The application of extracts of various medicinal plants as a traditional medicine for varieties of human’s illnesses has been very common among many nations of the world since centuries. The vast majority of the rural people in the world completely rely on locally available plants with medicinal values. Ethiopia is one of the six centers of biodiversity in the world. Traditional medicine performs a tremendous role in Ethiopia, in which massive majority of Ethiopia’s population lives in rural regions with little access to health offerings. In recent years, numerous Ethiopian medicinal plants have been validated in a scientific empirical framework through phytochemical analysis and subsequent bioassays. It is understood that 25% of the modern medicines enters the market utilizing either directly or indirectly traditional medicinal plant parts. However, pharmaceutical manufactures use about 60% of these local plants. The medicinal plant species are used to treat number of diseases . Echinops Sp., a 2nd ranked medicinal vegetation in Ethiopia, is valued primarily for its root parts and its medicinal uses are documented in the ancient medico-religious pharmacopoeia . The roots of Echinops Sp., have been used in the preparation of medicines against migraine, mental illness, heart pain, leprosy, kidney disease, malaria and syphilis. It grows up to a height of 1.2 M with leafy stem. In order to explore the influence of biomolecules of medicinal plant on the eco-friendly synthesis of Ag NPs and to evaluate their cumulative antibacterial effect, Echinops Sp. plant has been chosen for our work.
The research on the fabrication of plant mediated Ag NPs for antimicrobial applications has gained significance in the recent years. The green Ag NPs have been used for photocatalytic, electrocatalytic, industrial dye degradation, nano medicinal, environmental and catalytic applications since many decades . These Ag nanoparticles such as nanoparticles, nanocrystals, nanorods, nanotubes, and nanosheets exhibit versatile properties and hence found to exhibit inhibitory activity against many microorganisms and bacterial strains .
A very few medicinal plants such as Azadirachta indica , Dioscorea bulbifera , Caesalpinia bonducella , Hagenia abyssinica , Mentha longifolia , Cocos nucifera , Syzygium cumini , and Amazon Fruits  have been applied to synthesize silver, gold , copper and their oxides  in the recent past for multifunctional applications. The commonly adopted synthetic routes for nanoparticles includes green synthesis , sol-gel synthesis , biosynthesis , microwave assisted solvent-free synthesis, micro emulsion method, etc.
Despite all advantages, no significant work has been performed on using medicinal plant root extracts to reduce and cap silver ions towards the synthesis of Ag NPs in Ethiopia for biomedical, photocatalytic, electrochemical sensor and antibacterial applications. Thus, the present work was attempted for the eco-friendly green synthesis of Ag NPs using medicinal plant Echinops Sp. plant root extract at low temperature to investigate synergistic influence of phytochemicals and Ag NPs on few selected bacterial strains. The synthesised Ag NPs have been characterised by all the advanced techniques and investigated for antibacterial properties.
Chemicals and reagents
The chemical compounds and reagents, AgNO3, C2H5OH, Mueller-Hinton agar solution, 0.5 McFarland standard, chloramphenicol discs, dimethyl sulfoxide, indigo carmine and malachite green dyes used during the experiments were of analytical grade (procured from Merck company).
Collection and authentication of plant materials
Echinops Sp. plant roots have been collected from the agricultural plots of Wondo Genet Agricultural Research Centre, Oromia, Ethiopia, after accomplishing the sector inspection. The authentication of Echinops Sp. (Code EB005) plant roots was conducted at Herbarium, Addis Ababa University.
Preparation of aqueous plant root extract
The roots of Echinops Sp. were washed repeatedly with water followed by rinsing with distilled water and then shade dried for 2 weeks to remove moisture content from the roots. The procedure followed is similar to the synthetic procedure reported in our earlier work  and thus not discussed in detail here. The prepared Echinops Sp. plant root (EcS-PR) extract was stored at 4 °C for future experiments.
Green synthesis of EcS-Ag NPs
100 mL of EcS-PR extract was mixed with AgNO3 (400 mL of 0.5 M solution) in a 500 mL flask which was incubated for 24 hr at room temperature. The obtained brownish colored solution was centrifuged for 30 min at 8000 rpm to get black Ag NPs. These EcS-Ag NPs (Fig. 1) were washed, dried, ground and stored .
X-ray diffractometer (Shimadzu-50 kV and 20 mA with Cu Ka radiation, λ = 1.541 Å) was used for crystal structure analysis. Chemical bonding interactions were explored using Shimadzu FTIR spectrophotometer (IR Affinity 1S). Shimadzu UV-visible spectrophotometer was utilized to evaluate the optical properties. Morphological analysis of the samples was conducted using JEOL, JEM-2100 (accelerating voltage up to 200 kV, LaB6 filament) EDS-1.5 Å TEM resolution. The evaluation of interplanar spacings (IPS) of lattice fringes was done using Gatan Digital Micrograph software ,.
Method of antimicrobial evaluation
All the antibacterial tests were conducted at Oromia Regional Laboratory, Adama, Ethiopia. Agar disc-diffusion method was adopted for the evaluation of in-vitro antibacterial properties of EcS-Ag NPs verses S. aureus, E. coli, P. aeruginosa, and E. aerogenes. The effectively growing bacterial cultures were dispersed on Mueller-Hinton Agar (MHA) plate (turbidity was adjusted with Tryptone Soy Broth, TSB to match 0.5 McFarland standard) The extract of the NPs was prepared with four different concentrations in Dimethyl Sulfoxide (DMSO). Four concentrations (6.25, 12.5, 25 and 50 µg/µl) of the synthesized NPs were added to the respectively labelled discs. The antibiotic discs of 6 mm diameter were applied to agar surface using forceps with gentle pressure and then impregnated with the dissolved extract. The positive and negative controls taken were chloramphenicol and DMSO, respectively. The plates were incubated at 35 ±2°C in an ambient air incubator for 18-24 hours. The zone of inhibition was measured to the nearest millimeters (mm) using a ruler and recorded for all the samples.
RESULTS AND DISCUSSION
Synthesis of EcS-Ag NPs
The synthesis of EcS-Ag NPs was successfully performed using the EcS-PR extract to reduce and cap silver ions to silver. The alkaloids, tannins, flavonoids and terpenoids were found in EcS-PR extract during the phytochemical analysis. The list of the phytochemicals present in the extract are as given in Table 1.
Three steps involved in the origin of NPs include: reduction of metal ions, formation of cluster and growth of nanoparticles. The biomolecules of the extract basically behave as antioxidants. In addition, the enzymes of EcS-PR extract also assist silver ions to get reduced and form protein capped silver NPs. It is worth noting that that phytochemicals act as the organic ligands assisting the reduction of silver ions to silver NPs. In addition, these compounds also influence the size of the nanoparticle as reported by the earlier researchers .
Characterization of EcS-Ag NPs
The EcS-Ag NPs were characterized by employing UV-visible, UV-DRS, FT-IR, XRD, SEM, EDXA, TEM, HRTEM and SAED techniques.
The UV-visible absorbance spectrum of instantaneously synthesised EcS-Ag NPs revealed λmax of 452 as shown in Fig. 2a, just after 10 min of mixing plant extract with silver nitrate solution. The absorbance spectrum recorded after 20, 30, 40, 50 and 60 minutes of forming homogeneous mixture exhibited identical absorbance bands at λmax of 454 nm (Fig. 2b).
The enhanced absorbance in the consecutive bands clearly confirms the increased concentration of nanoparticles. As the time passes on, the reduction of silver ions is followed by nucleation of small cluster of silver atoms to form nanoparticles in the presence of biomolecules of plant extract that may act as a reducing and stabilising agent.
Almost identical results were observed during the analysis of the synthesized Ag NPs using the Lustrum lucidum leaf extract  and Persea americana seed extract . The surface plasmon absorbance presents a set of different λmax values for NPs synthesised using different plant extracts which is possibly due to morphological features of the NPs. The UV-Vis-DRS spectrum was recorded (Fig. 2c) for NPs. For the most part of the plot, the Kubelka - Munk capacity F(R) is modified over the diffused reflectance into equivalent absorption coefficient and utilized for investigating the powders as given by equation (1),
ere R is the reflectance, and F(R) is Kubelka-Munk function. The optical energy gap was calculated using Tauc relation as in equation (2),
F(R) hn = A (hn –Eg)n(2)
where n = 1/2 and 2 are for direct and indirect transitions, respectively, for indirect band and direct band . The optical energy gap was calculated using Tauc relation as in equation (2) thereby giving direct band . The Tauc plot (Fig. 2d) was utilized to compute the band gap energy of EcS-Ag NPs. The energy gap (Eg) of 2.22 eV was deduced for EcS-Ag NPs.
The XRD analysis was executed to explore the in-depth details of crystal structure of EcS-Ag NPs. The XRD spectrum of EcS-Ag NPs (Fig. 3) demonstrates a total of 4 prominent peaks. These peaks with 2θ values of 38.14o, 44.7o, and 64.50o, and 77.42o conform to 111, 200, 220 and 311 crystal lattice planes of silver (fcc structure) and the diffraction data is in compliance with the data of ICPD card No. 004-7383 (Fm3m) .
The average crystallite size (D) obtained for EcS-Ag NPs was deduced to be 21.47 nm respectively by using Scherrer’s formula .
D= Kλ / βcosθ (3)
where, K is a constant, λ is wavelength of X-rays and β is full width at half maximum (FWHM).
The FTIR spectroscopy was helpful in revealing the bonding features of both EcS-PL extract and EcS-Ag NPs. The intense peaks, shown in Fig. 4, at 3395 cm-1 correspond to–OH stretching frequencies. The peak at 1718 cm-1 arises due to C=O vibration of ketonic groups. The small peak at 2925 cm−1 was correlated to the alkane C−H stretching mode. The vibration of −COO group of carboxylic acid was found to appear at 1449 cm−1. A moderately intense band at around 1035 cm-1 confirms the stretching of C-O-C bond. The presence of prominent peaks at 3395 cm-1, 1718 cm−1, 1449 cm−1, 1035 cm-1 and 601 cm-1 in the FTIR spectra of both plant root extract and NPs clearly indicate the presence of bioactive molecules around the NPs. These bioactive molecules have shown a sizable function in the nucleation and growth of EcS-Ag NPs. The bending vibrations of Ag–O–H bonds resulted in a small peak at 601 cm−1, possibly due to Ag–O bond. The FTIR spectral inspection confirmed the presence of phytochemicals (phenolics, tannins, glycosides and proteins) in EcS-PR extract and their roles as a reducing and stabilizing agent during the synthesis of EcS-Ag NPs. Especially the phenolics were reported to be strong candidates for binding with silver NPs .
The electron microscopy was applied to investigate the morphological features of EcS-Ag NPs. The SEM micrographs of NPs in Fig. 5a-b depict varieties of nanoparticles with diverse shapes and sizes.
The presence of mixed type of NPs is possibly due to the nature and amount of capping agents around the particles. The average grain size of Ag NPs was found to be in the range of 5–50 nm . The presence of less agglomerated Ag NPs due to high surface area yielded small size particles. The Ag NPs were found to be as small as 6.5 nm, clearly confirming the efficient role of biomolecules as stabilizing agents preventing the growth of silver clusters to bigger NPs.
The EDAX analysis revealed the elemental composition of the EcS-Ag NPs as depicted in Fig. 5c. The elements, Ag, C, O, Si, S and Cl have been identified in the spectrum and additional peaks appeared due to the usage of standard during the analysis as well as impurities.
In order to gain deep insight about the shape, size and structure of the EcS-Ag NPs, TEM-HRTEM-SAED technical micrographs and patterns have been well utilized . As shown in Fig. 6a-d, the HRTEM images (at different magnifications) affirm that as-synthesized EcS-Ag NPs are mostly spherical, regular as well as irregular in geometry. This is possibly due to the dual role (reducing and stabilizing) played by the bioactive molecules of EcS-PL extract.
The existence of nanosized particles (6.5 nm) approves the efficient role of bioactive components of the extract as capping and stabilizing agents, otherwise agglomerated particles are formed.
The TEM images of EcS-Ag NPs are depicted in Fig. 6a-d. A mixture of diversely shaped particles including cylindrical, hexagonal, and triangular shapes were found in these images. It is worth to note that the particle size and shape control still remains a great challenge as no external constraints were imposed during the experimentation. This result, indicating the formation of various sizes and shapes, is in close agreement with the earlier findings . The spherical structures with various sizes ranging from 6.5 nm to 42.1 nm with the average size of 33.86 nm are shown in Fig. 7a-b.
The five spots appeared on the SAED pattern were correlated to specific crystal planes of EcS-Ag NPs as shown in Fig. 7. The most prominent 4 spots shown in colored concentric circles represent (111), (200), (220) and (311) planes. The interplanar spacing (IPS) value of 0.242 nm for Ag (111) plane is deduced from Fig. 7d. Figs. 8a, 8b and 8c respectively show the HRTEM micrographs of EcS-Ag NPs with enhanced lattice fringes, IFFT, and profile of IFFT with interplanar spacing (IPS) value for a specified plane. The IPS values for a specific parallel crystal planes on the surface of Ag NPs were found to be 0.2418 nm at a specific point.
Table 2 provides the IPS values for the 5 spots appeared on the SAED pattern (Fig. 7c) of EcS-Ag NPs. Each spot on the SAED pattern corresponds to a specific set of lattice planes. The IPS values of 0.2417 nm, 0.2056 nm, 0.1471 nm, 0.2040 nm and 0.1254 nm, derived for spots 1, 3, 4 and 5, correspond to crystal planes 111(Ag), 200(Ag), 220(Ag) and 311(Ag) that are in accordance with the IPS values of Ag crystal structure . The presence of other one spot (2) is possibly due to trace amount of crystalline AgCl. The presence of Cl peak in the EDAX spectrum of Ag NPs further confirms the possibility of AgCl formation.
This SAED analysis using HRTEM images is in compliance with the previously discussed XRD results for the EcS-Ag NPs . The IPS value of lattice fringes at the surface of the EcS-Ag NPs was found to be 0.2418 nm, that is similar to the dhkl value of 0.24 nm for (111) plane of fcc structured Ag obtained in the previously reported work .
The EcS-Ag NPs demonstrated superior antibacterial activities versus all the tested pathogens; S. aureus, E. coli, P. aeruginosa, and E. aerogenes. The present work evaluated synergistic influence of biomolecules with NPs against all these 4 pathogens. The zone of inhibitions (ZoI) for chloramphenicol, DMSO and NPs with four concentrations (6.25, 12.5, 25 and 50 µg/µl) are shown in Fig. 9. EcS-Ag NPs were found to show better antimicrobial activity against gram-negative bacterial strain E. aerogenes than the other gram-positive bacterial strains that could be attributed to the structural differences in the cell walls of bacteria .
The antimicrobial activity of NPs was highly appreciable against S. aureus with ZoI of 18 mm. The maximum zone of inhibitions (mm) inscribed by EcS-Ag NPs against E. coli, P. Aeruginosa and E. aerogenes bacteria are 14 mm, 16 mm and 15 mm, respectively (Table 3). In addition, the lowest zone of inhibition was exhibited by E. coli bacteria remained at 9 mm for the most concentrated solution of NPs. In addition, two gram-negative bacteria E. coli and E. aerogenes exhibited the opposite trend of decreased antibacterial activity with increase in the concentrations of Ag NPs as against gram-positive bacteria. In the case of E. coli, the ZoI decreased from 14 mm to 9 mm on moving from 12.5 µg/µl to 50 µg/µl concentration. But gram-positive bacteria S. Aureus exhibited the opposite trend, where antibacterial activity increased with an increase in the concentration of A NPs. This behaviour is basically due to the structural differences between two types of bacteria as well as differences in the morphological features of Ag NPs.
The antimicrobial resistance recorded for EcS-Ag NPs was found to be superior when compared with few of the earlier results (Table 4) presented by many researchers, even though few other workers reported higher inhibitions that is believed to be due to the usage of higher concentrations of NPs or presence of higher concentrations of bioactive compounds. The highest zone of inhibition (mm) recorded with Ag NPs against bacteria was 18 mm. Thus, it can be concluded that the cumulative effect of Ag NPs coupled with phytochemicals (alkaloids, tannins, flavonoids and terpenoids) of EcS-PR extract proved to be detrimental for bacterial strains.
Even though many antimicrobial mechanisms were proposed by earlier researchers, the action of EcS-Ag NPs on the bacteria is unknown and yet to be exploited completely. Nanoparticles were found to cause the death of bacteria adopting direct or indirect ways by attacking bacteria’s cell wall, inhibiting RNA synthesis and preventing DNA replication. It is assumed that the positive Ag+ in the NPs adsorb directly onto the cell wall of bacteria interacting with negatively charged species. This result in disruption of the cell wall of bacteria and via the generation of reactive oxygen species (ROS) by the effect of visible/UV light radiation . It is also assumed that the cumulative effect of EcS-Ag NPs and bioactive compounds of EcS-PL extract displayed magnificent influence on bacteria as suggested by the recent studies .
The mechanism of NPs action basically differs as there exist significant structural differences between gram-negative and gram-positive bacteria . In addition, electrochemical charge variations across the cell membrane influence the interactions with the released Ag+ ions by EcS-Ag NPs deteriorating the structural integrity of the membrane . The maximum antibacterial activity was recorded for gram-positive bacteria compared to the gram-negative bacteria due to their differences in their cell structures as well as the potentiality of nanoparticles. .
The application of medicinal plant Echinops sp. root extract towards the green synthesis of silver (EcS-Ag) nanoparticles was successful. The UV-visible spectra, XRD pattern, and FTIR spectra approved the formation of crystalline EcS-Ag NPs in the presence of biomolecules (alkaloids, tannins, flavonoids and terpenoids) of EcS-PL extract. SEM-TEM-HRTEM-SAED analyses corroborated the existence of silver by the observation of Ag (111), (200), (220) and (311) lattice fringes in EcS-Ag NPs with the calculated interplanar spacing values matching exactly with the standard value for Ag. The synergistic influence of bioactive compounds and EcS-Ag NPs proved to exhibit highly effective antibacterial mechanism against pathogens, S. aureus, E. coli, P. aeruginosa, and E. aerogenes with the highest inhibition zone of 18 mm. It can be concluded that the cumulative effect of biomolecules of plant and silver NPs can be detrimental to disease causing pathogens.
The author would like to thank the staff, Department of chemistry and Management of Adama Science and Technology University, Ethiopia for allowing me to use their facilities. This work has been funded through the project (ANSD/04/0453/11-2018) approved by Research and Technology Transfer Office, sanctioned by Adama Science and Technology University, Ethiopia.
CONFLICT OF INTEREST
The author declares that there is no conflict of interest.