Green synthesis of silver nanoparticles by Rivina humilis leaf extract to tackle growth of Brucella species and other perilous pathogens.

Novel approaches are obligatory to treat chronic intracellular bacterial infectious diseases like Brucellosis specifically, are very complicated to deal with. The aim of the study to take upon nanotechnology approach to exploit the efficacy of the synthesized nanoparticles, to overcome barriers for treatment of Brucella species and other pathogens. Present study used Rivina humilis extract as reductant of silver ions for synthesis of silver nanoparticles for the first time. Rh-AgNP's was characterized by UV-visible spectroscopy, DLS, FT-IR, SEM, EDS, TEM and XRD. Radical scavenging, antibrucellosis, bactericidal activity was evaluated. Clinical application was assessed by Rate of haemolysis, fibrinolytic and Hemagglutination activity. UV-visible spectrum of synthesized Rh-AgNP's showed maximum peak at 440 nm indicating the formation of nanoparticles. TEM showed that the average particle size of nanoparticles 51 nm with spherical shape, DLS depicted monodisperse state in water; EDS confirmed the presence of silver metal. Rh-AgNP's exhibited potential antibrucellosis activity against B. abortus, B. melitensis and B. suis effective inhibition at 800 μg/mL. The bio-compatibility of Rh-AgNP's was established by rate of haemolysis, hemagglutination and fibrinolytic activity. For the first time it has been proved that Rh-AgNP's have efficacy as antimicrobial agent with potential application in the biological domain.

Introduction

Brucellosis is a neglected remerging bacterial zoonotic infectious disease, that distresses domesticated animals, wild animals and humans. The overall occurrence of human brucellosis is estimated to go beyond 800,000 cases annually, of which 40% leading to chronic infection (Kirk et al., 2015). Preferred treatment specified for human brucellosis caused by Brucella melitensis field strains is a combination of antibiotics (Grilló et al., 2006). However, due to emerging resistance there is high rate of disease relapse and treatment failure, the major concern for the treatment of Brucellosis is that the disease is pathognomonic (Raghava et al., 2017). Thus, new approach is becoming obligatory for brucellosis treatment.

Nanotechnology advancement is promising approach leading to varied scope of applications, has a great potential to deliver the therapeutic agents to the target in the medical treatments (Vilchis-Nestor et al., 2008). Nanotechnology is the method of alteration /assembly of the atoms and molecules in which the resulted structures and material leads to different and novel properties (Hamedi et al., 2017, Pishahang et al., 2018). Lately, the progress of efficient green chemistry methods like plant based synthesis of nanoparticles has received more consideration as a substitute approach to synthesize the metal nanoparticles by minimizing the toxic and hazardous waste materials by establishing sustainability (Selvan et al., 2018, Soshnikova et al., 2018). Medicinal plants have always been vital sources for new drug discovery has they readily synthesize substances themselves to defend/ inhibit against herbivores and microorganisms (Aboaba et al., 2006). World’s 80% population mainly relies on phytoproducts for health care and also for the clinically required drug sources (Werka et al., 2007). Hence in our study we applied nanotechnology for the synthesis of potential nanoparticles by using plant source Rivina humilis. R. humilis belongs to family phytocaccaceae is popularly known as pigeon berry grows well in tropical America and Africa and now widely naturalized in indo-malaysia and pacific islands. Seeds are propagated through dispersal by birds (Tseng et al., 2008, Harsha et al., 2012). Even though many researchers have synthesized silver nanoparticles using plants very scarce have shown antibrucellosis activity. In the present study, Rh-AgNP’s were synthesized, were further characterized using different analytical instruments. In addition, the antioxidant efficacy and biocompatibility was assessed by rate of haemolysis, haemagglutination and fibrinolytic activity.

Materials and methods

Bacterial reference strains

Brucella strains were procured from IVRI, Uttar Pradesh, India. Other bacterial strains were obtained from MTCC, Chandigarh, India and ATCC. Staphylococcus aureus (96), Escherichia coli (1610), Bacillus cereus (430), Shigella flexneri (1457), Bacillus subtilis (6939) and Enterobacter aerogenes (13048) strains.

Preparation of the plant extracts

The plant Rivina humilis was collected from the Western Ghats of Karnataka, India and identified by the former Dr. G. R. Shivamurthy, Professor, Department of Botany, University of Mysore, Mysuru, India (Fig. 1). Leaves were shade dried for about twenty days and ground into fine powder. Fifty grams of finely powdered R. humilis material was used to get the aqueous extract in 500 mL and were filtered with Whatman No.1 filter paper and collected in collection tubes and refrigerated.

Fig. 1

Plant material Rivina humilis collected from Western Ghats region of Karnataka, India.

Synthesis of silver nanoparticles

Ten ml of leaf plant aqueous extract was added to 90 mL of freshly prepared 1 mM silver nitrate solution (AgNO3). The mixture was kept under direct sunlight the gradual colour change was noted as an indication of Rivina humilis silver nanoparticle (Rh-AgNPs) formation (Brahmachari et al., 2014, Rastogi and Arunachalam, 2011)

Characterization of Rh-AgNP’s

UV–Visible Spectroscopy

Absorption wavelength range of nanoparticles was confirmed using spectrophotometer (Hitachi U 3000, Japan). Exactly, 1 mL of the diluted supernatant of the synthesized nanoparticles were placed in a quartz cuvette with a 1 cm path length and inserted in a UV–Vis spectrophotometer to obtain the UV–Visible spectra of the sample in the 300–800 nm wavelength range against water as the reference. The surface Plasmon resonance of Rh-AgNP’s was observed.

Dynamic light scattering (DLS) and zeta potential

The Nanotrac wave (USA) particle size analyzer was used to evaluate the particle size distribution and stability of Rh-AgNP’s. The analyser gives the size measurement of the synthesized particle, the state of aggregation and zeta potential value of the synthesized Rh-AgNP’s.

Fourier Transform Infrared Spectroscopy (FT‑IR)

The functional groups on the Rh-AgNP’s were studied by the FT-IR analysis. The Rh-AgNP’s synthesized were analysed by Bruker, Massachusetts, (USA) to reveal the distribution of biological macromolecules at spectrum ranging from wave number 400 to 4000 cm−1. The number of scans and spectral resolution were set to 24 and 4 cm − 1, respectively.

Scanning electron microscopy (SEM) of Rh-AgNP’s and Energy Dispersive X-ray Spectroscopy (EDS) of Rh-AgNP’s

A drop of biosynthesized Rh-AgNP’s was placed on a carbon-coated copper and allowed to air dry. After drying the image captured using HITACHI (S-3400 N, Japan) at 10 kV acceleration voltages. The Energy Dispersive X-ray Spectroscopy (EDS) analysis was carried out using HITACHI (Noran System 7, USA) system attached to SEM for the detection of elements on the sample in the AgNPs.

Transmission electron microscopic (TEM) for Rh-AgNP’s

For morphological analysis of Rh-AgNP’s, transmission electron microscopy (TEM) was performed. A drop of AgNPs solution was placed on a copper grid coated by an amorphous carbon film and dried under vacuum for further observation. More characteristics of the AgNP’s including their morphology and size were identified using a high-resolution transmission electron microscope (JEM-1230, Tokyo, Japan) at an accelerating voltage of 100.0 kV.

X‑ray diffraction (XRD) analysis

The dry powder of the silver nanoparticles was analyzed by XRD. The diffracted intensities were recorded from 20 to 80° at 2 theta angles by using X-ray diffractometer XRD-6000 (shimadzu, Rigaku Corporation, Tokyo, Japan). The obtained peaks were compared with the standards. The size of the particle was calculated using the Scherer’s formula.

DPPH radical scavenging activity

The radical scavenging activity of the synthesized nanoparticles was estimated using the stable DPPH radical scavenging assay described by Odeyemi et al. (2015) with slight modifications. DPPH solution was mixed with synthesized Rh-AgNP’s (different concentration) and incubated for 30 min. The absorbance was measured at 517 nm. The scavenging percentage of DPPH was calculated using the formula

Where, AS- absorption of the test sample and AC: DPPH solution absorbance without the test sample. All the experiments were carried out in triplicates and repeated twice.

In vitro antimicrobial efficacy of Rh-AgNP’s against Brucella species

The bacterial strains were subjected to disc diffusion method according to Ghanwate et al., 2016 with slight modifications to deduce the Rh-AgNP’s susceptibility. Antimicrobial study was carried out with the reference bacterial strains obtained from Indian Veterinary Research Institute, Izatnagar, India. The Brucella culture suspension was prepared and 1x106 CFU/mL cells was inoculated on to Muller Hilton agar supplemented with 1% sheep’s blood, then sterile disc (6 mm) was loaded 5 μL of different serial dilutions of Rh-AgNP’s (25, 50, 100, 200, 400 and 800 μg/mL). The plates were incubated at 37 °C under 7%– 10% CO2 for 72 h. The zone of growth inhibition was measured (Ghaderkhani et al., 2019). The test was performed in triplicates and repeated thrice.

Bactericidal effect of Rh- AgNPs on other perilous pathogen

Disc diffusion method was carried out according to Manukumar et al. (2017) with slight modifications to check the Rh-AgNP’s efficacy to inhibit the growth of perilous pathogens. Gram positive S. aureus, B. cereus, B. subtilis and Gram negative E. aerogenes, E. coli, S. flexneri pathogens. The bacterial suspension was prepared from the overnight culture and 1x106 CFU/mL cells inoculated on to nutrient agar, then sterile disc (6 mm) was loaded with 5 μL of different serial dilutions of Rh-AgNP’s (25, 50, 100, 200, 400, 800 and 1200 μg/mL). Sterile distilled water and Streptomycin (10 μg) served as negative and positive control, respectively. The plates were sealed and incubated at 37 °C for 24 h to examine zone of inhibition. Assay was performed in triplicates and repeated thrice.

Biocompatibility assay

Rate of haemolysis

Different concentrations (1, 5, 10, 20, 40, 80, 100, 200 400, 800 and 1200 μg/mL−1) of Rh-AgNP’s and freshly collected human blood was mixed and incubated at 37 °C for 30 min. Centrifuged, supernatant was collected and absorbance was measured at 545 nm (Manukumar et al., 2017, Huang et al., 2016). Assay was performed in triplicates and repeated thrice. Haemolysis rate was determined by:

Fibrinolytic activity

Citrated sheep plasma 100 μL and 0.25 M CaCl2 30 μL was mixed to form a soft clot fibrin at 37 °C for 3 h, then washed, resuspended with PBS (pH 7.4) and mixed with 500 μL Rh-AgNP’s in Tris-HCl buffer (pH 8.5) incubated for 2 h at 37 °C. Finally, 750 μL of 0.44 M TCA was added to cause the precipitation of the undigested clot according to protocol of Harish et al. (2015) Centrifuged and protein content in the supernatant was determined using Folin-Ciocaltues reagent to know the fibrinolytic activity.

Hemagglutination assay

100 µL Phosphate Buffer Saline of pH 7.4 and 100 µL Rh-AgNP’s were added into microtiter plate and mixed different concentration from 1 to 1200 µg/mL were added till column 12. Then 100 µL of red blood cells was added to each well and incubated at room temperature for 30–60 min (Amruthraj et al., 2015).

Statistical analysis

ORIGIN, SPSS software (version 15) used for statistical analysis and T-test to compare the groups. P-value of < 0.05 was considered significant. All the tests were done in triplicates and repeated thrice.

Results and discussion

Synthesis of Rh-AgNP’s

Synthesis of Rh-AgNP’s arising from reduction of AgNO3 by constituents of Rivina humilis leaf aqueous extract was indicated by an observable change in colour to a reddish brown Similar observations have been presented by other investigators using plant extracts as sources of reductants of ionic silver into nanoparticles. Upon exposure of Ocimum sanctum extract and silver nitrate solution in the sunlight, a dark brown-reddish colour mixture was witnessed (Brahmachari et al., 2014). Use of P. longum by the Jayapriya et al., (2019) adduced similar observation.

Characterization of nanoparticles

UV–visible spectroscopy

Plasmonic metallic nanoparticles possess fascinating optical properties. Therefore, 300–800 nm UV–visible spectroscopy range can be used to gather useful information about metal nanoparticles within 2 to 100 nm dimensions’ interval (Singh et al., 2016). Typical of Ag-NPs having λmax values in the visible range of 440 – 460 nm (Thangaraju et al., 2012, Manukumar et al., 2017, Muthuraman et al., 2019). The position of a Surface Plasmon Resonance (SPR) peak arising from oscillation of conduction band electrons has a bearing on the nature, shape and size of AgNPs (Bastús et al., 2015). The SPR peak for Rh-AgNP’s was located 440 nm (Fig. 2).

Fig. 2

UV- Vis Spectra of Synthesized Rh-AgNP's.

DLS and zeta potential

Dynamic light scattering size analysis gave an average particle diameter of 51 nm. The distribution is displayed in Fig. 3. The Rh-AgNP’s had a zeta potential of +4.3 mV. This gave moderate stability to the biosynthesized nanoparticles due to electrostatic repulsion. Surface charge is an important determinant of nanoparticle interactions in biological systems. Chinnappan et al. (2018) have reported similar results for the nanoparticles synthesized by the Bauhinia purpurea.

Fig. 3

The hydro dynamic size distribution determined for Rh-AgNP's by dynamic light scattering(DLS); The synthesized Rh-AgNP's analysed for the nanoparticle size distribution.

FT-IR spectra of Rh-AgNP’s

Fourier Transform Infrared Spectroscopy was used to examine the molecular species present in R. humilis and which amongst them participated in stabilization of Rh-AgNP’s. The broad peak at 3400 cm−1 from the aqueous extract which could be assigned to water O-H group stretch. The Rh-AgNP’s spectrum had peaks at 1739 cm−1 which attributes to C = 0 stretching frequency, 1638 cm−1 which corresponds to C = C alkane chain, 1367 cm−1 peak corresponds to CH3 chain split umbrella mode, 1216 cm−1 attributes to C-O-C vibration peaks and 625 cm−1 corresponds to the SO42- ions which occurs at 580–660 cm−1. (Fig. 4). Reduction of ionic silver and formation of Rh-AgNP’s was most probably effected by the aforementioned functional groups arising from the plant extract. while by –OH group stretching broad band at 3349 cm − 1 confirms participation of molecules in R. humilis extract. Similar results were reported by the Devaraj et al., 2013, Goswami et al., 2018.

Fig. 4

FT-IR spectra of Rh -AgNP’s.

Scanning electron microscope and Energy Dispersive X-ray Spectroscopy

Scanning electron microscopy assessment of Rh-AgNP’s nanoparticles size and morphological characteristics at the nanometer to micrometer scale is displayed in Fig. 5B and C. It shows approximate spherical nanoparticles in clusters. Energy Dispersive X-ray Spectroscopy (EDS) spectrum displays the elemental composition of the nanocrystals with presence of an identifiable silver peak. Similar study was reported by Hyllested et al., 2015. larger silver particles may be due to the aggregation of the smaller ones.

Fig. 5

Transmission Electron Microscopy (A), Scanning Electron Microscopic image (B) and Energy Dispersive X-ray Spectroscopy spectrum (C) of biosynthesized Rh-AgNP’s from R. humilis. The TEM analysis indicates the spherical shape of the nanoparticles with the average size of 69 nm. The SEM of Rh-AgNP’s observed results showed that, the nanoparticles were spherical in shape. The energy dispersive X-ray spectroscopy (EDS) spectrum (B) of Rh-AgNP’s showing presence of silver.

Transmission electron microscopic (TEM) was used to complement SEM in obtaining the finer details of biosynthesized R. humilis silver nanoparticles size and shape (Fig. 5A). The particle had an average sized of 51 nm. findings were similar to other researchers (Singh et al., 2016, Mittal et al., 2013).

X- ray diffraction spectral analysis

X- ray diffraction spectrum of the synthesized silver nanoparticles is given in Fig. 6. The peaks at position 38.07, 44.22, 64.39 and 77.30 in the plot of Intensity count verses 2θ correspond to (1 1 1), (2 0 0), (2 2 0) and (3 1 1) hkl lattice planes of face center cubic (fcc) structure of metallic silver respectively. This confirms that the nanoparticles predominantly were consisted of elemental silver. There were no other conspicuous peaks to signify distinguishable contamination. The results were similar to reports of Anandalakshmi et al. (2016) using Pedalium murex to synthesize silver nanoparticles and Siddiqui et al. (2018) using honey has a reducing agent to synthesis silver nanoparticles.

Fig. 6

XRD pattern of Rh-AgNP’s- The Synthesized Rh-AgNP’s was analysed for crystalline structure of the Rh-AgNP’s. The obtained results peak positions were compared with standard. The results showed 2θ peak values at 38.07, 44.22, 64.39 and 77.30 corresponds to (1 1 1), (2 0 0), (2 2 0) and (3 1 1) respectively.

DPPH radical scavenging activity of Rh-AgNP’s

DPPH activity of silver nanoparticles is measured quantitatively by changes in absorbance (Ajayi & Afolayan, 2017). The dose dependent radical scavenging activity significantly elevated with the concentration of Rh-AgNP’s (Fig. 7). The experiment was conducted thrice. The significant (p ≤ 0.05) results was observed in the (Fig. 7). The Scavenging activity of Rh-AgNP’s from 0 to 50% with the IC50 value of 100 μg/ml compared to standard ascorbic acid which was taken as a control showed RSA of 72% at 50 μg/ml) (Manukumar et al., 2017, Elblbesy, 2016). Earlier reports of Sriranjani et al., 2016, Kharat and Mendhulkar, 2016 revealed that plant mediated nanoparticles have enhanced the antioxidant activity.

Fig. 7

DPPH radical scavenging activity of Rh-AgNP’s; Results show dose dependent radical scavenging property of Rh-AgNP’s and exhibited 50% inhibition at the concentration of 100 μg/mL concentration.

Antibrucellosis activity by the Rh-AgNP’s

Different dilution concentration of the synthesized Rh-AgNP’s were assessed by disc diffusion method and compared with the negative control in dose dependent manner for different Brucella cultures. The inhibition zone was observed on the plates and compared with the antibiotics (Fig. 8). The highest antibacterial action exhibited at concentration of 800 ug/mL compared to the standard antibiotic. Similar study was carried out by synthesizing the nanoparticles against B. melitensis by Alizadeh et al., 2013.

Fig. 8

Antibrucellosis activity by the Rh-AgNP’s - Zone of Inhibition from Rh-AgNP’s at different concentration 10, 25, 50, 100, 200, 400, 800 μg /mL was tested against B. abortus, B. melitensis and B. suis, Compared to standard antibiotics SM- Streptomycin. The MIC concentration was found to be 800 μg/mL compared to standard SM (10 μg). All experiments were done in triplicates to assess the veracity of the results.

Currently, due to inappropriate use of antibiotics, drug resistant microorganisms are the foremost concern globally. Different concentration of synthesized Rh-AgNP’s was used to assess bactericidal efficacy against different bacteria which resulted in the dose dependent bacterial inhibition. Rh-AgNP exhibited significant inhibition zone against bacteria compared to antibiotics (Table 1). Gram positive S. aureus (11 ± 0.1 mm), B. cereus (12 ± 0.06 mm), B. subtilis (10 ± 0.09 mm) and Gram negative E. aerogenes (12 ± 0.06 mm), E. coli (12.50.1 ± mm), S. flexneri (13 ± 0.02 mm). Dehghanizade et al., 2018. reported the Anthemis atropatana AgNPs showing activity against S. aureus, S. pyogenes, P. aerugenosa and E. coli. Moodley et al., 2018 reported AgNP’s activity against E. coli, E. facealis, K pneumonia, P. aeruginosa and E. coli. Similar report has been documented by Manukumar et al., 2017, Alfuraydi et al., 2019.

Table 1

Inhibition zone (mm) showing antibacterial activities of the nanoparticles derived from R. humilis with the standard drug Streptomycin against bacterial test organisms.

Concentration in µg/ml	25 µg/ml	50 µg/ml	100 µg/ml	200 µg/ml	400 µg/ml	800 µg/ml	1200 µg/ml	SM Std 10 µg
B. cereus	3.4 ± 0.04	4.7 ± 0.04	6 ± 0.02	8.9 ± 0.2	10.5 ± 0.06	11.7 ± 0.1	12 ± 0.06	10 ± 0.2
S. aureus	–	3 ± 0.04	4 ± 0.05	5 ± 0.02	7 ± 0.03	8.5 ± 0.5	11 ± 0.1	12.7 ± 0.09
B. subtilis	–	–	3 ± 0.04	4 ± 0.05	6 ± 0.07	8 ± 0.2	10 ± 0.09	12.2 ± 0.4
E. aeroginosa	3 ± 0.04	4.5 ± 0.04	6 ± 0.02	7 ± 0.06	9.5 ± 0.03	10.8 ± 0.06	12 ± 0.06	12 ± 0.06
S. flexineri	4.6 ± 0.04	6 ± 0.01	7.5 ± 0.03	8.5 ± 0.03	10 ± 0.2	11 ± 0.09	13 ± 0.02	13.4 ± 0.1
E. coli	4 ± 0.04	5 ± 0.05	7.5 ± 0.04	8.4 ± 0.03	9.5 ± 0.2	10.5 ± 0.06	12.5 ± 0.1	11.8 ± 0.06

Haemolysis assay

The Rh-AgNP’s haemolysis activity (<5%) on erythrocytes at different dilutions was evaluated (Fig. 9). Even the small difference in osmolarity and physical change in blood causes their haemolysis resulting in the release of haemoglobin and measured by colorimetric assays. Results indicated that upto 800 μg/mL of Rh-AgNP’s can be used for the biological application (Kim and Shin, 2014, Huang et al., 2016). Similarly, Khan et al. (2012) also reported that the R. humilis berry juice does not affect the growth and normal biochemical homeostasis hence it is safe to consume without any adverse effect. Selvakumar et al., 2018 have reported that AgNPs of Acalypha hispida have similar biocompatibility activity.

Fig. 9

The biocompatibility on RBCs. The Rh-AgNP’s samples treated with citrated blood for different concentrations. Rh-AgNP’s did not show any effect up to 400 ug/mL, but 800 ug onwards shows a significant effect on RBCs to cause haemolysis. Before and after centrifugation shows an effect of Rh-AgNP’s for haemolysis, saline and distilled water used as a negative and positive control.

Fibrinolytic assay

Rh-AgNP’s depicted plasmin-like activity by considerable fibrin clot hydrolysis in a dose-dependent manner (Fig. 10A). Stability of nanoparticles is very significant to for the biological application. Rh-AgNP’s in the presence of chelator EDTA and EGTA did not show any defect in blood coagulation and rate of re-clarification of citrated plasma (Fig. 10B) (Lateef et al., 2016).

Fig. 10

Fibrinolytic and hemagglutination assay- The lysis of fibrin clot and clotting time of citrated plasma at various concentrations of Rh-AgNP’s is depicted in (A and B) the hemagglutination assay(C) depicted that the agglutination of red blood cells by Rh-AgNP’s.

Hemagglutination assay revealed that the Rh-AgNP’s at a concentration of 1200 and 800 µg/mL agglutinated the RBC cells (Fig. 10(C)). Below 400 µg/ml dilutions did not show agglutinating activity. It increases efficacy of silver nanoparticles applications in biological domain. From the present study we can say that, the Rh-AgNP’s are biocompatible with the red blood cells. Similar results were observed for Capsaicin-capped silver nanoparticles by Amruthraj et al., 2015. Hence, the Rh-AgNP’s can be used has a potential drug after the invivo studies. Based on the above observation the plausible molecular action of green synthesized silver nanoparticle against bacteria (Fig. 11).

Fig. 11

Plausible molecular mechanism of the Green synthesized silver nanoparticles against bacteria.

Conclusion

The silver nanoparticles synthesized by R. humilis is cost effective, efficient and environmental friendly process. Morphologically distinct silver nanoparticles synthesized were characterized. Formation of bio-functionalized spherical AgNP’s with a size mean of 51 nm was confirmed using UV–VIS, FTIR, SEM, EDS, and TEM. XRD and DLS, Zeta potential confirms the stability of the formed AgNPs. Rh-AgNP’s showed a dose-dependent radical scavenging activity. Antibacterial results show that the Rh-AgNP’s were inhibiting the growth of Brucella species. The biocompatibility of synthesized Rh-AgNP’s by rate of haemolysis, Fibrinolytic activity and Hemagglutination assay showed the efficacy of synthesized Rh-AgNP’s in the biological domain. This study revealed that the Rh-AgNP’s can be used in biological application.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.