein vitro antibacterial activities and the clinical efficacies of several antibacterial agents towards the eradication ofH. pylori related infections [32]. Some earlier researchers [33] have reported that an ingestion up to 16 mg of silver is well tolerated in humans. Furthermore, it has low toxicity and minimal side effects when ingested since at most 2–4% is retained in tissues after absorption by the body [34]. Development of antimicrobial agents for the eradication of multidrug-resistant (MDR) microbes is a challenge for the synthetic chemists [11]. It has been reported that the efficacy of the triple therapy regime is decreasing to unacceptable levels (i.e., ≤80%) [11, 12] due to the antibiotic resistance inH. pylori [11–15]. Almost similar resistance patterns were found in the present research in case of TET and CLA. However, no drug resistance was found inH. pylori after prolonged exposure to S5. Metallic silver and its compounds have been used as antimicrobial agents and disinfectants because of their mild toxicity to humans [3]. However, with the development of synthetic antimicrobials for the treatment of infectious diseases, the use of silver in the clinical setting had been restricted solely to the topical use [3]. Due to the emergence of drug-resistant bacteria, there has been a resurgence of the promotion of silver-NPs as alternate antibiotics [8]. Therefore, silver-NPs find extensive applications in the field of medicine as anti-inflammatory agents [35], in wound healing [3, 11], and as antimicrobial agents against various classes of Gram-positive and Gram-negative bacteria [20].In vivoanti-H. pylori activity of silver-NPs may be conferred from the small size, preferential penetrability to the target site, and potent wound healing properties [11]. One possible explanation for the accordance betweenin vitro MICs andin vivo efficacy of S5 may be its stability under acidic conditions. Some metals complexes have been reported for possessing bactericidal activities againstH. pylori[11, 16]. Among these bismuth compounds like bismuth subsalicylate, bismuth subcitrate, and ranitidine bismuth citrate have officially been recommended as a part of triple therapy [36]. Due to toxic effects of bismuth on human cells [17, 35] some other metals and their NPs including Ag-NPs can be explored as a possible treatment for treatment of gastrointestinal andH. pylori related infections.


## 4. Conclusions

A green method has been reported for the synthesis of Ag-NPs usingP. harmala L. seeds extract as reducing and capping agent. It was found that the size and the shape of the Ag-NPs could be tailored by optimizing the reaction temperature, time, and pH of the media. The optimized extract pH value, temperature, and molar ratio of the reactants improved the size and the shape of Ag-NPs. The adopted method is compatible with green chemistry approaches as theP. harmala L. seeds extract serves as a matrix for both reduction and stabilization of the synthesized NPs. These NPs due to biocompatibility and bactericidal potency againstH. pylori may be exploited as an anti-H. pylori agent capable of replacing the existing triple and quadruple therapy regimens.



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*Source: 135824-2014-08-06.xml*