Phytogenic-mediated silver nanoparticles using Persicaria hydropiper extracts and its catalytic activity against multidrug resistant bacteria

1 Introduction

Nanotechnology or nanoscale technology is the field of material science which deals in the synthesis of particles having size below 100 nm. Moreover, these particles have different chemical properties that’s why they have been extensively used in different fields such as optics, mechanics, photo electrochemical sciences, medical sciences, chemical industry, catalysis, drug-gene delivery, energy sciences, electronics and nonlinear optical sciences (Quintanilla-Carvajal et al., 2010). Silver (Ag) is an inorganic compound, available in powder form and under normal condition is insoluble in water. It is extensively used in the formulation of different products such as pigments ceramics, lubricants, adhesives tapes, ointment and batteries (Bharadwaj et al., 2021). Furthermore, silver is a non-toxic and biocompatible semiconductor used in the biological fields i.e., nano-medicine due to their extensive antimicrobial properties (Parveen et al., 2012; Xu, 2020).

Different methods such as chemical, physical, and biological methods are available now-a days to prepare nanoparticles. Chemical and physical methods are effective with respect to time and production rate. However, in chemical methods, different chemicals are used in the production process that may leads to the formation of toxic by-product and hence increase nanoparticles recovery and purification cost. On the other hand, physical methods are highly expensive due to use of energetic radiations that may leads to changes in the physicochemical property of nanoparticles (Viorica et al., 2017). On the other hand, biological methods are safe and cost effective as compared to chemical and physical methods (Xu, 2020). Biological synthesis or green synthesis used naturally occurring reducing agent such as plant extract, microorganisms (yeast, fungi, and bacteria) and biological particles like enzyme, protein, peptides, polysaccharide etc. In literature, different bacterial strains such as Lactococcus lactis (LCLB56-AgNCs) and actinobacteria strain (CGG11n) were reported as cost effective and novel reducing agents for the synthesis of AgNPs (Railean‐Plugaru et al., 2016; Viorica et al., 2017).

During biological synthesis of nanoparticles, different factors are needed to be considered and these include cell culture maintenance, intracellular synthesis, and multi-purification steps. Bioinspired green synthesis of silver nanoparticles (AgNPs) using plants has become a reliable method (Bharadwaj et al., 2021). Plants provide eco-friendly, safe, and beneficial approach for the synthesis of nanoparticle because plants are easily accessible to produce nanoparticles on large scale. In additions, the rate of production is faster as compared to other biological models like algae, fungi, and bacteria (Gurunathan et al., 2015).

Wide varieties of antibiotics are available in market for the treatment of infectious diseases and among them the local branded antibiotics have low potency towards pathogenic microorganisms. Therefore, usage of such antibiotics may lead to antibiotic resistance phenomenon (Gurskis et al., 2010). The bacterial plasmid showed role in the development of antibiotic’s resistance. In 1970 s, antibiotic’s resistance phenomenon was observed only in Enterobacteriaceae family such as Klebsiella pneumonae and Escherichia coli (Horan et al., 2008). It is now observed that most of the organisms which initially didn’t showed resistance to antibiotics, are now become resistance strains e.g., Klebsiella pneumonae and Escherichia coli caused infection in colonization with other pathogenic organisms within hospitalized patients and showed resistance to antibiotics (Akova, 2016).

Persicaria hydropiper is an annual plant of 40–70 cm tall which belongs to the family of Polygonaceae. It is distributed in Asia, Europe, Northern Africa, and Australia (Huq et al., 2014). The plant generally grows in wet areas at water sides in marshes and is usually predominant in agricultural fields. It is also commonly distributed to high land sites with highly organic, moist, or salty areas (Miyazawa and Tamura, 2007). P. hydropiper also has a wide range of traditional uses for medicinal purposes. In Europe, the plant has been used as diuretic and regulates menstrual irregularities (Ayaz et al., 2020). In addition, the whole plant either alone or mixed with other medicinal plants, is also given for menstrual bleeding, hypersensitivity, and hemorrhoids. Several reports on pharmacological properties of P. hydropiper are available to support the ethno-medicinal uses of the plant including antimicrobial, antifungal, anti-parasitic, anti-inflammatory, anti-fertility, anti-cholinesterase, and toxicological effects (Huq et al., 2014).

Silver nanoparticles (AgNPs) synthesized from the root, stem and leaves extracts of plants has also proven to be used as antimicrobial agent against number of bacterial species (Ahmed et al., 2016; Hernández-Morales et al., 2019). It has also been reported that AgNPs have strong antimicrobial properties against variety of microorganisms as they are small so they can easily penetrate into the cell and destroy synthesis machinery within cell and ultimately caused death of the target organisms (Xu, 2020: Nieto-Maldonado et al., 2022). In addition to this, AgNPs are also used in combination with other pharmaceutical products especially antibiotics to catalyze or enhance their activity against MDR bacterial species (Bankar et al., 2010; Dos Santos et al., 2014: Caloca et al., 2017).

The aim of the current study was to utilize the P. hydropiper root, stem, and leaves extracts for the synthesis of AgNPs and then determined its bio-catalytic potential to enhance the activity of commercially available antibiotics against multi drug resistance (MDR) bacterial species. As reported, extracts of P. hydropiper contained many compounds like flavonoids such as epicatechin, hyperin, isoquercitrin, isorhamnetin, kaempferol, quercetin, quercetrin, rhemnazin and rutein, which had strong antibacterial activity against bacterial species (Hawrył and Waksmundzka-Hajnos, 2011; Khurana et al., 2014). As the phenomenon of multi drug resistance increases day by day, therefore by utilizing AgNPs (fabricated from P. hydropiper extracts) in combination with commercially available antibiotics will provide an alternate therapy for the treatment of bacterial infections caused by MDR bacterial species.

2 Materials and methods

3 Results

3.1

3.1 Characterization of AgNPs

3.1.1

3.1.1 Visual analysis of the synthesized AgNPs

In the present study, visual confirmation of the AgNPs was done by noticing the change in the color of mixture of plant extract and nanoparticle. When AgNO₃ was added to plant extracts, the color of the extract changes from yellowish to reddish brownish (Fig. 1a).

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Fig. 1

(a) Visual confirmation of silver nanoparticles i.e., silver nitrate solution and AgNPs formation; (b) UV–Vis spectrum of AgNPs synthesized from the leaves of P. hydropiper; (c) UV–Vis spectrum of AgNPs synthesized from the stem of P. hydropiper; (d) UV–Vis spectrum of AgNPs synthesized from the roots of P. hydropiper.

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3.1.2

3.1.2 UV– vis spectrophotometric analysis of the synthesized AgNPs

In Fig. 1b, 1c and 1d, absorbance (on the vertical axis) is just a measure of the amount of light absorbed. The higher the value, the more of a particular wavelength is being absorbed. It was depicted that the UV–Vis spectrum of AgNP_S synthesized from the leaves, stem, and roots of P. hydropiper showed strong absorption (OD) at 370 nm (lambda-max) with no other peak displayed high purity of the synthesized AgNPs.

3.1.3

3.1.3 Fourier transformed infrared spectroscopy (FTIR) of leaves extracts and AgNPs

In the FTIR spectrum of the P. hydropiper leaves extract, different absorption peaks were observed indicating the presence of different functional groups i.e., peak value at 3311.78 cm⁻¹ was due to vibration of stretch O-H having carboxylic acid, at 2945.30 cm⁻¹ was due to C-H stretch of alkane, at 1568.13 cm⁻¹, 1556.55 cm⁻¹ and 1606.70 cm⁻¹ were due to C-C stretching having aromatic compounds. Similarly, peak value at 1319.45 cm⁻¹ was due to C-N stretching of aromatics amines, at 1286.52 cm⁻¹ was due to band of C-H of alkyl halides, while peaks values at 1238.38 cm⁻¹ and 1199.72 cm⁻¹ showed C-N stretch in aliphatic amines (Fig. 2a).

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Fig. 2

(a) FTIR spectrum of P. hydropiper leaves extract; (b) FTIR spectrum of AgNPS synthesized from P. hydropiper leaves extract.

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In addition to this, the FTIR spectrum of AgNP_S (synthesized from the leaves extract) showed slight variations in wave numbers. The missing waves spectra at 1568.13 cm⁻¹ and 1568.55 cm⁻¹ suggested that the rings aromatics compounds might account for the AgNP_S production. A minute change of the wave statistics was perceived at the wider stretch of 3311.78 cm⁻¹ to 3300.20 cm⁻¹, 1049.28 cm⁻¹ to 1022.27 cm⁻¹, 657.73 cm⁻¹ to 655.80 cm⁻¹, while stretches at 2900 cm⁻¹, 1568.13 cm⁻¹, 1556.55 cm⁻¹, 1554.03 cm¹, 1417.68 cm⁻¹ and 1049.28 cm⁻¹ were almost disappeared in the FTIR spectrum of AgNP_S as shown in (Fig. 2b).

3.1.4

3.1.4 Fourier transform infrared spectroscopy (FTIR) of stem extract and AgNPs

In the FTIR spectrum of the P. hydropiper stem extract, absorption peak at 3356.14 cm⁻¹ was due to vibration of stretch N-H having amine and amides groups of proteins, peak value at 2945.30 cm⁻¹ was due to C-H stretch of alkane, while peak at 1635.64 cm⁻¹ displaying N-C bend having amines. Similarly, peaks at 1361.74 cm⁻¹ and 1203.58 cm⁻¹ displaying C-O symmetric stretch of nitro compounds, C-N stretch in aliphatic amines respectively. Moreover, all the three peaks at 1049.28 cm⁻¹, 1029.99 cm⁻¹ and 1199.72 cm⁻¹ showing C-N stretch in aliphatic amines (Fig. 3a).

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Fig. 3

(a) FTIR spectrum of stem extract; (b) FTIR spectrum of AgNPS synthesized from P. hydropiper stem extract.

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Moreover, the FTIR spectrum of AgNP_S (fabricated using P. hydropiper stem extract) showed slight variations in wave numbers. Peaks of synthesized AgNP_S suggested that the aromatics compounds might account for the AgNP_S production. A minute’s change of the wave statistics was perceived at the wider stretch of 3356.14 cm⁻¹ to 3331.07 cm⁻¹, 2945.30 cm⁻¹ to 2916.37 cm⁻¹, 1049.38 cm⁻¹ to 1031.92 cm⁻¹ while, peak at 1556.55 cm⁻¹ almost disappeared in the FTIR spectrum of AgNP_S as shown in Fig. 3b.

3.1.5

3.1.5 Fourier transforms infrared spectroscopy (FTIR) of root extract and AgNPs

In the FTIR spectrum of the P. hydropiper roots extract, the absorption peaks at 3329.71 cm⁻¹, 2900.73 cm⁻¹, 1606.70 cm⁻¹, 1319.58 cm⁻¹ and 1238.38 cm⁻¹ were due to vibration of O-H stretch having carboxylic acid, C-H stretch of alkane, C-C stretching having aromatic compounds, C-N stretching of aromatics amines, and C-H alkyl halides respectively as shown in Fig. 4a.

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Fig. 4

(a) FTIR spectrum of root extract; (b) FTIR spectrum of AgNPS synthesized from P. hydropiper root extract.

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On the other hand, FTIR spectrum of AgNPs (fabricated using P. hydropiper roots extracts) displayed slight variations in wave numbers. Peaks of synthesized AgNP_S suggested that the aromatics compounds might account for the AgNP_S production. Further, a minute’s change of the wave statistics was perceived at the wider stretch of 3329.71 cm⁻¹ to 3311.78 cm⁻¹, 2922.16 cm⁻¹ to 2900.73 cm⁻¹, 1222.87 cm⁻¹ to 1199.17 cm⁻¹ while, peak at 1575.15 cm⁻¹ almost disappeared in the FTIR spectrum of AgNP_S as shown in Fig. 4b.

3.1.6

3.1.6 SEM analysis of the synthesized AgNPs

Scanning electron microscopy (SEM) (Philips SEM, CMC-300 KV model, Netherlands) was used to specify the size and morphology of synthesized AgNP_S. SEM images of the leaves, stem and root synthesized-AgNPs displaying tetrahedron, spherical and oval shapes which were the characteric shapes of AgNPs (Figs. 5-7). Furthermore, the average size of the leaves, stem and root synthesized-AgNPs was determined by Image-J software and it was 32, 77 and 39 nm respectively.

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Fig. 5

SEM images of the leaves synthesized AgNPs nanoparticles (a) X10,000; (b) X20,000; (c) X30,000; (d) X60,000.

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Fig. 6

SEM images of the stem synthesized AgNPs nanoparticles (a) X250; (b) X500; (c) X30,000; (d) X60,000.

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Fig. 7

SEM images of the roots synthesized AgNPs nanoparticles (a) X10,000; (b) X20,000; (c) X20,000; (d) X30,000.

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4 Discussion

In this study, AgNPs were synthesized from P. hydropiper leaves, stem, and roots extracts to determine their bio-catalytic potential to enhance the activity of commercially available antibiotics against MDR bacteria. For this purpose, ecofriendly process of green synthesis was used as prescribed by Shaik et al. (2018). Panáček et al. (2006) also accredited green synthesis method in their study as compared to physical and chemical methods for fabrication of AgNP_S. In the current study, synthesis of AgNPs from the leaves, stem, and roots of P. hydropiper were visually confirmed by noticing the change in color from yellowish green to reddish brown. Furthermore, it was confirmed by UV–VIS spectroscopy, where a characteristics peak (surface plasmon resonance intensification) of synthesized AgNPs was observed at 370 nm. Krithiga et al. (2015) determined absorption peaks at 420 and 440 nm in their study for Clitoria ternatea and Solanum nigrum based AgNPs respectively. Furthermore, they reported that the lambda max of silver was 346 nm (UV spectrum) while the UV–Visible spectrum of synthesized AgNPs found in the range of 360 nm to 470 nm depending on their shape and size. Nasir et al. (2016) also found absorption peak of AgNP_S synthesized from the olive leaves extracts at 419 nm.

Further, FTIR spectroscopy was used for the identification of different functional groups responsible for reducing and stabilizing of silver nanoparticles. The FT-IR spectra were recorded for extract and AgNP_S. It was observed that the FTIR spectra of leaves extract displaying different bands representing different functional groups however, the position of functional groups changed in the FTIR spectrum of leaves extracts synthesized AgNPs. Changes were recorded at 3300.20 cm⁻¹ to 3311.78 cm⁻¹, 1029.99 cm⁻¹ to 1022.27 cm⁻¹, 657.73 cm⁻¹ to 655.80 cm⁻¹ whereas the peaks at 2900 cm⁻¹,15 68.13 cm^−1, 1556.55 cm⁻¹ 1554.03 cm⁻¹,1417.68 cm⁻¹ and 1049.28 cm⁻¹ were almost disappeared. The missing peaks of 1568.13 cm⁻¹, 1556.55 cm⁻¹ and 1568.55 cm⁻¹ in FTIR spectrum of synthesized AgNP_S suggested that the rings aromatics compounds might account for the AgNP_S production (Fig. 2). Khodadadi et al., (2021) conducted similar study and found two new peaks at 1638 cm^-1and 3449 cm⁻¹ confirmed the presence of oxygenated compounds in the FTIR analysis of AgNPs synthesized from the fruit extract of Vaccinium arctostaphylos. In comparison, Maity et al., (2019) also performed FTIR analysis of synthesized AgNPs using mangrove fruit polysaccharide extract and found peaks around 3430 cm⁻¹, 2,910 cm⁻¹, 1600 cm⁻¹, 1450 cm⁻¹, and 1070 cm⁻¹ which confirmed the presence of O-H stretching, aldehydic C-H stretching, C = C group, –COO stretching, and -C-O-C- stretching, respectively (Fig. 2). Another study conducted by Raja et al. (2017) revealed that various functional groups were responsible for reduction of silver nanoparticle in Calliandra haematocephala leaves extracts when analyzed by FTIR spectroscopy.

The FTIR spectrum of AgNPs synthesized from the stem extract displayed changed in the position of functional groups as compared FTIR spectrum of stem extract. Even some of the groups in stem extract FTIR spectrum i.e., 1556.55 cm⁻¹ peak was missed in the FTIR spectrum of AgNPs recommended its production by the rings aromatics compounds (Fig. 3). In comparison, Begum et al. (2009) conducted study on the synthesis of gold (Au) and AgNPs using black tea leaf extracts and also reported the peak bands in the FTIR spectrum of AgNPs at 1697, 1618, 1514, 1332 and 1226 cm⁻¹ which were related with the stretching vibrations for –C = C–C O, –C = C– aromatic, –C–C– aromatic, C–O (esters, ethers) and C–O (polyols), respectively. Balachandar et al., (2019) also found similar changed in the functional group of AgNPs synthesized from the stem extract of Phyllanthus pinnatus. While the FTIR peaks of AgNP_S synthesized from the root extract were recorded at different vibration bands displaying O-H stretch having carboxylic acid, C-H stretch of alkane, C-C stretching having aromatic compounds, C-N stretching of aromatics amines and C-H alkyl halides respectively. However, in comparison to FTIR spectrum of root extract, some of the peaks were missed in the FTIR spectrum of AgNPs suggested that the rings aromatics compounds might account for the AgNP_S production (Fig. 4). In similar report of Logaranjan et al. (2016), the FTIR analysis observed the peak values of AgNPs at 1587.6 cm⁻¹ (C = C groups or from aromatic rings), 1386.4 cm⁻¹ (geminal methyl), and 1076 cm⁻¹ (ether linkages) indicated the presence of flavanones or terpenoids. Following by another study in which AgNPs was synthesized from the root extract of Phoenix dactylifera where, the FT-IR spectrum revealed peak at 3896 cm⁻¹ indicated the binding of Ag with N-H and or O-H group, the peak band at 1395 to 1658 cm⁻¹ indicated C-H stretching vibrations of alkene whereas the peak at 1129 cm⁻¹ revealed the C-H in plane bending of alkenes, alcohols, carboxylic acids, esters, and ethers respectively (Oves et al., 2018).

In addition, SEM analysis was performed for overall appearance of the sample. The particle dimension and its shape were determined by SEM image-j software for the calculation of size and shapes of synthesized AgNP_S. SEM micrograph of AgNP_S showed that these nanoparticles present in various shapes (spherical, tetrahedron and oval) and sizes. The size of the AgNPs synthesized from the extracts of leaves, stem, and roots was 32, 77 and 39 nm respectively. Size comparison demonstrates that the manufactured AgNP_S were of diverse sizes (Figs. 5-7). These results are correlated with study performed by Somashekarappa (2021), where spherical shaped AgNPs with agglomerated appearance were recorded in the SEM image.

Moreover, in the present study, the synergetic effect of silver nanoparticles coated antibiotics were increased against Gram-positive and Gram-negative multidrug resistance bacteria determined by the disc diffusion assay. It was observed that the potency of antibiotics against tested MDR bacteria increased after coating with leaves, stem, and root synthesized AgNPs (Table 1-3). These results agreed with previous studies indicating that AgNPs fabricated by using Aloe extracts had a high inhibitory action against E. coli (42 mm ZI) at low concentration (Zhang et al., 2010). Similar study was also performed by Gogoi et al. (2006) and found that plant extracts synthesized AgNP_S exhibited high inhibitory effects against E. coli (22 mm) and Salmonella (19 mm) while moderate activity was observed against Pseudomonas sp., and Bacillus sp. Furthermore, Boateng and Catanzano (2020) reported that AgNPs coated antibiotics inhibit the bacterial growth at very low concentration than non-coated antibiotics and up till now no side effects were reported for coated antibiotics. Almalah et al. (2019) found similar results against multidrug resistance bacteria using disc diffusion method. It has been stated that the biological activity of AgNPs is constantly related with their antimicrobial properties and hence considered an effective agent, inhibiting the growth of different Gram-positive and Gram-negative bacterial pathogens (Pryshchepa et al., 2020). AgNPs promotes the efficacy of antibiotics towards the site of action in bacteria, as it increases cell permeability that facilitate the intracellular access of antibiotics and hence greatly affect bacterial cells by altering metabolic pathways, DNA, protein synthesis and the disruption of cell wall and cell membrane (Vazquez-Muñoz et al., 2019; Aabed and Mohammed et al., 2021). Vazquez-Muñoz et al. 2019 suggested that enhancement in antimicrobial activity could be attributed to the NPs-antibiotic combined chemical interaction, however, further studies are still needed to justify this phenomenon. Railean-Plugaru et al. (2016) also conducted similar study and found that the growth of E. coli, Salm. infantis and K. Pneumonia was synergistically inhibited by antibiotics coated with AgNPs. Rafińska et al. (2019) also found synergistic effect of synthesized AgNPs with antibiotics against B. subtilis.

5 Conclusions

It was concluded that phytogenic mediated approach for the synthesis of AgNP_S using P. hydropiper extracts was a rapid and effective method. Moreover, synthesized AgNPs displaying excellent synergistic effect to enhance the activity of antibiotics against MDR bacterial species. Absorption peak at 370 nm using UV–VIS spectroscopy confirmed the presence of AgNPs which was further confirmed by FTIR spectrum and SEM analysis. It was concluded that FTIR spectrum at peak values of 1554.03 cm⁻¹, 1556.55 cm⁻¹ and 1568.13 cm⁻¹ confirmed the AgNPs formation while tetrahedron, spherical and oval shape of nanoparticles in SEM images confirmed the characteristic shape of AgNPs. In addition to this catalytic activity of AgNPs to enhance the activity of commercially available antibiotics against MDR bacterial isolates was also determined using disc diffusion assay. It was concluded the potency of antibiotics against MDR bacteria increased after coating with synthesized AgNPs i.e., the potency of Ceftazidime and Ciprofloxacin increased up to 450% and 500% against Bacillus respectively while, the potency of Gentamicin, Vancomycin and Linezolid increased up to 150%, 200% and 58% against Bacillus, Staphylococcus, and Proteus species respectively. Additionally, it is recommended that further research work is needed to use other metals (zinc, gold, and iron) against resistant bacterial and fungal pathogens. While in vivo models of mice should be used to observe phytogenic mediated AgNPs activity against bacterial pathogens.

CRediT authorship contribution statement

Ghadir Ali: Conceptualization, Methodology. Aftab Khan: . Asim Shahzad: Formal analysis, Writing – original draft. Aiyeshah Alhodaib: Writing – review & editing. Muhammad Qasim: Writing – review & editing. Iffat Naz: Formal analysis, Writing – original draft, Writing – review & editing. Abdul Rehman: Conceptualization, Methodology, Supervision.