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SN Applied Sciences

, 1:681 | Cite as

Silver–zinc oxide nanocomposite antiseptic from the extract of Bidens pilosa

  • Hilda Dinah KyomuhimboEmail author
  • Immaculate Nyambura Michira
  • Francis B. Mwaura
  • Solomon Derese
  • Usisipho Feleni
  • Emmanuel I. Iwuoha
Research Article
  • 117 Downloads
Part of the following topical collections:
  1. 4. Materials (general)

Abstract

Silver nanoparticles (Ag-NPs), zinc oxide (ZnO-NPs) and zinc oxide–silver (ZnO–Ag-NPs) were biosynthesized based on the rich matrix of alkaloids, flavones, tannins capping/stabilizing agents present in Bidens pilosa extract. Different plant parts-root, leaf and seed ware used to prepare the plant extract for synthesis. Also, zinc and silver nitrate salts were used as precursor materials. The surface plasmon peaks (SPR) based on the UV–Vis results for the Ag-NPs, ZnO-NPs were located between 408–411 and 365–450 nm respectively. The SPR peaks for the Ag–ZnO-NPs occurred at 300–450 nm indicating both blue and red shifts. The Ag–ZnO-NPs SPR shifts were associated with possible nanoparticle size reduction and change in dielectric constant of the synthesis medium. Raman measurement peaks at 356, 484, 1350, 1578, 2435 cm−1 associated with OH, –C==C–, –C–O, S=O, =C–H moieties indicated successful capping. Nanoparticle yield was temperature dependent and optimal yield could not be tied to a particular plant part as source of extract. Tunneling electron microscope results showed Ag-NPs and ZnO-NPs were globular/spherical with a diameter range of 2–20 nm. Interestingly, ZnO-NPs TEM displayed isolated miniaturized globular nanoparticles (< 2 nm) which then joined up to form a large donut shaped structure indicating different formation mechanisms for the nanoparticles. XRD results showed the Ag-NPs, ZnO-NPs and the Ag–ZnO-NPs particles were crystalline in nature. The high signal/noise in XRD originated from possible crystalline biomaterials in the extracts. Energy dispersive spectroscopy (EDS) elemental composition results confirmed successful formation of the nanoparticles. Anti-Microbial activity of the synthesized Ag-NPs, ZnO-NPs and ZnO–Ag-NPs were studied against gram negative bacteria Escherichia coli (E. coli), gram positive bacteria Staphylococcus aureus and fungus Candida albicans. Different ZnO: Ag-NPs nanocomposite ratios were used to test for antimicrobial activity. Optimal antimicrobial activity was attained at Ag-NPs:ZnO-NPs ratio of 4:1 which also displayed the least minimum inhibition concentration (MIC) and therefore was used as the active ingredient in formulating a hand sanitizing antiseptic. The formulated antiseptic exhibited good antimicrobial activity.

Keywords

Silver nanoparticles Zinc oxide nanoparticles Zinc oxide–silver nanoparticles Antimicrobial activity Hand sanitizing antiseptic 

1 Introduction

The rampant spread of infectious diseases globally cannot be overlooked [1, 2]. Antimicrobial drugs, disinfectants and antiseptics have been introduced to counter the cause and spread of these infectious diseases, however, the resistance of microbial strains against the antimicrobial drugs is increasing at an alarming rate due to inappropriate use of antimicrobial drugs [3, 4, 5, 6, 7]. Therefore there is a need in the use of an agent that does not generate resistance, presents good antimicrobial property and without cytotoxic effects on humans and other species [8, 9, 10]. Nanosized antimicrobial agents have gained popularity due to their high stability and enhanced antimicrobial activities [9, 11]. The versatility of nanoparticles in rendering themselves to many applications is currently being explored. Top in the list are silver nanoparticles (Ag-NPs) and zinc oxide nanoparticles (ZnO-NPs) which find applications in medicine, sensors, renewable energies, cosmetology, environmental remediation, bio-therapeutic devices, clothing, surface disinfection and antimicrobial applications [12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23]. This could be attributed to their small size and large surface arear to volume ratio which gives them improved physical, biological and chemical properties as compared to their large scale counter parts [24, 25]. Ag-NPs and ZnO-NPs have been proved to have effective antimicrobial activity at very low concentrations even to resistant strains of microbes [26, 27]. This is because they destroy many biological pathways in a cell and it would necessitate the microorganism to go through many concurrent transmutations so as to develop resistance [7, 28]. More so they need very short contact time to cause a long lasting cell growth inhibition [29, 30]. Of recent, the Ag/ZnO nanocomposite has been found to show enhanced antimicrobial activity against the common microbes as compared to the individual nanoparticles [31]. This is because the properties of the hybrid are not just the sum of the individual advantages of both nanoparticles but are derived from their synergic effect which creates a new class of hybrid-nanomaterials [11]. Also the synergic effect of Ag and ZnO is beneficial for mass production of Reactive Oxygen Species such as H2O2, hence increased antimicrobial activity of nanocomposite [32, 33]. Several nanoparticle synthesis methodologies have been employed including chemical methods [34], electrochemical methods [35], gamma radiation methods [36], photochemical [37] as well as laser ablation techniques [38]. A major impeding barrier as to employment of most of these conventional methods in nano-production is that they are cost prohibitive and also that some make use of harmful solvents and synthetic reactants which are environmental non-friendly [39, 40, 41, 42, 43, 44, 45, 46, 47].

Green nanotechnology on the other hand makes use of environmentally benign materials which are readily available at minimal or no cost [2, 21]. The nanoparticles produced by this method are usually stable, hydrophilic and have very small diameter [48, 49, 50, 51]. In addition, plant extract precursors, have an extensive variety of active ingredients which aid in the reducing and stabilizing process and also act as templates for the modulation of nanostructure formation. The biosynthesizing precursors are not only safe to handle but the process easily renders itself to scaling up without use of energy, high temperatures or toxic reagents thereby giving an environmentally friendly alternative to physical and chemical syntheses [2, 19, 45, 52, 53, 54].

Several plant extracts have been used as stabilizers, reducing and templating or structure directing agents for silver and zinc oxide nanoparticles synthesis. Camellia sinesis [55], Hibiscus rosa-sinensis [56], Rumex hymenosepalus [57], Cissus quadrangularis [58], Olea rupea [59], Octinum sanctum [46], Ocimun tenuiflorum [51, 60] and Panicum virgatum [61] amongst others have been used. The presence of many polyphenols [2, 13, 34, 62] in the extracts not only allow them to exhibit high antioxidant activity but to act as strong reducing agents owing to the presence of many hydroxyl (OH) groups in their structural composition [46].

Bidens pilosa contains several bio-molecules of the polyphenols type (Scheme 1) [55, 56, 57, 58, 59, 60, 61, 63, 64, 65, 66, 67, 68, 69]. It is believed that these biomolecules are the stabilizing, reducing and nanostructurizing media that guide the formation of the various nanoparticles.
Scheme 1

Structures of some of the compounds found in Bidens pilosa

So far no work on nanotechnology has been carried out using Bidens pilosa. In this work, silver, zinc oxide and silver–zinc oxide nanoparticles stabilized/capped by Bidens pilosa ligands were synthesized. The antimicrobial properties of Ag-NPs-ZnO-NPs hybrid nanoparticles and possible application in formulating a hand antiseptic are discussed. The efficacy of the formulated antiseptic against E. coli, S. aureus and C. albicans is explored.

2 Materials and methods

Bidens pilosa plants were obtained from University of Nairobi compound. Silver nitrate, Zinc nitrate and guar gum were purchased from Sigma Aldrich. Microorganisms of E. coli, S. aureus, and C. albicans were obtained from Department of Microbiology, University of Nairobi. Chloramphenicol and Fluconazole were purchased from a nearby pharmacy around the University. Pure glycerin, chantia fragrance and α-tocopherol were purchased from a nearby pharmacy around the University of Nairobi.

2.1 Sample collection and leaf extract preparation

The plant (Bidens pilosa) was collected from Nairobi County. The seeds, leaves and roots were plucked separately from the plant and washed with distilled water, dried under shed and ground to powder. 2 g of each of the seed, leaf and root powders were boiled in 250 mL of distilled water at 90 °C for 10 min, allowed to cool under nitrogen gas and filtered using a filter paper. The residues were discarded. The filtrates were centrifuged at 14,500 rpm for 10 min to obtain the seed, leaf and root extracts. The above procedure was repeated at temperatures of 65, 45 and 25 °C to demonstrate the effect of varying extraction temperature on the spectroscopic properties and yield of the silver and zinc oxide nanoparticles.

2.2 Synthesis of Ag-NPs, ZnO-NPs and ZnO–Ag-NPs

20 mL of the seed extract obtained at 90 °C were transferred to a beaker into which 80 mL of 1 mM silver nitrate solution was added drop wisely. The mixture was allowed to stand for 2 h under nitrogen gas blanket. Exactly 1 mL of this mixture was transferred to a cuvette for UV–Vis analysis. The rest of the mixture was centrifuged at 14,500 rpm for 10 min. The supernatant obtained after centrifugation was transferred to the oven where it was dried at 60 °C for 8 h. The nanoparticles were removed and transferred to a closed container and kept for further analysis. The above procedure was carried out for seed, leaf and root extracts obtained at 90 °C, 65 °C, 45 °C and 25 °C.

For synthesis of ZnO-NPs, the above procedure was repeated with 80 mL of 1 mM zinc nitrate solution. For the ZnO–Ag-NPs, the above procedure was repeated with 80 mL of varying volumes of silver nitrate and zinc nitrate solutions to obtain various compositions of AgNO3/Zn(NO3)2 solutions. The loading of Ag was based on volume to volume ratios of silver nitrate solution to zinc nitrate solution. AgNO3:Zn(NO3)2 volume ratios used were between 0:1 that is; 0.2 (16 mL AgNO3, 64 mL Zn(NO3)2), 0.4(32 mL AgNO3, 48 mL Zn(NO3)2), 0.5(40 mL AgNO3, 40 mL Zn(NO3)2), 0.6(48 mL AgNO3, 32 mL Zn(NO3)2) and 0.8 (64 mL AgNO3, 16 mL Zn(NO3)2).

2.3 Characterization of Ag-NPs, ZnO-NPs and ZnO–Ag-NPs

The Surface Plasmon Resonance (SPR) properties and spectral analysis of the nanoparticles were analyzed using UV–visible spectrometer [UV-1700 pharmaspec UV–Vis spectrophotometer (shimadzu)]. The extracts, Ag-NPs, ZnO-NPs and ZnO–Ag-NPs were subjected to Fourier Transform Infrared (FTIR-JASCO 4100) to determine capping of biomolecule on the surface of the nanoparticles. The Ag-NPs, ZnO-NPs and ZnO–Ag-NPs were subjected to Raman spectroscopy (Renishaw RM1000) to further identify the functional groups responsible for their capping and stabilization. The Ag-NPs, ZnO-NPs and ZnO–Ag-NPs powders were characterized by X-ray Diffraction (SIEMENS D5005 Diffractometer) and Transmission Electron Microscopy (Philips Technai-F12) and Scanning Electron Microscopy (Hitachi S-3000N).

2.4 Antimicrobial activity of Ag-NPs

The antimicrobial activity of Ag-NPs, ZnO-NPs and ZnO–Ag-NPs was evaluated against gram positive Staphylococcus aureus, gram negative Escherichia coli and fungus Candida albicans. Microorganisms of E. coli, S. aureus, and C. albicans were obtained from Department of Microbiology, University of Nairobi. These microorganisms were chosen as representative strains of gram positive and gram negative bacteria owing to their unique shell envelop structure. They also are not only easy to culture in the laboratory but also live on the human skin surface meaning a successful study would be a step towards human health problems [3].

Fresh overnight inoculums (50 µL) of each culture were spread on to agar plates. Wells of 6 mm diameter were made in the agar pates using a sterile coke borer. 5 mL of Ag-NPs solution of concentration 100 µg/mL was prepared by dispersing Ag-NPs in distilled water. 45 µL, of Ag-NPs solution was poured into the wells. The E. coli and S. aureus plates were incubated at 37 °C for 24 h. The plates with C. albicans were left to stand at room temperature for 48 h. The diameters of zones of inhibitions were recorded. The above procedure was repeated for ZnO-NPs and ZnO–Ag-NPs.

2.5 Determination of minimum inhibitory concentration

Exactly 13 g of nutrient broth was weighed in a beaker into which 100 mL of distilled water was added while stirring. The medium was then transferred to 1 L medium sterilizing bottle which was brought to a boil and then allowed to cool. The cooled medium was then poured into 15 mL tubes each tube receiving 9 mL. This was followed by autoclaving the medium containing tubes at a pressure of 15 Psi for 15 min in order to kill any microorganisms present.

To make an Ag-NPs/medium stock solution, one autoclaved medium containing tube was selected. Into this tube 1 mL Ag-NPs solution was transferred using a micropipette and the mixture thoroughly shaken to ensure formation of a uniform mixture. This was the 10 µg/mL Ag-NPs concentration. From this stock solution, a 1 µg/mL Ag-NPs concentration was made by drawing out an aliquot of 1 mL again from the stock solution and adding it to a new autoclaved medium containing tube followed by thorough shaking. By micro pipetting 1 mL of the 1 µg/mL Ag-NPs solution and placing it in another autoclaved medium, a 0.1 µg/mL Ag-NP concentration was made.

In order to test the effect of the different concentrations of the nanoparticles (10, 1, 0.1 µg/mL Ag-NPs) E. coli media was scooped and transferred to each of the three tubes and the extent of inhibition determined. The above procedure was repeated for S. aureus and C. albicans. Similar procedure was followed for ZnO-NPs and the Ag-NPs/ZnO-NPs nanohybrid.

2.6 Formation of hand sanitizing antiseptic

77.1 g of deionized water was placed into a beaker followed by 0.7 g of guar gum (gelling agent). The solution was heated to a temperature of 50 °C while stirring at 1000 rpm for 30 min. The solution was then allowed to cool to room temperature and 2 g of α-tocopherol (emollient) and 0.1 g of ZnO–Ag-NPs (0.8Ag-NPs/0.2 ZnO-NPs—active ingredient) was added while stirring at 1000 rpm. Lastly 7 g of pure glycerin (humectant) followed by 0.2 g of fragrance (Chantia) were added to the mixture. The mixture was stirred for more 30 min at 1000 rpm at room temperature. The antiseptic/hand sanitizer was then allowed to stand for 2 h and then tested for antimicrobial activity.

3 Results and discussion

The formation of the Ag-NPs, ZnO-NPs and Ag-NPs/ZnO-NPs nanohybrids were marked by an onset of colour changes upon mixing the reactants. The colour changed from a bright yellow to brown and then to dark brown after 5 min with precipitation signaling formation of ZnO-NPs. For silver nanoparticles, the final colour turned into a cloudy dark grey after 5 min. Interestingly the final colour of the ZnO–Ag-NPs depending on the ratio lay between yellow and grey with the majority having a tinge of brown colour. In fact the colour of the ZnO–Ag-NPs was an intermediate of the colour of ZnO-NP solution and Ag-NP solution that is a brown colour (Fig. 1)
Fig. 1

a Seed extract, b leaf extract, c root extract, d silver nitrate, e zinc nitrate, f solution containing ZnO-NPs, g solution containing Ag-NPs and h solutions containing ZnO–Ag-NPs

A UV–Vis scan of the formed Ag-NPs gave rise to the surface pasmon band in almost the same wavelength (408–411 nm) for each extract type indicating each of the selected plant parts yielded adequate polyphenolic materials for the nanostructurizing work Fig. 2a. This surface plasmon band results from the interaction between the conduction electrons and the striking radiation. A similar observation was made for the ZnO-NPs nanocomposites whose plasmon band ranges were 365–372 nm Fig. 2b. A double band at around 300 and 450 nm was observed for the ZnO–Ag-NPs (Fig. 2c) nanoparticles a clear indication, of the successful formation of the hybrid. In the hybrid the band corresponding to ZnO-NPs showed a significant blue shift while that corresponding to Ag-NPs showed a significant red shift. This could be due to electromagnetic coupling between the Ag and ZnO which leads to inphase interactions between adjacent colloidal nanoparticles hence the blue and red shift in ZnO-NPs and Ag-NPs respectively. The red shift of the Ag-NPs band could be attributed to increased charge magnitude on mixing the two and possible different interaction with synthesis medium [70, 71, 72]. Elsewhere blue shift has been associated with nanoparticle size reduction as well as change in the synthesizing media dielectic constant [73].
Fig. 2

UV–Vis spectra for Ag-NPs (a), ZnO-NPs (b) and Ag-NPs-ZnO-NPs (c) showing the surface plasmon band(s)

Variation of the synthesis temperature indicated nanoparticle yield increased with increase in temperature. This was the general observation observed across all the nanoparticle types whether Ag-NPs, ZnO-NPs or ZnO–Ag-NPs irrespective of the part of plant used as source of the stabilizing agents. The masses of the product nanoparticles were calculated using Eq. 1
$$\% \;yield\; of\;AgNPS = \frac{{{\text{Mass}}\;{\text{of }}\;{\text{Ag}} - {\text{NPs}}}}{{{\text{Mass }}\;{\text{of }}\;{\text{Ag }}\;{\text{ion}}\; {\text{in}}\; {\text{solution}}}} \times 100$$
(1)

3.1 Effect of temperature on the yield

Figure 3a–c shows an upward yield trend for all the nanoparticles with temperature although the actual plant part that led to maximum yeild was erratic. For all temperatures > 80 °C, the nanoparticle yield irrespective of the source of the stabilizing agents was well above 80%.
Fig. 3

Showing yield dependence on extraction temperature for Ag-NPs (a), ZnO-NPs (b) and Ag-NPs-ZnO-NPs (c)

An increase in nanoparticle yield with temperature could mean the mechanism for nanoparticle formation is in line with arrhenius theory which highlights that reaction rates rise with temperature. An increase in temperature increases the kinetic energy of the precursor materials hence enhancing the rate of collision of the reacting spieces with a higher part of collisions exceeding the threshold of activation energy. This in turn increases the amount of nanoparticles produced as the temperature of the extract increases [74, 75]. For all the cases it was found maximum nanoparticle yield was obtained at a maximal temperature of 90 °C. However, each part of the plant gave different yields for the various nanoparticles, that is, the seed extract gave the highest yield for ZnONPs; the leaf extract gave the highest yield for AgNPs while the root extract gave the highest yield for the ZnO–AgNPs.

Since Ag-NPs and ZnO-NPs have been known to exhibit antimicrobial properties, the aim of the research was to produce ZnO–Ag-NPs nanohybrids and test for their antiseptic effect. Through experimentation by varying the composition of the Ag-NPs in the hybrid, it was established that an Ag-NPs:ZnO-NPs ratio of 0.8:0.2 (4:1) gave rise to highest absorbance—a typical result is presented in Fig. 4. Further analysis of the nano-products was based on samples produced at optimal conditions.
Fig. 4

UV–Vis spectra of Ag-NPs-ZnO-NPs from root extract of Bidens pilosa at varying composition of Ag-NPs

Further characterization was based on results obtained at these optimal conditions.

3.2 Raman and FTIR results

The functional groups mainly OH, –C=C–, C–O, S=O, =C–H bending and aromatic C–H bending derived from heterocyclic compounds or alkanols e.g. alkaloid, or flavones and tannins are present in Bidens pilosa extracts and are the capping ligands of the nanoparticles. This shows that the compounds from the extracts cap and stabilize the nanoparticles during the synthesis process.

Figure 5a–c compares and contrasts the Raman spectra of the Ag-NPs, ZnO-NPs and the Ag-NPs-ZnO-NPs nanoparticles.
Fig. 5

Raman spectra of the Ag-NPs (a), ZnO-NPs (b) and the nanohybrid Ag-NPs-ZnO-NPs (c) at a ratio of 0.8:0.2

There is a general appearance of some broad Raman bands indicating some presence of some degree of amorphousness across the three spectra (Fig. 5a–c). Broad bands are as a result of distortions of interatomic bond angles and the consequent overcrowding due to the presence of many such distortions [76]. Interestingly, the occurrence of Raman shifts as low as 158 cm−1 is indicative of Ag lattice vibrations indicative of some degree of crystallinity. Other bands for Ag-NPs that were obtained include; 356 cm−1 corresponding to C–C bending vibration of aliphatic chains, 484 cm−1 corresponding to S–S bonds, 1350 cm−1 corresponding to carboxylate salt, 1578 cm−1 corresponding to aromatic ring and 2435 cm−1 corresponding to thiol group. The functional groups attached to the silver nanoparticles confirm that the leaf extract contains compounds that reduce the Ag + to Ag0 and also stabilize the Ag-NPs formed. Similar functional groups were confirmed from the FTIR results (Table 1).
Table 1

Values for IR bands for the extracts and nanoparticles obtained using FTIR

Extract (wave number/cm−1)

Nanoparticles (wave number/cm−1)

Functional group

Leaf extract

AgNPs

 
 

3448

Free O–H

2772

 

O–H stretch for carboxylic acid

 

1560

Aromatic C=C stretch

 

1489

C–H bending alkane—methyl group

 

1307

C–O stretching aromatic ester

 

1298

S=O stretching sulfone

1151

112

C–O stretch

912

 

=C–H bending

728

 

Aromatic C–H bending

Seed extract

ZnONPs

 
 

3432

Free O–H

2772

 

O–H stretch for carboxylic acid

 

1559

C=C stretch

 

1489

C–H bending alkane—methyl group

 

1307

C–O stretching aromatic ester

 

1298

S=O stretching sulfone

1151

 

C–O stretch

912

 

=C–H bending

728

 

Aromatic C–H bending

603

 

C–I, C–Br stretching for halo compound

Root extract

ZnO–AgNPs

 
 

3432

Free O–H

2928

 

O–H stretch for carboxylic acid

 

1564

C=C stretch

 

1490

C–H bending alkane—methyl group

 

1307

C–O stretching aromatic ester

 

1292

S=O stretching sulfone

1151

 

C–O stretching

912

 

=C–H bending

728

 

Aromatic C–H bending

3.3 TEM results

Tunneling electron microscopy results show that the prepared nanoparticles exhibit diameters of between 2 and 20 nm (Fig. 6a–c). EDS results (Fig. 6d–f) of the Ag-NPs nanoparticles indicated presence of elemental silver at 3.2 and 2.9 keV due to Ag L line transitions while as those of ZnO-NPs were observed 1, 8.6, 9.5 keV corresponding to the Zn L lines, Zn Kα and Zn Kβ line respectively [77, 78]. The appearance of peaks due to P, S, O, Na was associated with plant extracts origin because these elements can be absorbed from soil, while as Cu is from the TEM grid. The Ag-NPs-ZnO-NPs nanohybrid clearly indicates the presence of both Ag and Zn nanoparticles a successful indication of the formation of the nanohybrid. Charging effects due to build-up of electrons in the sample is evident both in the TEM and in the SEM results, a characteristic of the presence of poor conducting, stabilizing organic moieties.
Fig. 6

Tunneling electron micrographs for Ag-NPs (a), ZnO-NPs (c) and Ag-NPs-ZnO-NPs hybrid (e). Energy dispersive spectra for Ag-NPs (b), ZnO-NPs (d) and Ag-NPs-ZnO-NPs (f). Scanning electron micrographs for Ag-NPs (g), ZnO-NPs (h) and Ag-NPs-ZnO-NPs (i)

The Ag-NPs exhibited globular/spherical shape as compared to that of the ZnO-NPs. The latter exhibited miniature isolated globular nanoparticles (< 2 nm) which then joined up to form larger globules (Fig. 6g–i). Interestingly these giant globular aggregates joined up to form a large donut shaped structure with a hollow center. Looking at the rear of the donut crystal, co-joint segments boundaries are visible probably indicating stepwise formation of this donut structure. Similar observations were made by Buthysusnatao et al. [79] who concluded that the crystal growth is bidirectional while working on Mg-OH crystals. It appears the magnitude of the charge on a particle influences the resultant morphology. However, this donut morphology is not distinct in the Ag-NPs-ZnO-NPs hybrid probably because the sample was a 0.8–0.2, Ag-NPs-ZnO-NPs respectively ratio.

3.4 XRD results

Three intense peaks at 2θ = 38.05°, 45.35° and 64.42° and can be indexed to the (111), (200) and (220) for the face-centered cubic silver as per the JCPDS card no. 89–3722 [80]. The fact that the XRD results displayed a high/signal to noise ratio could be instrumental but can also be due to the presence of crystalline biological materials from plant extract origin [81]. The Ag-NPs formed by the reduction of Ag+ ions using Bidens pilosa leaf extract were crystalline in nature although the broad noisy base could be associated with the amorphous nature of the stabilizing/reducing agents. The intense peaks for ZnO-NPs occurring at 2θ = 30.15°, 35.74°, 47.16° and 56.67° correspond to 100, 101, 102 and 110 planes of the Bragg’s reflection of zinc. The peaks confirm ZnO hexagonal phase as compared to JCPDS card No. 89-7102 [82, 83]. The well resolved and sharp peaks show that the nanoparticles are crystalline in nature. XRD analysis showed distinct diffraction peaks at 2θ = 38.05°, 45.35° and 64.42° of (111), (200) and (220) for the face-centered cubic silver as per the JCPDS card no. 89–3722 and 30.15°, 35.74°, 47.16° and 56.67° corresponding to 100, 101, 102 and 110 for hexagonal ZnO-NPs as per JCPDS card No. 89-7102 respectively [84, 85]. The peaks and the EDS elemental results which indicate the compositions of the various nanocomposites clearly indicate successful synthesis (Fig. 7).
Fig. 7

ac XRD results for Ag-NPs (a), ZnO-NPs (b) and Ag-NPs-ZnO-NPs (c)

3.5 Antimicrobial activity of the nanohybrids

In the testing of antimicrobial activity of the nanoparticles, two bacteria and one fungus were used. The Gram positive bacteria, S. aureus and the Gram negative bacteria E. coli were used as test microbes. The fungus, C. albicans was also used. For comparison purposes different nanoparticle ratios were used. In all the tests, clear halos indicating antimicrobial properties were evident. The strength of the antimicrobial activity depending on the nanoparticle composition ratio was fitted into zones of inhibition curves. Figures 8a–c show optimal antimicrobial activity was attained at an Ag-NPs:ZnO-NPs ratio of 0.8:0.2 nanoparticle composition translating into a 4:1 ratio. This result was so irrespective of the origin of the stabilizing reductants—leaf, root or seed as shown in Fig. 8a–c.
Fig. 8

ac A graph of average values of zones of inhibition of (a) C. Albicans for nanoparticles obtained using seed, leaf and root extract against composition of silver nanoparticles b E. coli, c S. Aureus

During this testing, chloramphenicol was used as positive control for E. coli and S. aureus. Fluconazole was the positive control for the fungus C. albicans. No inhibition zones were observed for E. coli on the positive control (Chloramphenicol). This implies that the culture of E. coli that was used was resistant to the positive control. For the S. aureus, the positive control gave a zone of inhibition of 26.25 ± 0.17 mm. The positive control used for C. albicans, fluconazole gave an average zone of inhibition of 24.75 ± 0.17 mm Fig. 9. Distilled water served as the negative control for all the micro-organisms since it was used to disperse the nanoparticles. No inhibition zones emanated from use of distilled water.
Fig. 9

Zones of inhibition for E. coli, S. aureus and C. albicans for the various nanoparticles

From the above results, the nanoparticles that exhibited highest antimicrobial activity were used to determine their minimum inhibition concentration (MIC). This is the lowest concentration of the nanoparticles that would inhibit the growth of the testing agents namely—E. coli, S. aureus and C. albicans. The Ag-NPs-ZnO-NPs used were all in the optimal ratio of 0.8 Ag-NPs: 0.2 ZnO-NPs and prepared at an optimal temperature of 90 °C. Results were compared across the various plants parts source of stabilizing agents as follows; leaf extract originating nanoparticles denoted as (L90-0.8), seed extract originating nanoparticles denoted as (S 90-0.8) and root extract originating nanoparticles denoted as (R-90-0.8). The results for MIC are as shown in the Table 2.
Table 2

Comparison of minimum inhibition concentration (MIC) for various nanoparticles

Microorganism

MIC (µg/mL)

S90-0.8

L90-0.8

R90-0.8

E. coli

10

10

1

S. aureus

1

1

1

C. albicans

1

1

1

According to the table, the seed originating nanoparticles (S90-0.8) and leaf originating nanoparticles (L90-0.8) exhibited a minimum inhibition concentration of 10 µg/mL for the E. coli and 1 µg/mL each for S. aureus and C. albicans. The nanoparticles of root origin, R90-0.8 showed an MIC of 1 µg/mL for all testing agents. Generally, this implied that, irrespective of the source, the nanoparticles were more effective on the S. aureus and C. albicans as compared to the E. coli. From the above results the ZnO–Ag-NPs of root origin were used to make a nanohybrid based antiseptic.

3.6 Formulation of a nanohybrid based antiseptic

Table 3 gives the % composition (% w/w) of the formulated antiseptic. % composition of each component was worked out based on the formula;
$$\% composition\; of\;component = \frac{Mass \;of\; component}{Mass\; of\; the \;antiseptic}$$
(2)
While as only the nanohybrids exhibited anti-microbial/anti-fungal activity, other ingredients were added to improve the aesthetic nature of the yellowish foam forming antiseptic (Fig. 10).
Table 3

The Ingredients of the formulated antiseptic (% w/w)

Component

Mass (g)

Composition (%w/w)

Deionized water

77.10

88.52

Guar gum (gelling agent)

0.70

0.80

α-tocopherol (emollient)

2.00

2.30

ZnO–AgNPs (active ingredient)

0.10

0.11

Glycerin (humectant)

7.00

8.04

Fragrance

0.20

0.23

Total

87.10

100.00

Fig. 10

Hand sanitizing antiseptic containing ZnO-Ag-NPs as active ingredient before shaking and after shaking

3.6.1 Antimicrobial activity of the formulated antiseptic

In the testing of antimicrobial activity of the antiseptic, again the three test agents were used namely—the Gram positive S. aureus bacteria, the Gram negative E. coli bacteria, and the fungus C. albicans. In all the tests carried out, clear halos were observed as shown in Fig. 11, thus indicating inhibition of microbial growth by the antiseptic. The values of zones of inhibition were 14 mm for E. coli, 20.5 mm for S. aureus and 24.5 mm for C. albicans. These values for the zones of inhibition indicate that the hand sanitizer exhibited strong antimicrobial activity.
Fig. 11

Zones of inhibition for a E. coli, b S. aureus and c C. albicans for the hand sanitizing antiseptic formulated

4 Conclusion

AgNPs, ZnONPs and ZnO–AgNPs were synthesized using the seed, leaf and root extracts of Bidens pilosa. Although nanoparticle yield depended on temperature maximum yield attainment could not be associated to a particular plant extract source-root, leaf, seed. FT-IR and Raman spectra of the nanoparticles revealed the presence of functional groups O–H, C=C, C–H aromatic, and =C–H which were responsible for reduction and stabilization of the nanoparticles. UV–Vis results for the for Ag-NPs displayed SPR bands at 408–411 nm but a red shift of the Ag-NPs band in the Ag–ZnO-NPs hybrid was associated with changes in the dielectric constant of the reaction media. The UV–visible results for the ZnO-NPs observed at 365–372 nm shifted to high energy region for the Ag–ZnO-NPs probably indicating nanoparticle size reduction. The particles were found to have average mean size of 2–20 nm and were spherical in shape without significant agglomeration as revealed from the SEM and TEM analysis. EDX spectrum revealed the strong signal in the silver region confirming the presence of Ag and in the zinc region confirming the presence of ZnO. The synthesized nanoparticles exhibited face-centered cubic crystals as demonstrated from XRD studies.

The results confirmed that in the medium treated with AgNPs, ZnONPs and ZnO–AgNPs, E. coli, S. aureus and C. albicans growth was inhibited. The ZnO–AgNPs with composition of 0.8 Ag/0.2 ZnO gave the highest antimicrobial activity regardless of the extract used. From the ZnO–AgNPs of the three extracts, those originating from the root extract showed the lowest Minimum Inhibition Concentration.

The root extract ZnO–AgNPs (0.2:0.8) were used as active ingredients to formulate a hand sanitizing antiseptic. The antiseptic formed exhibited strong antimicrobial activity. Inhibition zones indicating antimicrobial activity of the hand antiseptic were 14 mm E. coli, 20.5 mm S. aureas and 24.5 mm C. albicans.

Notes

Acknowledgements

The study was conducted from University of Nairobi, Department of Chemistry, Department of Physics, Department of Biotechnology, and School of Microbiology and also at University of Western Cape, Sensor Lab—Department of Chemistry. It was funded by Deutscher Akademischer Austauschdienst (DAAD), REF: 57221138 and Kenya/South Africa collaborative grant REF: NCST/5/003/2/53, for the project titled, ‘Nanobiosensor Chips for Anti-Tuberculosis’.

Compliance with ethical standards

Conflict of interest

On behalf of all the authors, the corresponding author states that there is no conflict of interest.

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© Springer Nature Switzerland AG 2019

Authors and Affiliations

  1. 1.Department of ChemistryUniversity of NairobiNairobiKenya
  2. 2.Department of MicrobiologyUniversity of NairobiNairobiKenya
  3. 3.Sensor Lab, Department of ChemistryUniversity of Western CapeCape TownSouth Africa

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