A multi-target therapeutic potential of Prunus domestica gum stabilized nanoparticles exhibited prospective anticancer, antibacterial, urease-inhibition, anti-inflammatory and analgesic properties.

Background Phytotherapeutics exhibit diverse pharmacological effects that are based on the combined action of a mixture of phytoconstituents. In this study, Prunus domestica gum-loaded, stabilized gold and silver nanoparticles (Au/Ag-NPs) were evaluated for their prospective anticancer, antibacterial, urease-inhibition, anti-inflammatory, and analgesic properties. Methods Au/Ag-NPs were biosynthesized and characterized with UV-Vis, FTIR, SEM, EDX, and XRD techniques. The effect of gum and metal ion concentration, reaction temperature, and time on the synthetic stability of nanoparticles was studied along with their post-synthetic stability against varying pH and salt concentrations, long-term storage and extremes of temperature. Nanoparticles were tested for anticancer (HeLa cervical cancer cells), antibacterial (Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa), urease inhibition (jack-bean urease), anti-inflammatory (carrageenan-induced paw edema), and antinociceptive (abdominal constriction response) activities. Results The nanoparticles were mostly spherical with an average particle size between 7 and 30 nm (Au-NPs) and 5–30 nm (Ag-NPs). Au/Ag-NPs maintained their colloidal stability and nanoscale characteristics against variations in physicochemical factors. Au/Ag-NPs have potent anticancer potential (IC50 = 2.14 ± 0.15 μg/mL and 3.45 ± 0.23 μg/mL). Au/Ag-NPs selectively suppressed the growth of S. aureus (10.5 ± 0.6 mm, 19.7 ± 0.4 mm), E. coli (10 ± 0.4 mm, 14.4 ± 0.7 mm), and P. aeruginosa (8.2 ± 0.3 mm, 13.1 ± 0.2 mm), as well as showed preferential inhibition against jack-bean urease (19.2 ± 0.86%, 21.5 ± 1.17%). At doses of 40 and 80 mg/kg, Au-NPs significantly ameliorated the increase in paw edema during the 1st h (P < 0.05, P < 0.01) and 2–5 h (P < 0.001) of carrageenan-induced inflammation compared to the 200 and 400 mg/kg doses of P. domestica gum (P < 0.05, P < 0.001). At similar doses, Au-NPs also significantly abolished (P < 0.01) the tonic visceral, chemically-induced nociception, which was comparable to that of P. domestica gum (200 mg/kg; P < 0.05, 400 mg/kg; P < 0.01).


Conclusions
The Prunus domestica gum-integrated nanoparticles have multi-target therapeutic capabilities and thus possess an advantage in combating multigenic diseases that affect multiple tissues or cell types.

Background
Drugs designed to act against individual molecular targets cannot usually combat multigenic diseases or diseases that affect multiple tissues or cell types. Combination drugs that impact multiple targets simultaneously are better at controlling complex disease systems, are less prone to drug resistance, and are the standard of care in many important therapeutic areas [1]. The multiple target therapeutic approach increasingly is used to treat many types of diseases, including AIDS, atherosclerosis, cancer, and depression [2]. The low affinity of multi-target drugs is more likely to induce synergistic therapeutic effects by the combination of various mechanistic actions. The therapeutic efficacy of phytotherapy is based on the combined action of a mixture of constituents and offers new treatment opportunities [3].
Compared to conventional systems of traditional medicine, the incorporation of the nano-traditional concept has several advantages, including (1) improvement of the biological availability and therefore saves the limited resources of the Materia Medica; (2) strengthening of the target-oriented therapeutic effects; (3) provide pharmaceutical preparation choices; and (4) promote the standardization and internationalization of the drug preparation. This concept has been successfully implemented in the Chinese Materia Medica and has shown many advantages [4]. The combination of nanotechnology with traditional herbal medicine therefore provides a very useful tool in designing future herbal medicine with an improved bioavailability profile and less toxicity. This new approach is increasing the interest of a number of scientists to improve and to accelerate the joint drug discovery and development of novel nano-delivery systems for herbal extracts [5].
Gums are water-soluble polysaccharides (including modified polysaccharides), which produce viscous aqueous systems, generally at low concentrations. The gums are apparently not normal products of plant metabolism, but probably are more or less pathological products formed by plants when injured or diseased or under adverse climatic conditions [17]. Generally, plant gum exudates contain galactose, arabinose, rhamnose, uronic acids, galacturonic acid, protein, Ca and Mg as major structure constituents as well as, glucose, xylose, mannose, protein, and fat as minor constituents [18]. Natural gums along with mucilages constitute a structurally diverse class of biological macromolecules with a broad range of physicochemical properties, which are widely used for various applications in pharmacy and medicine [19]. There is a huge scope of natural gums as a novel natural polymer for the development of different drug delivery systems. In this study we evaluated the P. domestica gum-loaded, stabilized gold and silver nanoparticles for their prospective in vitro anticancer, antibacterial, and urease inhibition activities. Moreover, the P. domestica gum-loaded gold nanoparticles were assessed for in vivo anti-inflammatory and analgesic properties. Gold nanoparticles show several features that make them well suited for biomedical applications including their ease of synthesis, high surface area, stability and low inherent toxicity [20,21], compared to silver nanoparticles, which are toxic to mammalian cells and produce adverse-effects in different organs [22].

Materials
Tetrachloroauric acid trihydrate (HAuCl 4 .3H 2 O, 99.5%) and silver nitrate (AgNO 3 , 99.9%) were purchased from Merck, Germany. Prunus domestica fresh gum was purchased from the local market in April 2013 and was formally identified (RA-85) prior to its use by Prof. Dr.
Samen Jan of Department of Botany, Islamia College University, Peshawar, Pakistan. Water was purified through a Milli-Q-SP ultra pure water purification system.

Synthesis of gold and silver nanoparticles
The P. domestica gum-mediated biosynthesis of gold and silver nanoparticles was carried out by utilizing the stock solutions of tetrachloroauric acid trihydrate/silver nitrate and P. domestica gum at concentrations of 1 mM and 0.5% w/v, respectively. These solutions were centrifuged at 5000×g for 10 min to remove bulk impurities. The aqueous solutions of tetrachloroauric acid and silver nitrate were reduced by mixing with 0.5% P. domestica gum solution in differing ratios and stirred gently at temperatures of 20, 40, 60 and 80°C. The optimized product having surface plasmon resonance (SPR) at 555 nm for the gold nanoparticles was obtained by mixing 8 mL of tetrachloroauric acid solution (1 mM) and 5 mL of 0.5% w/v P. domestica gum solution at a temperature of 80°C and a reaction time of 5 h. Similarly, the optimized silver nanoparticles having SPR at 450 nm were obtained by use of 20 mL of silver nitrate solution (1 mM) and 8 mL of 0.5% w/v P. domestica gum solution at a temperature of 80°C.

Assessment of stability
The effect of gum concentration on the synthesis of gold and silver nanoparticles was studied by heating different concentrations (0.1-0.5%) of gum solutions containing 1 mM of tetrachloroauric acid and silver nitrate solutions, respectively, for 1 h. The effects of gold or silver ions were evaluated by changing their concentration from 1 to 5 mM and then heating at 80°C for 3 h. The thermal stability was studied by keeping the nanoparticles solution at 20, 40, 60, 80°C, each for 3 h. The effect of varying reaction time (1-5 h) was assessed with 0.5% gum at 1 mM gold or silver salt solution. The salt stability was checked by adding 20, 40 and 60 μL of sodium chloride solution (1 M) to 3 mL colloidal solution of gold and silver nanoparticles under continuous mixing for 3 h. The resistance to varying pH conditions was measured at different pH values (2-3, 4-5, 6-7, 8-9, 10-11, 12-13) by drop-wise addition of 1 M HCl or NaOH solution. The long term stability was estimated by keeping the nanoparticles at room temperature for eight months. The extreme thermal stability was evaluated by heating the nanoparticles at 100°C for 30 min.
In vitro biological assays Cytotoxicity assay The HeLa cervical cancer cell line (HeLa cells) was cultured in RPMI-1640, having heat-inactivated fetal bovine serum (10%), glutamine (2 mM), pyruvate (1 mM), 100 U/mL penicillin, and 100 μg/mL streptomycin, in T-75 cm 2 sterile tissue culture flasks in a 5% CO 2 incubator at 37°C. For experiments, 96-well plates were used for growing HeLa cells by inoculating 5 × 10 4 cells per 100 μL per well, and plates were incubated at 37°C for 24 h in a humidified atmosphere containing 5% CO 2 . Within 24 h, a uniform monolayer was formed, which was used for experiments. To perform the cytotoxicity assay, a previously described method [23] was adapted with small modifications [24]. Briefly, cells were cultured in different 96-well plates for 24 h. Initially, 1 mg/mL of P. domestica gum solution and the nanoparticles were inoculated in test wells. Further these solutions screened for different concentrations (1 mg/mL -1 ng/mL) were inoculated in test wells. Cisplatin was used as a positive control. The well containing culture media with cells having no compound or drug was taken as blank. All the plates were then incubated for 48 h. After that, cells were fixed with 50 μL of 50% ice cold trichloroacetic acid solution (TCA), and plates were incubated at 4°C for 1 h. Subsequently, plates were washed five times with phosphate-buffered saline (PBS) and air dried. Fixed cells were further treated with 0.4% w/v sulforhodamine B dye (prepared in 1% acetic acid solution) and left at room temperature for 30 min. The plates were rinsed with 1% acetic acid solution and allowed to dry. In order to solubilize the dye, the dried plates were treated with 10 mM Tris base solution for 10 min at room temperature. The absorbance was measured at 490 nm subtracting the background (blank) measurement at 630 nm [25]. All experiments were performed in triplicate. The IC 50 values of potential inhibitors (≥50%) were determined with the help of the non-linear regression analysis program of GraphPad Prism 5.0 Software Inc., San Diego, CA, USA [24].

Urease inhibition assay
Urease inhibition activity of the synthesized compounds was determined by the indophenol method [26] with small modifications [27]. Reaction mixtures comprised of 40 μL of buffer (100 mM urea, 0.01 M K 2 HPO 4 , 1 mM EDTA and 0.01 M LiCl 2 , pH 8.2) and 10 μL of jack-bean urease enzyme (5 U/mL) were incubated with 10 μL of P. domestica gum solution and the nanoparticles (1 mg/mL) at 37°C for 30 min in 96-well plates. Urease inhibitory activity was calculated by the indophenol method based on the production of ammonia. The phenol reagent (40 μL, 1% w/v phenol, 0.005%, w/v sodium nitroprusside) and alkali reagent (40 μL 0.5% w/v NaOH, 0.1% active chloride NaOCl) were added to each well, and, after 10 min of incubation at 37°C, the absorbance was measured at 630 nm with a microplate reader (Bio-TekELx 800™, Instruments, Inc. Winooski, VT, USA). Thiourea was used as the standard inhibitor. All experiments were performed in triplicate. The percent inhibition was calculated according to the equation, Percent inhibition = 100 -[absorbance of nanoparticles/ absorbance of control] × 100.

Antibacterial assay
The antibacterial activity of P. domestica-loaded gold and silver nanoparticles was evaluated against Grampositive [Staphylococcus aureus (ATCC 25923)] and Gram-negative [Escherichia coli (ATCC 25922), and Pseudomonas aeruginosa (ATCC 27853)] strains of bacteria, by the disc diffusion method. Three independent experiments were carried out for each bacterial strain with streptomycin as the positive control. Au/Ag-NPs (5 μg) were dissolved in DMSO and incubated at 30°C for 24 h. The final DMSO concentration was kept below 1%.

In vivo biological assays Animals
BALB/c mice of either sex weighing 25-30 g were purchased from the National Institute of Health (NIH), Islamabad, for the in vivo experiments. The animals were acclimatized at 22 ± 2°C for one week prior to experiments. The animals have free access to food and water during this period. Prior to experiments, the animals were fasted for 2 and 4 h, respectively, for antinociceptive and anti-inflammatory assay. All experimental procedures on animals were performed in accordance with the NIH guidelines for the care and use of laboratory animals. The experimental protocols conformed to the Animal Research: Reporting In Vivo experiments (ARRIVE) guidelines. The study was approved by the Graduate Studies Committee (GSC) of the Institute of Chemical Sciences (ICS), University of Peshawar (reference letter number: 942-51/ICS). Animals were randomly assigned to different treatment groups, with each group consisting of six animals.

Antinociceptive assay
The antinociceptive efficacy afforded by P. domestica gum-loaded gold nanoparticles against tonic visceral chemically-induced nociception was determined by the acetic acid-induced abdominal constriction assay [28]. All drugs were dissolved in normal saline and were administered through an oral gavage tube, except diclofenac sodium, which was given as an intraperitoneal injection. Animals were randomly divided into six groups, with each group containing 6 mice. Group I received normal saline and served as control. Group II was administered with the positive control, diclofenac sodium (50 mg/kg, i.p). Group III and IV received P. domestica gum at 200 and 400 mg/kg, while group V and VI were treated with P. domestica gum-loaded gold nanoparticles at doses of 40 and 80 mg/kg, respectively. After 30 min of drugs treatment, the animals were injected with 1% acetic acid (10 mL/kg, i.p) and the abdominal writhes were counted for 20 min.

Anti-inflammatory assay
The inhibitory effect produced by P. domestica gumloaded gold nanoparticles against a phlogistic agentmediated paw swelling was evaluated by the carrageenan induced paw edema assay [29]. Animals were divided into seven groups, with each group containing 6 mice. P. domestica gum and its gold nanoparticles were administered by an oral gavage tube. Diclofenac sodium was used as positive control and was injected i.p at a dose of 50 mg/kg. After 30 min of treatment, all animals were challenged with 50 μL of 1% solution of carrageenan, injected into the plantar surface of the left hind paw. The anti-inflammatory effect was evaluated by measuring the paw volume of each animal with a digital plethysmometer after each hour of the 5 h study duration.

Statistical analysis
Data were expressed as mean ± SD or SEM. Statistical analysis was done by one-way ANOVA followed by Dunnett's or Tukey's post hoc test where appropriate with GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA, USA).

Results
Characterization of synthesized gold and silver nanoparticles UV-Vis spectroscopy UV-Vis absorption spectra of P. domestica gum-loaded gold and silver nanoparticles solutions were recorded against P. domestica gum and pure metal salt solutions. The typical SPR peaks for gold and silver nanoparticles were obtained at 555 and 450 nm, respectively, however, no SPR peaks were observed for the pure chlorauric acid, silver nitrate, and gum solutions. The biosynthesis of gold and silver nanoparticles were further confirmed from the appearance of wine red and light yellow color respectively, while the pure gum, chloroauric acid, and silver nitrate solutions appeared colorless (Fig. 1).
FTIR P. domestica gum has mainly polysaccharide compounds, which, along with glucuronic acid and its 4-Omethyl ether having O-H, −COOH, hemiacetal and ether groups, showed a broad O-H stretching band at 3352 cm −1 (Fig. 2a). A weak peak at 2947 cm −1 was due to C-H stretching. The peak observed at 1734 cm −1 corresponds to the C = O stretching of -COOH. Similarly, a strong peak at 1614 cm −1 can be attributed to the conjugated C = C bond stretching, which might be due to the presence of flavonoids and carotenoids. Coupled vibrations due to C-O stretching and O-H deformations were observed at 1417 and 1373 cm −1 . The strong peak at 1018 cm −1 corresponds to C-O stretching vibrations. In case of the gold nanoparticles (Fig. 2b), the O-H bond stretching shifted from 3352 cm −1 to the lower frequency of 3288 cm −1 , and this shows the involvement of the OH groups during the process of reduction and capping. A sharp carbonyl carbon peak at 1734 cm −1 disappeared completely, thus demonstrating the participation of carboxylic acid groups in nanoparticle formation as a reducing and capping agent. The bending vibrations at 1373, 1361, 1313, 1246, and 1220 cm −1 also disappeared due to possible attraction by the gold nanometal. A shift in the C-O acyclic bond stretching from 1018 to 1020 cm −1 can be attributed to attraction by gold metal during the capping process. Almost similar FTIR spectrum was observed for the P. domestica gumloaded silver nanoparticles (Fig. 2c).
SEM P. domestica gum-loaded gold and silver nanoparticles were in the size range of 7-30 and 5-30 nm, respectively, with most particles were spherical in shape, but a small number of anisotropic nanostructures, such as nanotriangles, a few nanorods, and hexagonal and polygonal nanoprisms were also present (Fig. 3). The uniform distribution of gold and silver nanoparticles indicates the complete stabilization of nanoparticles by capping agents, while the large sizes and anisotropic shapes of some particles might be due to the aggregation of the smaller nanoparticles.

EDX and XRD
The EDX analysis (Fig. 4) indicated the presence of gold and silver in the P. domestica gum-loaded gold and silver nanoparticles. Strong signals were observed from Au atoms in the gold nanoparticles at approximately 0.6 and 2.6 keV, while a weak signal was observed at 9.7 keV. In the case of silver nanoparticles, strong signals were observed at 0.6 and 2.6 keV, while a weak signal was observed at 3.7 keV. The appearance of the Si signal corresponds to the use of a silicon grid in the EDX analysis. The signal for N indicates the presence of nitrogen-containing organic compounds, while the signals for K and Mg correspond to the X-ray emission from different biomolecules of the gum. Similarly, the other strong signals for C and O also were due to the presence of bio-organic molecules capping the gold and silver nanoparticles. The presence of the Cl atom in the EDX spectra of gold nanoparticles was due to its presence in the tetrachloroauric acid trihydrate molecule. The appearance of elemental Au and Ag in the EDX analysis supports the XRD results, which provides confirmation of the reduction of metal cations to elemental form.
In the XRD spectrum, no peaks were observed for the P. domestica gum, thus indicating their non-crystalline (amorphous) nature. However, the XRD patterns of gum-loaded gold and silver nanoparticles have characteristic peaks, indicating their crystalline nature. The gold nanoparticles (Fig. 5) exhibited characteristic peaks  The diffraction peak at 38 o was the only intense peak among the observed peaks for both the gold and silver nanoparticles. In addition, the ratio between the (2 2 0) and (1 1 1) peaks was lower than the standard value (0.1 vs. 0.4). The mean particle diameters of the gold and silver nanoparticles were calculated from the XRD data. Diameters were derived from the Debye Scherrer eq. (D = k λ/ β½ cosθ). This equation exploits the reference peak width at an angle θ, where λ is the X-ray wavelength (1.5418), β½ is the width of the XRD peak at half height and k is the shape factor. Average particle size was around 20 and 17 nm for gold and silver nanoparticles, respectively.

TGA
The thermogram obtained from the thermogravimetric analysis showed three successive weight losses for gum and nanoparticles within the temperature range of 50-800°C (Fig. 6). The first observed weight loss was attributed to the loss of entrapped water molecules from the polymer matrix. The second weight loss might be due to the thermal decomposition of the polymer as well as the polymer capping around the nanoparticles. The third weight loss could be attributed to the conversion of remaining polymer to carbon residue. A low percent weight loss was observed for the gum after nanoparticle synthesis.

Stability assessment Effect of gum concentration
An increase in the absorption intensity at 555 and 450 nm was observed, as the concentration of gum was increased. However, the characteristic SPR peaks for both the nanoparticles were slightly red shifted (Fig. 7).
These changes indicate an enhancement in the nanoparticles concentration and an increase in the nanoparticle size. Both size and concentration of nanoparticles affect the absorbance. The concentration of gum affects the nanoparticle size distribution. The broad particle size distribution at high gum concentration, could be due to  the increased intermolecular force of gum molecules, while, at lower gum concentration, it might be due to insufficient protection. In addition, a color change also indicates an increase in nanoparticle concentration due to increasing gum concentration. As concentration of gum was increased (0.1-0.5%), the colors of the obtained gold and silver nanoparticles were also intensified from light red to dark red and light yellow to dark yellow, respectively, indicating an increase in nanoparticle concentration.

Effect of Au (III) and Ag + ions concentration
As indicated from the morphologies of the UV-Vis spectra (Fig. 8), an increase in the concentration of metal precursor, produced an increase in nanoparticles concentration, and a slight change in the particle shape and diameter. The increased number of colloidal particles in the gum network might be due to an increase in the number of metal nuclei with an increase in metal precursor concentration. The linear increase in the absorption intensity was due to an enhancement in the nuclei formation, which resulted in an increase in nanoparticle concentration, while the shift in absorption intensity can be attributed to an increase in the particle size. At high Au (III) and Ag + ion concentrations (5 mM), quite larger particle size distributions were observed. This might be due to the less protective effects of the gum and account for the longitudinal plasmon resonance around 560 and 455 nm.

Effect of reaction temperature
With an increase in the reaction temperature, there was a linear increase in absorption intensity without any change in the position of the SPR peak (Fig. 9), indicating an increase in nanoparticle concentration of similar size. The maximum intensity of the characteristic absorption band was found at 80°C, which is indicative of a maximum nanoparticle concentration and therefore the optimum temperature for nanoparticles synthesis.

Effect of reaction time
An increase in the reduction capacity of the gum was observed with a change in the reaction time (1-5 h). The SPR peak occurs at 555 and 450 nm for gold and silver nanoparticles, respectively, and the intensity of absorption increases linearly without any shift in wavelength with time, thus indicating a continuous reduction of Au (III) and Ag + ions, which led to an increase in the number of colloidal particles. However, after 4 h, no significant change in the intensity of the SPR peak was  observed and is an indication of completion of the synthesis reaction (Fig. 10).

Effect of salt
No obvious color change was observed, when different volumes of NaCl solutions (20, 40, and 60 μL) were added to the colloidal solutions of gold and silver nanoparticles. Moreover, no changes were observed in the position of SPR peaks with varying salt volumes. However, a decrease in their peak intensity was noted, which might be due to the dilution effect, as a similar decrease was also recorded for the salt-free nanoparticles solutions (Fig. 11).

Effect of pH
Optimized samples of both gold and silver nanoparticles are slightly acidic, having a pH value of 6-7, and showed maximum absorption in the UV-Vis spectra (Fig. 12). It was observed that the gold nanoparticles were stable in acidic media as there were less significant shifts in the UV-Vis absorption spectra except at pH 2-3, where the absorption intensity was lowered with a blue shift. The P. domestica gum-loaded gold nanoparticles were unstable in basic media, as significant peak lessening and blue shift were observed in their UV-Vis spectra. Similarly, the P. domestica gum-loaded silver nanoparticles also exhibited stability in neutral-to-acidic media; however, at pH 2-3, low absorption intensity was observed in the UV-Vis spectrum. In basic media, the silver nanoparticles were unstable except at pH 8-9, where the shift was similar to that of pH 2-3, but with low absorption intensity.

Storage stability
P. domestica gum-loaded gold and silver nanoparticles solutions were stored at room temperature for six months. There was no change in color. Visual aggregation was observed during this storage period. From the UV-Vis spectra, a slight red shift was observed in the position of the SPR peak and a lessen absorption value, indicating an increase in the size of nanoparticles size due to aggregation (Fig. 13).

Thermal stability
Colloidal solutions of P. domestica gum-loaded gold and silver nanoparticles were heated at 100°C for 30 min, and no significant changes were observed in the position of the SPR peaks (Fig. 14). This high stability of gold and silver nanoparticles can be reasonably attributed to the protective effect of biomolecules on the nanoparticle surface.
Prunus domestica gum-loaded nanoparticles inhibit cancer cells P. domestica gum-loaded gold and silver nanoparticles were tested to evaluate their cytotoxic potential against human cervical cancer cells (HeLa) ( Table 1). A potential anticancer effect was afforded by P. domestica gumloaded gold nanoparticles, having an IC 50 value of 2.14 ± 0.15 μg/mL, followed by P. domestica gumloaded silver nanoparticles (3.45 ± 0.23 μg/mL) and P. domestica gum (4.92 ± 0.31 μg/mL). The standard anticancer drug cisplatin has the lowest IC 50 value (1.89 ± 0.12), and therefore exhibited a robust inhibitory effect on the HeLa cancer cells.

Prunus domestica gum-loaded nanoparticles suppressed pathogenic bacteria
The disk diffusion method was used to evaluate the potential antibacterial activity of P. domestica gum-loaded gold and silver nanoparticles against the pathological stains of Gram-positive and Gram-negative bacteria ( Table 2). Prominent growth suppression was observed in plates loaded with nanoparticles, while the negative control plates and the P. domestica gum did not produce any visible zone of inhibition. The potential antibacterial effect was observed with P. domestica gum-loaded silver nanoparticles against Gram-positive strain of S. aureus (19.7 ± 0.4 mm) and Gram negative strains of E. coli (14.4 ± 0.7 mm), and P. aeruginosa (13.1 ± 0.2 mm). Similarly, these bacterial strains were also inhibited by P. domestica gum-loaded gold nanoparticles but with  Prunus domestica gum-loaded nanoparticles modify the enzymatic activity of urease P. domestica gum-loaded gold and silver nanoparticles were tested in vitro for their potential effect as an inhibitor of urease. Both the gum and the loadednanoparticles were able to inhibit the jack-bean urease ( Table 3). An increased inhibitory effect was demonstrated by the P. domestica gum (26.7 ± 0.49%), followed by P. domestica gum-loaded silver (21.5 ± 1.17%) and gold (19.2 ± 0.86%) nanoparticles. The positive control, thiourea showed a robust efficacy by producing a potent inhibition (78.3 ± 2.33%) of urease.

Prunus domestica gum-loaded gold nanoparticles ameliorate inflammation
As shown in Table 4, the subplantar injection of carrageenan significantly elevated (P < 0.001) the normal paw volume. After 1 h, significant reduction in the carrageenan-induced paw edema was observed with P. domestica gum at doses of 200 mg/kg (P < 0.05) and 400 mg/kg (P < 0.001) as well as with the P. domestica gum-loaded gold nanoparticles at 40 mg/kg (P < 0.05) and 80 mg/kg (P < 0.01). Moreover, there was a significant antagonism (P < 0.001) of the phlogistic agent (carrageenan)-induced increase in paw volume by all the tested doses of P. domestica gum (200 and 400 mg/kg) and P. domestica gum-loaded gold nanoparticles (40 and 80 mg/ kg) during the subsequent 2-5 h of experiment. A similar anti-inflammatory profile was shown by the positive control, diclofenac sodium, at a dose of 50 mg/kg during the 1st (P < 0.05) and 2-5 h (P < 0.001) of the study period.

Prunus domestica gum-loaded gold nanoparticles deescalate nociception
A potent antinocicpetive effect was demonstrated by the P. domestica gum and its gold nanoparticles (Fig. 15). Significant attenuation of chemically-induced nociception was observed with P. domestica gum at 200 (P < 0.05) and 400 mg/kg (P < 0.01). Similarly, the tested doses (40 and 80 mg/kg) of P. domestica gum-loaded gold nanoparticles significantly abolished (P < 0.01) the acetic acid-induced writhes. The single dose of the positive control, diclofenac sodium (50 mg/kg), also produced an efficient analgesic effect by significantly alleviating (P < 0.01) the chemicallyinduced nociceptive pain.

Discussion
In this study, stabilized Prunus domestica gum-loaded gold and silver nanoparticles were evaluated for their multi-target therapeutic potential as an anticancer, antibacterial, urease inhibitor, anti-inflammatory, and antinociceptive agent. Nanoparticles can be loaded with a wide range of therapeutic agents, which together with a targeted delivery, increases the amount of drug accumulation at the pathological site, and decreases toxicity to normal tissues [30]. The use of natural gums for pharmaceutical applications is attractive as they are economical, readily available, nontoxic, capable of chemical modifications, potentially biodegradable, and also biocompatible. Natural gums have been widely employed as reducing, and stabilizing agents for the efficient synthesis of size-controlled nanoparticles that display greater stability, and better biological activities [31][32][33]. Phytotherapy offers new treatment opportunities as the therapeutic efficacy may based on the synergistic or antagonistic interaction of a mixture of phytochemicals [3].
Nano-based drug delivery systems can potentiate the action of plant extracts, promote sustained release of active constituents, reduce the required dose, decrease side effects, improve activity, and promote self-targeting at the infected pathological area [5].
Prunus domestica gum-loaded gold and silver nanoparticles were stable in different concentrations of NaCl (1-3 M), neutral to acidic pH (4-7) and did not show long-term storage (six months) or thermally (100°C)-induced deterioration changes. Regarding the nanoparticles synthetic stability, 0.2 and 0.3% w/v gum, 3 and 4 mM Au/Ag ion solution, reaction temperature of 60-80°C, and reaction time of 4 h were suitable for the efficient synthesis of P. domestica gum-loaded gold and silver nanoparticles. The biological utility of nanoparticles often stems from their unique physicochemical properties that are often a function of their surface interactions with other nanoparticles or biological entities. Therefore, apart from key attributes required for their successful use in biology, such as low toxicity, biocompatibility, and effective biological clearance, the colloidal or dispersion stability of these nanoparticles is also crucial to  preserve their intended physicochemical behavior and hence their utility in the physiological environment [34].
Cancer remains a high unmet medical need. Conventional chemotherapy employs drugs that are known to kill cancer cells effectively; however, these cytotoxic drugs kill healthy cells in addition to tumor cells, leading to serious adverse side effects that limit their clinical effectiveness.
Recently, single-target chemotherapy is fading in favor of a multi-target approach [35]. Coupling multiple targetedagents or using an agent that hits an individual target in several independent locations in the disease-causing pathway(s) may be the best approach to treat different cancers [36]. In this study, the P. domestica gum-loaded gold and silver nanoparticles showed potential inhibitory propensity towards human cervical cancer cells (HeLa), and the anticancer effect was similar to that of the standard cisplatin. Nanotechnology can play an important role by allowing the appropriate combination of agents that act as "smart nanogrenades" by targeting cancer lesions and therefore eliminate them without collateral effects on healthy tissue [37]. Integration of nanocarriers as a novel drug delivery system in the traditional system of medicine can be proved beneficial to conflict chronic notorious diseases like cancer [38].  Infectious diseases remain among the leading cause of death and produce an extremely significant impact on global health and economies [39]. Infections caused by bacteria represent a major public health burden, not just in terms of morbidity and mortality, but also in terms of increased expenditure on patient management and implementation of infection control measures. Conventional drugs usually provide effective antibiotic therapy for bacterial infections, but there is an increasing problem of antibiotic resistance and a continuing need for new solutions. Immediate actions should be taken to counter the antibiotic resistance and reduce the development and spread of life-threatening infections [40]. Several herbal preparations with antibacterial activity have shown efficacy in different clinical trials against various pathogenic species of bacteria [41]. Our study shows that the P. domestica gum-loaded gold and silver nanoparticles possessed antibacterial properties, while the P. domestica gum itself was unable to produce any zone of inhibition. Preferential inhibition was demonstrated by the P. domestica gum-loaded silver nanoparticles against both the Gram-positive (S. aureus) and Gram-negative (E. coi, P. aeruginosa) pathogenic strains, and the effect was comparable to that of streptomycin, used as positive control. Nanotechnology can provide important tools for designing and fabricating a new generation of substrates with specific antimicrobial properties [42]. Herbal drugs incorporated into nano-based drug delivery systems have potential benefit for use in the treatment of different bacterial infections [43].
Ureases are metalloenzymes that hydrolyze urea into ammonia and carbon dioxide. Urease is a virulence factor found in various pathogenic bacteria and is essential for the host organism in the maintenance of bacterial cells in tissues [44]. The production of urease by Helicobacter pylori, a Gram-negative bacterium, plays a key role in protecting this bacterium from the fatal acidic environment of stomach. The colonization of H. pylori in the human stomach leads to gastric ulcer or even gastric carcinoma, if left untreated [45]. There is an urgent need of novel urease inhibitors for counteracting the catastrophe of H. pylori infection on the eve of rising antibiotic resistance. Our study showed that both the P. domestica gum and its gold nanoparticles possessed promising urease inhibitory potency. There is a great potential of plant secondary metabolites of different classes to negatively affect the activity of ureases, the knowledge of which can contribute to the design of novel, safe, and less-costly urease inhibitors with the aim to improve human life by fighting urease-related diseases [46]. Utilization of plant extract-fabricated nanoparticles to inhibit urease can be a boon for the development of new drugs to treat multidrug-resistant H. pylori by means of approaches such as their encapsulation with drugs or making synergistic combinations with standard drugs [47].
Inflammation is a complex set of interactions among soluble factors and cells that can arise in any tissue in response to traumatic, infectious, post-ischemic, toxic, or autoimmune injury. The process normally leads to recovery from infection and to healing; however, if targeted destruction and assisted repair are not properly phased, inflammation can lead to persistent tissue damage by leukocytes, lymphocytes, or collagen. Inflammation per se remains one of the main therapeutic targets in diverse disorders with a staggering collective impact [48]. Natural products play a significant role in human health in relation to the prevention and treatment of inflammatory conditions [49]. Inflammation induced by carrageenan is acute, nonimmune, and produces the cardinal signs of inflammation, i.e., edema, hyperalgesia, and erythema, which develop immediately following subcutaneous injection, resulting from the action of proinflammatory agents, including bradykinin, histamine, tachykinins, complement, and reactive oxygen, and nitrogen species. Such agents can be generated in situ at the site of insult or by infiltrating cells. Neutrophils readily migrate to sites of inflammation and can generate proinflammatory reactive oxygen and other species. The  inflammatory response is usually quantified by an increase in paw size (edema), which is maximal around 5 h post-carrageenan injection and is modulated by inhibitors of specific molecules within the inflammatory cascade [29]. In this study, P. domestica gum (200 and 400 mg/kg) inhibited the carrageenan-induced biphasic paw edema response. Similarly, the P. domestica gumloaded gold nanoparticles also exhibited a similar antiinflammatory profile; however the beneficial effect was observed at much lower doses (40 and 80 mg/kg) compared to P. domestica gum, and the effect was comparable to that of the standard anti-inflammatory drug, diclofenac sodium. Current management of inflammatory processes can be improved by the use of biodegradable nanoplatforms that specifically deliver antiinflammatory molecules to inflamed tissues [50]. Nanobiomaterials based on gold nanoparticles conjugated with biomolecules possessed unique anti-inflammatory properties by reducing the leukocyte-endothelium interaction and leukocyte influx to adjacent tissues after leukotriene B4 stimulation in vivo as well as producing a marked reduction of chemotaxis and oxidative burst activation in vitro [51].
Pain is an unpleasant sensory and emotional experience associated with actual or potential tissue damage or described in terms of such damage [52]. Pain is usually elicited by the activation of specific nociceptors (nociceptive pain). It may also result from injury to sensory fibers or from damage to the CNS itself (neuropathic pain) [53]. Writhing, which is an overt response to the intense pain induced by acetic acid through nociceptors, is characterized by episodes of retraction of the abdomen and stretching of the hind limbs. The signals transmitted to the central nervous system in response to pain due to irritation cause release of mediators, such as prostaglandins, which contribute to the increased sensitivity of nociceptors [54]. The acetic acid-induced nociceptive test is sensitive to analgesics [55], and sensory afferents in the peritoneum that carry various receptors on their terminals [56] are activated by appropriate agonists and therefore depress the generation of pain impulses [57,58]. P. domestica gum has potential peripheral antinociceptive properties since a marked reduction in chemically-induced nociceptive response was noted at doses of 200 and 400 mg/kg. The extent to which P. domestica gum abolished the tonic visceral chemicallyinduced nociception was also exhibited by P. domestica gum-loaded gold nanoparticles. However, the effect was observed at much smaller doses (40 and 80 mg/kg) of gold nanoparticles and was similar to diclofenac sodium used as a standard analgesic. Chronic pain, resulting from disease or injury, constitutes an enormous burden for the individual and society. The effectiveness of current pain therapies is limited by the extent of pain relief provided and the occurrence of significant side effects. Nanotechnology has the potential to address multiple, major, unmet problems in the diagnosis, treatment, and symptom management of a large variety of diseases and conditions, such as cancer, which are accompanied by pain [59]. Gold nanoparticles have shown effectiveness to be useful for enhancing the analgesic effects of medicinal plant extracts with potential antinociceptive properties [60][61][62]. Nanomedicine offers unprecedented opportunities in the development of novel painrelieving therapies that change the frowning face of pain to a smile of relief [63].  Group 3 Diclofenac (50 mg/kg) 0.287 ± 0.029** 0.265 ± 0.019*** 0.265 ± 0.031*** 0.287 ± 0.025*** 0.305 ± 0.012*** Group 4 Gum (200 mg/kg) 0.302 ± 0.009* 0.290 ± 0.008*** 0.295 ± 0.013*** 0.305 ± 0.013*** 0.315 ± 0.019*** Group 5 Gum (400 mg/kg) 0.282 ± 0.017*** 0.292 ± 0.012*** 0.287 ± 0.012*** 0.280 ± 0.022*** 0.290 ± 0.018*** Group 6 Gold nanoparticles (40 mg/kg) 0.305 ± 0.021* 0.307 ± 0.012*** 0.305 ± 0.012*** 0.302 ± 0.015*** 0.317 ± 0.026*** Group 7 Gold nanoparticles (80 mg/kg) 0.295 ± 0.013** 0.287 ± 0.017*** 0.295 ± 0.013*** 0.287 ± 0.022*** 0.300 ± 0.018*** Values expressed as mean paw volume in mL ± SD. ### P <0.001 compared to group 1. *P <0.05, **P <0.01, ***P <0.001 compared to group 2. One-way ANOVA followed by Tukey's post hoc test. Results are the means of 6 animals per group Fig. 15 Antinociceptive effect of P. domestica gum-loaded gold nanoparticles. *P < 0.05, **P < 0.01 compared to saline treated group. One-way ANOVA followed by Dunnett's post hoc test. Results are the means of 6 animals per group