The optical properties of AgNps were calculated by UV-Vis absorption spectroscopy, an important and most commonly used technique, to ascertain the formation stability of metal nanoparticles. Due to surface plasmon resonance (SPR), a strong absorption of electromagnetic waves is exhibited by metal nanoparticles in the visible range. The SPR phenomenon arises when nanoparticles are irradiated with visible light, because of the collective oscillations of the conduction electrons. It is well known that AgNps exhibit a yellowish-brown color in aqueous solution due to the excitation in UV-visible spectrum depending upon the particle size [47].

The SPR bands in the UV–Vis spectra of AgNps colloidal suspension S1, S2, S3, S4 and S5 appeared at 406, 412, 428, 425 and 433 nm, respectively, Figure 1(a). For colloid S5 the SPR at 433 nm was broad. By increasing the concentration of SXE, the SPR bands for colloids S4, S3, S2 and S1 became sharper and for the typical sample S1 SPR appeared at 406 nm. The broad SPR at lower quantities of the SXE was due mainly to the formation of large anisotropic particles. This perhaps may occur because by lowering the quantity of SXE, the concentration of groups/molecules such as phenolics, alkaloids and sugars, etc. responsible for capping and stabilizing of nanoparticle reduces. Similar results have already been reported in the case of the stabilization effect of biological extracts on the formation of metal nanoparticles [14–15,22]. The increased bandwidth of colloid S5 is due to the fact that beyond a limit the bio-molecules in an extract cease to act as a capping agent. The fairly sharp and symmetrical SPR band observed for colloid S1 at 406 nm is indicative of small spherical nanoparticles.

Figure 1(b) shows the effect of temperature on the synthesis of typical sample S1. A broad peak of less intensity was observed at 433 nm for the colloidal suspension obtained after heating the reaction mixture at 25 °C for 2–3 h. With increase in temperature from 25 °C to 45 °C, the SPR peaks became sharper and sharper and for the yellowish brown solution, obtained after 30 min of stirring at 45 °C, an absorption band at 406 nm was obtained which suggested the formation of silver nanoparticles. It can be observed that an optimum temperature is required for the completion of reaction due to the instability of formed silver nanoparticles. The optimum temperature required for the completion of reaction was investigated to be 45 °C. Upon a further increase in temperature (up to 50 °C), a red-shift appeared from 406 nm to 450 nm due to increase in AgNps size. Further increase in temperature caused the broadening of the peak revealing the increased size of nanoparticles. This temperature dependent increase in the peak intensity showed the dependence of the silver ion reduction on the reaction temperature. It was observed that reduction rate of silver ions increased by increasing temperature. Similar results were reported by Pastoriza-Santos and Liz-Marzan [9]. This sharpness in absorbance peak depends on the size of the synthesized nanoparticle, as with higher temperature particle size may be smaller, which results in sharpness of the plasmon resonance band of AgNPs [48]. Initially, the size was reduced due to the reduction in aggregation of the growing nanoparticles. Increasing the temperature beyond 45 °C aids the growth of the crystal around the nucleus resulting in a decrease in absorption.

Effect of time on the completion of reaction was studied by UV-Vis spectroscopy for colloidal suspension S1. An absorption band of very low intensity appeared at 406 nm for the colloid after 10 min of stirring, Figure 1(c), which changed into a clearly visible peak after 30 min at the same absorbance, indicating the presence of spherical AgNps. The absorption peak intensity increased rapidly with increase in reaction time from 10 to 30 min due to the continuous formation of AgNps in the reaction system. It was, therefore, observed that an optimum time is required for the completion of reaction due to the instability of formed silver nanoparticles. The optimum time required for the completion of reaction was recorded to be 30 min.

pH is another important factor affecting the reduction of silver ions. The effect of pH on the reduction of silver ions was studied by UV-Vis spectroscopy and is shown in Figure 1(d,e). At pH 4.0, no absorption peak was observed in the range of 400–450 nm for the colloidal suspension of all samples even after 24 h of the reaction. However, an absorption band appeared at about 440 nm when pH increased from 4 to 5 indicating the formation of AgNps, Figure 1(d). It was observed that the absorption peak intensity increased gradually with an increase in pH, suggesting that the reduction rate of silver ions increases with an increase in pH. The formation of AgNps was suppressed by acidic conditions and enhanced by basic conditions. At lower pH (pH 5), larger nanoparticles were formed, whereas, at higher pH (pH 9), smaller and highly dispersed nanoparticles formed, Figure 1(e).

The XRD pattern of dried silver nanoparticles S1 is shown in Figure 2(a). The four diffraction peaks at 38.2°, 44.1°, 64.3° and 78° are indexed as (111), (200), (220) and (311) planes of face centered cubic silver. The data obtained was matched with the database of Joint Committee on Powder Diffraction Standards (JCPDS) file No. 04-0783. The (200), (220) and (311) Bragg reflections are weak and broadened relative to the intense (111) reflection. This feature indicates that the nanocrystals are (111)-oriented [49] as confirmed by TEM measurements. The crystalline nature of S1 was further evidenced by the selected area electron diffraction (SAED) pattern Figure 2(b) with bright circular spots corresponding to (111), (200), (220) and (311) Bragg reflection planes [50]. This indicates that the prepared silver nanoparticles may be enriched in (111) facets and thus the (111) plane seems to be preferentially oriented parallel to the surface of the supporting substrate. The average particle size of AgNps was calculated by the use of full width at half maximum (FWHM) of face-centered cubic (111) using the Debye–Scherrer equation, K λ/βcosθ, where K is the Scherrer constant with value from 0.9 to 1, λ is the wavelength of the X-ray, β is the full width at half maximum and θ is the Bragg angle in radians. From the Scherrer equation the average crystallite size of silver nanoparticles S1 and S2 was found to be about 10 nm and 15 nm, respectively.

Typical TEM images of colloid S1 and S2 with size distribution histograms are shown in Figure 2(c,d), respectively. It is evident from the Figure 2(c) that the morphology of S1 is almost spherical, which is in agreement with the shape of the SPR band in the UV–Vis spectra. The average particle size calculated for S1 was 10 nm with size ranging from 4 to 18 nm. Whereas the average particle size for sample S2 was calculated as 14 nm with an average size ranging from 7 to 18 nm. These results are in accordance with the shape of the SPR band and with the particle size calculated from XRD analysis.

MIC₉₀ values for a typical AgNp sample named as S1, silver nitrate and the standard drugs are listed in Table 1. The sample S1 exhibited stronger anti-H. pylori activity (MICs 2–8 μg mL⁻¹) than silver nitrate (MIC 16–64 μg mL⁻¹), TET (MIC 0.25–64 μg mL⁻¹), and MNZ (2–512 μg mL⁻¹), however it was found to be less potent than AMX (0.125–4 μg mL⁻¹) and CLT (0.125–8 μg mL⁻¹). The antibacterial activity of S1 against AMX-resistant, CLT-resistant, TET-resistant and MNZ-resistant clinical isolates was nearly comparable to those against AMX, CLT, TET, and MNZ susceptible isolates (Table 1). Furthermore, S1 was also found to be equally effective against the strains showing double (SA-1, SA-6, SA-7, SA-21, SA-28, SA-31 and SA-34) and triple (SA-3, SA-5 and SA-17) drug resistance. For some cases (SA-6, SA-31, MICs at 4 μg mL⁻¹) the isolate exhibiting a strong resistance to AMX was found to be susceptible to S1 with MIC at 2 μg mL⁻¹. This trend was frequently observed in the case of other standard drugs.

Urease inhibitory activity of silver nanoparticles S1 has been studied by the phenol red method. The results are shown in Table 2 in the form of percent inhibition. It was found that urease inhibitory activity increased linearly with increased concentration of S1. Under the conditions used in the present investigation, H. pylori urease inhibition by S1 followed Michaelis-Menten kinetics. Kinetic studies of the enzyme in the presence of S1 were presented by Lineweaver-Burk plots in which a reciprocal of enzyme activity was taken on the Y-axis and a reciprocal of substrate concentration on the X-axis. The trend of lines of different concentrations of the inhibitor gave the idea about inhibition.

Line weaver-Burk plots indicated that S1 is a non-competitive type of inhibitor. The plot of 1/V_o vs. 1/[s] (Figure 3) consisted of a family of straight lines that intersected each other on the same point on X-axis depicting the same K_m values with varying values of V_max with the concentration of the inhibitor. Furthermore, it was also clear from the graphs that inhibition increased almost linearly. Therefore, it may be suggested that the mechanism was non-competitive in which the inhibitor and substrate were both attached to the enzyme non-competitively [51].

Metal based nano-preparations have gained importance due to a variety of their applications in various fields [1–10]. The synthesis techniques involve the reduction of the corresponding metallic ion by a reducing agent. The newly synthesized nanoparticles are highly unstable and become aggregated in the absence of a suitable capping agent. Chemical techniques involving the use of chemicals and solvents are described as toxic and carcinogens, and have been suggested to be eliminated by the Conferences on Harmonization [10]. It is, therefore, necessary that the nanoparticles must be synthesized using such techniques which being benign to the environment must be cost-effective. The present investigation explored the use of fruit from an important medicinal plant S. xanthocarpum [31–34] as a template for the synthesis of AgNps by acting both as a reducing and a capping agent. The methanolic extract was found as an efficient capping as well as a stabilizing agent, whereas aqueous and acetone extracts did not exhibit good results (data not shown). The possible reason for this may be the dissolved phyto-chemicals present in the methanolic extract. During synthesis, it was found that an increase in concentration of S. xanthocarpum berry extract affected the morphology of the silver nanoparticles and the conditions were maintained such that finally 10 mL of the extract added to 20 mL of 1 mM silver nitrate solution resulted in a color change to golden brown, which is indicative of the formation of silver nanoparticles [16]. It has been established [52] that the optical absorption spectra of metal nanoparticles are dominated by SPR, which shift to longer wavelengths with increasing particle size. Therefore, a SPR band shift from 406 nm to 433nm (S1–S5) is indicative of the fact that an optimum concentration (10 mL) of extract is required for the synthesis of AgNps. Also, the shape of the SPR band of AgNps is strongly dependent on the particle size [53,54]. It has already been demonstrated [22] that only a single SPR band is expected in the absorption spectra of spherical nanoparticles, whereas anisotropic particles could give rise to two or more SPR bands depending on the shape of the particles. Furthermore, the number of SPR peaks increases as the symmetry of the nanoparticle decreases [54]. Therefore, the single symmetrical SPR band at 406 nm confirms the spherical morphology of the typical sample S1. S. xanthocarpum berries have been reported to contain several steroidal alkaloids like solanacarpine, solanacarpidine, solancarpine, solasonine, solamargine and other constituents like caffeic acid, coumarins like aesculetin and aesculin, steroids carpesterol, diosgenin, campesterol, daucosterol and triterpenes like cycloartanol and cycloartenol [55]. The flavonoids quercitrin and apigenin glycosides are the major chemical constituents present in S. xanthocarpum fruit [30]. Carbohydrates, flavonoids, terpenoids and certain proteins present in SXE may have a reducing and stabilizing effect in the nanoparticle synthesis. The synthesized AgNps were tested for their anti-H. pylori activity by the use of agar dilution method and their MICs were compared with silver nitrate and some of the standard drugs being used in anti-H. pylori therapy. AgNps exhibited more potent bactericidal activity than ionic silver (silver nitrate) and some of the standard drugs. The reason for much lower MICs and stronger antibacterial activities of S₁ than AgNO₃ might be due to smaller size and the presence of (111) lattice plane in the former [56]. Metal atoms in nano size provide a significantly large surface area in contact with the bacterial effluent. Considering a hypothetical case with spherical particles of uniform size, a reduction in the particle size from 10 μm to 10 nm would increase the contact surface area by 10⁹. Such a large contact surface is expected to enhance the extent of bacterial elimination. The size-dependent bactericidal activity of silver nanoparticles against gram-negative bacteria has already been reported [57,58]. Literature reports reveal the bactericidal activity of silver nanoparticles of either a simple or composite nature [59,60]. Multiple investigations have been performed to show the antibacterial activity of metals and metals chelated with some ligands against H. pylori [61].

To the best of our understanding, the present investigation can be considered to be the first of its kind reporting the anti-H. pylori activity of silver nanoparticles and their comparison with silver ions and some standard antibiotics being used in triple and quadruple therapy for the eradication of H. pylori. No standard susceptibility testing methods have yet been recommended for nanoparticles and metallic ions. Although broth dilution test and agar dilution test are both standard in vitro susceptibility tests recommended by the CLSI, due to some advantages, the agar dilution method has been adopted in the present investigation [59]. The exact mechanism of anti-H. pylori activity of silver nanoparticles is not clearly understood. However, there may be two possible explanations for this mechanism. In this direction, one theory speculates that a very small quantity of silver ions may enter into the bacteria to inhibit the microorganism’s respiratory system, electron transport system and enzymes, whilst another theory believes that silver ions may directly interfere with nickel in H. pylori urease, thereby making the enzyme inactive [59,62,63]. Urease, in H. pylori, converts urea into ammonia, which then counters the stomach acid, thereby creating a neutralizing environment for protecting H. pylori from the acid in the stomach. Gastric infection with H. pylori may lead to the onset of various gastric-related diseases. Most patients specifically with duodenal ulcer can be cured by killing H. pylori with antibiotics and proton pump inhibitors [35]. Several triple- or quadruple-antibiotic therapies with proton pump inhibitors have been shown to be effective in the eradication of H. pylori [36], but no single treatment regime is considered the final treatment of choice. Therefore, the specific inhibition of urease activity has been proposed as a possible strategy to inhibit H. pylori. [37,38].

Hence, there is a dire need for the eradication of H. pylori by single-therapy if possible. Bismuth is the only metal being widely used as antibacterial agent against H. pylori as a part of triple therapy. Due to potential toxic effects of bismuth, some other metals [37,38] including silver, having well defined antimicrobial activities, can be explored as possible treatments for gastrointestinal symptoms and infections. Silver compounds have been reported as anti-ulcer agents in animals and man, and their role in wound healing is well established [64]. Since H. pylori are a major peptic-ulcer causing agent, it raised the possibility that a part of the anti-ulcer activity of silver nanoparticles could be due to its effects on the growth of H. pylori.

Fully ripened S. xanthocarpum berries were collected from Soon Valley of the Punjab province of Pakistan in the month of June 2010 and brought to the Department of Biological Sciences, University of Sargodha, Sargodha, Pakistan. The specimens were further identified and authenticated by Amin Ullah Shah (Assistant Professor, Department of Biological Sciences, University of Sargodha (UoS), Sargodha, Pakistan). A specimen of the berries was also submitted to the herbarium of the UoS. The berries were dried under shade for 4–5 weeks.

Silver nitrate (AgNO₃, analytical grade) was purchased from Sigma–Aldrich. A total of 34 clinical isolates of H. pylori have already been isolated and characterized by the same group and published elsewhere [37]. Two reference strains of H. pylori NCTC 11637 and NCTC 11638 were purchased from the Health Protection Agency, London, UK.

Dried berries (50 g) were ground using an ordinary coffee grinder and extracted with methanol for 24 h using a conventional Orbital Shaker. After filtration, the residue was re-extracted with fresh solvent; both the extracts were pooled and then concentrated to dryness with a rotary evaporator (45 °C). The crude concentrated extract was quantitatively diluted with methanol and preserved at −4 °C for further experiments.

Silver nitrate (20 mL; 1 mM) was added dropwise into SXE (10 mL) while stirring and heated (45 °C) in a water bath at pH 9. The resulting solution became yellowish brown after 30 min of heating, indicating the formation of silver nanoparticles [8]. The colloidal suspension thus obtained was centrifuged at 4000 rpm for 30 min and the pellet obtained after discarding the supernatant was re-dispersed in deionized water. The centrifugation process was repeated 2 to 3 times for the removal of any adsorbed substances on the surface of silver nanoparticles (AgNps). The synthesized nanoparticles were lyophilized and recovered in powdered form using a Heto-Holten A/S, DK-3450 freeze dryer (Allerd, Denmark). Different samples of AgNps were synthesized by varying the concentration of SXE and silver nitrate solution as presented in Table 3.

In vitro anti-H. pylori activity of the synthesized AgNps was determined by the agar dilution method in accordance with the guidelines provided by CLSI [65]. The results were recorded as minimum inhibitory concentration (MICs). Different concentrations of the synthesized AgNps were prepared by the sample digestion method. Silver nanoparticles were centrifuged and the pellet thus obtained was washed several times with deionized water in order to remove un-reacted silver ions. The removal of un-reacted silver was further confirmed by treating each filtrate with 1 mM HCl until no silver chloride was precipitated. In order to determine the concentration of AgNps for susceptibility studies, the pellet free from silver ions was divided into two halves. One half was dissolved in aqua-regia and the solution evaporated near to dryness. The process of dilution and dryness was repeated three times. In this way, the AgNps were converted to AgNO₃. The concentration of silver ions was determined by atomic absorption spectrometry. The other half of the nanoparticles, having a concentration almost equal to the first half, was used for anti-H. pylori activities. Fourteen wells on a 96-wells plate were filled with a two-fold serially diluted colloidal suspension of silver nanoparticles in distilled water having final concentrations of 128 to 0.125 μg mL⁻¹. The control was filled with distilled water only. The final concentrations of other test compounds (AMX, TET, CLA) were also adjusted from 128 to 0.125 μg mL⁻¹ by dissolving in DMSO. The control was filled with DMSO only. These dilutions were transferred to molten Muller Hinton infusion agar plates.

The frozen clinical isolates were thawed, diluted using the Muller Hinton infusion broth (MHIB), and adjusted to 10⁷ CFU mL⁻¹. Culture (5 mL) was streaked over the agar plates. The plates were incubated under microaerophillic conditions in sealed bags at 37 °C for 72 or 96 h as appropriate. The MIC₉₀ were determined as per standard procedure. The breakpoints to define a resistant strain according to Megraud et al., [36] were found to be clarithromycin 1 μg mL⁻¹; and according to Wu et al., [66] 0.5 μg mL⁻¹ and 16 μg mL⁻¹ for amoxicillin and tetracycline, respectively. Each experiment was performed as triplicate.