Phytochemical Analysis
The phytochemical constituents of the plant extracts studied are summarized in Table 1. The concentration of phytochemicals in the tested plant extracts were qualitatively determined based on the color intensity observed in the reaction mixture as stipulated by the analytical procedure [24 - 27]. We noted that phenols, saponins, steroids, tannins, anthocyanins, coumarins, terpenoids, and flavonoids were present in all tested extracts of Ocimum gratissimum. In Achillea millefolium extract; phenols, saponins, anthraquinones, coumarins, flavonoids, and terpenoids were recovered. In Perilla frutescens; phenols, saponins, steroids, anthocyanins, and terpenoids were found. Carica papaya extract had phenols, saponins, steroids, coumarins, flavonoids, and terpenoids. While Phenols, saponins, steroids, coumarins, flavonoids, terpenoids, and carbohydrates were identified in Garcinia kola. Besides, flavonoids were found at moderate concentrations in Ocimum gratissimum extract, whereas coumarins were recovered at moderate concentration in Achillea millefolium and Carica papaya extracts than in other plant extracts. Also, steroids were found in moderate concentrations only in Garcinia kola (nuts) and tannins were at the highest concentrations in these nuts than any of the analyzed plants. Carbohydrates were only recovered in Garcinia kola. Even though glycosides were not found in any of the plant extracts, terpenoids, flavonoids, and phenols were instead present in all plant extracts tested. These phytochemical analytical results indicate that each plant contains a blend of different phytochemicals. These phytochemicals could be of important medical value and are asserted to play a major role in reducing and stabilizing Ag ions during the bioformulation of AgNPs [11]. Also, terpenoids, phenols, and flavonoids, which were affirmed present in all analyzed plant extracts, can be responsible for stabilizing and capping AgNPs [23]. Besides, the transformation of flavonoids to keto-enol tautomer might occur leading to the release of free active molecules of hydrogen, which might accelerate the reduction of Ag+ to Ag0 nanoparticles [32]. Moreover, some of the identified phytochemicals such as phenols and terpenoids have polar groups including OH and CO, which can bind to the surface of Ag ions. These groups (OH and CO) may also play a vital role in the mechanisms involved in the bioreduction of metallic Ag [33].
Table 1 Phytochemical results of the plants used in the biosynthesis of AgNPs
|
|
Plant
|
S. No.
|
Phytochemical
|
Ocimum gratissimum (African basil)
|
Achillea millefolium (Yarrow)
|
Perilla fructescens (Perilla)
|
Carica papaya (Pawpaw)
|
Garcinia kola (Bitter kola)
|
1
|
Phenols
|
+
|
+
|
+
|
+
|
+
|
2
|
Saponins
|
+
|
+
|
+
|
+
|
+
|
3
|
Steroids
|
+
|
-
|
+
|
+
|
++
|
4
|
Tannins
|
+
|
+
|
+
|
+
|
+++
|
5
|
Glycosides
|
-
|
-
|
-
|
-
|
-
|
6
|
Anthocyanins
|
+
|
-
|
+
|
-
|
-
|
7
|
Anthraquinones
|
-
|
+
|
-
|
-
|
-
|
8
|
Coumarins
|
+
|
++
|
-
|
++
|
+
|
9
|
Terpenoids
|
+
|
+
|
+
|
+
|
+
|
10
|
Flavonoids
|
++
|
+
|
+
|
+
|
+
|
11
|
Carbohydrates
|
-
|
-
|
-
|
-
|
+
|
(Notation: - = Absent of phytochemical, + = Presence of phytochemical, ++ = Moderate concentration of phytochemical, +++ = high concentration of phytochemical).
Color Change
Also, color change in the reaction mixture was observed. The first visible clue that nanoparticles are formed in a reaction mixture is a color change [34]. Changes of color in the reaction mixtures were perceived macroscopically within 1 to 60 minutes in all the reaction mixtures. The color intensity in each reaction mixture gradually changed from a colorless solution of AgNO3 to varying colors over time (Table 3 and Fig. 2).
Table 3 Color observed in the reaction mixture during biosynthesis of AgNPs
|
Before
|
1 to 60
|
24 hours
|
Solution
|
reduction
|
minutes after
|
after
|
Aqueous 5 mM solution of AgNO3
|
Colorless
|
-
|
-
|
Achillea millefolium leaf extract
|
Brown
|
-
|
-
|
Perilla frutescens leaf extract
|
Dark green
|
-
|
-
|
Garcinia kola extract
|
Yarrow
|
-
|
-
|
Carica papaya leaf extract
|
Dark green
|
-
|
-
|
Ocimum gratissimum leaf extract
|
Dark green
|
-
|
-
|
Achillea millefolium leaf extract + 5 mM AgNO3
|
-
|
Brown
|
Dark brown
|
Perilla fructescens leaf extract + 5 mM AgNO3
|
-
|
Brown
|
Dark
|
Garcinia kola extract + 5 mM AgNO3
|
-
|
Pale yarrow
|
Brown
|
Carica papaya leaf extract + 5 mM AgNO3
|
-
|
Yellow brown
|
Dark
|
Ocimum gratissimum leaf extract + 5 mM AgNO3
|
-
|
Brown
|
Dark brown
|
However, beyond 24 hours, no considerable color change was perceived in the reaction mixture (Fig 2). This insinuated that the reduction of Ag+ to Ag0 was complete. The final color observed in the presence of each plant extract after 24 hours was brown for Garcinia kola, dark brown for Achillea millefolium and Ocimum gratissimum, and dark for Achillea millefolium and Carica papaya.). The change in color in the reaction mixture during the synthesis of AgNPs using plant extracts is attributed to the fact that biologically active compounds found in plant extracts at different concentrations can reduce Ag+ metallic ions to Ag0. This is a clear evidence that AgNPs are formulated [28]. Besides, the induced stimulation of superficial plasmon vibrations of AgNPs can also be responsible for the development of color during the synthesis of AgNPs [35].
pH Variation
A general reduction in pH was noted in the mixture after 10 mL of each plant extract was added to 90 mL of 5 mM aqueous solution of AgNO3 (Table 2). The reduction in pH ranged from 0.10 to 0.97 with the greatest reduction recorded in the reaction mixture with Carica papaya leaf extract were noted. The decreases in pH indicate a reduction of AgNO3 to Ag0 nanoparticles in the presence of each plant extract. Also, decreases in pH in the reaction mixture have been reported during the formulation of AgNPs using leaf extracts of Syzygium cumini and Tecomella undulate. In the reaction mixture with Syzygium cumini extract, pH slightly dropped from 4.84 to 4.72 [36] whereas, with Tecomella undulate extract, pH decreased from 6.5 to 5.5 [28]. The variations in pH could also play an essential role in the formulation of nanoparticles with different sizes and shapes. For instance, Mishra et al. [37] substantiated that the synthesis of AgNPs using leaf extract of Averrhoa carambola led to the formation of spherical nanoparticles at pH range from 2.00 to 7.00. Whereas at higher pH of 8.00, nanoparticles with other shapes, which include hexagons, spheres, triangles, squares, and rectangles were synthesized [37]. These documented results correlate with those obtained in this study as the HRTEM analysis further confirmed the synthesized AgNPs had varying shapes and sizes.
Table 2 pH variation recorded during biosynthesis of AgNPs
pH of
|
pH before
|
pH after
|
|
Solution
|
Solution
|
reduction
|
reduction
|
Aqueous 5 mM solution of AgNO3
|
6.00
|
-
|
-
|
Carica papaya leaf extract
|
7.83
|
-
|
-
|
Achillea millefolium leaf extract
|
6.06
|
-
|
-
|
Perilla frutescens leaf extract
|
8.12
|
-
|
-
|
Ocimum gratissimum leaf extract
|
7.30
|
-
|
-
|
Garcinia kola extract
|
6.00
|
-
|
-
|
Carica papaya leaf extract + 5 mM AgNO3
|
-
|
6.00
|
5.03
|
Achillea millefolium leaf extract + 5 mM AgNO3
|
-
|
5.10
|
5.00
|
Perilla frutescens leaf extract + 5 mM AgNO3
|
-
|
7.00
|
6.21
|
Ocimum gratissimum leaf extract + 5 mM AgNO3
|
-
|
5.40
|
5.16
|
Garcinia kola extract + 5 mM AgNO3
|
-
|
5.00
|
4.82
|
UV-Vis Spectroscopy
The UV-Vis spectra obtained using colloidal solutions of biosynthesized AgNPs and the plant extracts (controls) are shown in Fig. 3 with single absorption peaks centered at 441, 429, 451, 438, and 447 nm. These peaks correspond to Achillea millefolium, Perilla frutescens, Garcina kola, Carica papaya, and Ocimum gratssimum AgNPs. These spectrum absorption peaks for the biosynthesized AgNPs correspond to the surface plasmon resonance (SPR) formed as oscillating electrons from AgNPs come in resonance with light waves [38, 39]. This is an indication that addition of each plant extract to AgNO3 aqueous solution stimulated a transition of electrons, resulting in Ag2+ ions being reduced to Ag0 [38,40]. Moreover, the broadening nature of peaks for all the formulated AgNPs, as can be seen in the UV-Vis spectra indicates that the nanoparticles are polydisperse and this greatly correlates with the HRTEM results.
FTIR Spectroscopy
The FTIR spectra of Achillea millefolium, Perilla frutescens, Ocimum gratissimum, Carica papaya, and Garcinia kola, alongside their biosynthesized AgNPs are shown in Fig. 4. The spectra of Achillea millefolium extract and Achillea millefolium AgNPs (Fig. 4A) showed peaks at 3400.04 and 3130.79 cm-1 (O‒H stretch from alcohol or phenol), 2930.02 cm-1 (C‒H stretch of alkanes), 1611.71 and 1596.05 cm-1 (N-H stretch of amine), 1400.86 and 1399.17 cm-1 (C-H stretch of alkanes), 1077.10 and 1079.74 cm-1 (C-OH stretch), 617.39, 539.38 and 531.55 cm-1 (C-Cl stretch of alkyl halides).
The IR spectra of Perilla frutescens extract and AgNPs from Perilla frutescens (Fig. 4B) have peaks at 3400.72 and 31778.67 cm-1 (O‒H stretch from alcohols or phenols), 2345.39 cm-1 (–C=C- stretch of alkynes), 1603.76 cm-1 (N-H stretch of amines), 1400.64 cm-1 (C-H stretch of alkanes), 1055.00 cm-1 (C-OH stretch) and 617.39 cm-1 (C-Cl stretch of alkyl halides). For Ocimum gratissimum extract and AgNPs synthesized using Ocimum gratissimum (Fig. 4C), the IR spectra have peaks at 3399.90, 3210.95, and 3129.70 cm-1 (O‒H stretch from alcohols or phenols), 2930.02 cm-1 (C‒H stretch of alkanes), 2345.69 cm-1 (–C=C- stretch of alkynes) 1610.36 and 1595.75 cm-1 (N-H stretch of amines), 1399.17 cm-1 (C-H stretch of alkanes), 1118.57, 1067.51 and 1032.16 cm-1 (C-OH stretch) 705.94, 618.35, 617.48, and 533.93 cm-1 (C-Cl stretch of alkyl halides).
In the case of Carica papaya extract and Carica papaya AgNPs and (Fig. 4D), IR spectra peaks appeared at 3357.42 and 3138.07 cm-1 (O‒H of alcohols or phenols), 2939.04. 2932.46 and 2848.17 cm-1 (C‒H stretch of alkanes), 2346.26 and 2342.87 cm-1 (–C=C- stretch of alkynes) 1647.54 and 1638.38 cm-1 (N-H stretch of amine), 1400.64 and 1396.92 cm-1 (C-H bend), 1257.59 cm-1 (C-N stretch of amines), 1058.55 and 1036.33 cm-1 (C-OH stretch), 917.74, 816.92 and 779.05 cm-1 (C-H bend), 670.12, 615.48 and 540.11 cm-1 (C-Cl stretch of alkyl halides).
The IR peaks for Garcinia kola extract and Garcinia kola AgNPs were seen at 3391.14 and 3136.50 cm-1 (O‒H of alcohols or phenol), 2937.14 and 2922.28 cm-1 (C‒H stretch of alkanes) 1634.22 cm-1 (N-H stretch of amide), 1401.17 cm-1 (C-H bend), 1260.08 cm-1 (C-N stretch amines), 1056.12 and 1024.04 cm-1 (C-OH stretch), 901.45, 831.01 and 705.08 cm-1 (C-H bend), 627.55, 562.84 and 527.10 cm-1 (C-Cl stretch of alkyl halides) (Fig. 4E).
The absorption peaks (Fig. 4A, B, C, D, and E) suggest that phytochemicals with functional groups such as OH (alcohol and phenol), C-H (alkanes), –C=C- (alkynes), =C-H (alkenes), N-H (amines), C-OH (carbonyls) and C-Cl (alkyl halides) were present in all the plant extracts and could be involved in the biosynthesis of AgNPs. These results are similar to previously reported in the literature that revealed phenols, alcohols, alkanes, alkynes, carbonyls, protein, and other metabolites inform extracts of some plants can interact with metallic ions during the formation of nanoparticles [33, 41, 42]. In addition, the results insinuate that the biosynthesized AgNPs could be enclosed by metabolites and proteins coming from the plants. For example, the identified IR peaks corresponding to the carbonyl (C-OH) group present in all samples analyzed, could be radiating from proteins and residual amino acids and are alluded to strongly bind to metal ions. Moreover, proteins can function as capping agents for AgNPs and hence, stabilize the emulsion and also prevent agglomeration, implying that bioactive functional molecules from the plant extracts have a dual function in the synthesis and stabilization of AgNPs [41].
When comparing the absorbance peaks of these plant extracts (control) to their biosynthesized AgNPs (Fig. 4A, B, C, D, and E), some shifts in peaks can be seen. This is probably due to the re-arrangement of functional groups found in the plant extracts as the synthesis of AgNPs takes place. For instance, the general shift (shift to a lower wavelength) in peaks of the hydroxyl group was noted and can be considered there was oxidation of this group during the formation of AgNPs with increased production of a carbonyl group [43]. Likewise, when comparing the IR peaks for the 5 biosynthesized AgNPs (Fig. 4.3F), intense absorbance peaks were seen around 3137.21 cm−1 (O‒H of alcohol or phenol), 1634.22 cm−1 (N-H stretch), 1400.64 cm−1 (C-H bend), and 1055.00 cm−1 (C-OH stretching). The functional groups associated with these peaks suggest that major phytochemicals such as flavonoids, terpenoids, and phenols could be considerably involved in the formulation of AgNPs [44]. Equally, phytochemical analysis confirmed the presence of flavonoids, terpenoids, and phenols in all plant extracts.
SEM and EDX Analysis
The SEM micrographs (Fig. 5a, c, e, g, and i) show clouds of well-capped and homogenous nanoparticles of different sizes and shapes. The larger nanoparticles seen in the micrographs can be due to the aggregation of smaller particles, which could be linked to the SEM analytical procedure used [45]. However, the HRTEM results of this study were then used to confirm the shapes and sizes of the synthesized nanoparticles. Besides, the EDX results (Fig. 5b, d, f, h, and j) further established that the clouds of nanoparticles seen in the SEM micrographs are AgNPs. This is because EDX data for similar samples had very strong optical absorption peaks at 3 keV, which corresponded to the surface plasmon resonance of metallic Ag. The strong peaks affirmed the formation and purity of the AgNPs. However, other weak optical peaks for C, O, Cl, Cu, and P were observed. These weak peaks may have originated from organic biomolecules in these plant extracts used during the biosynthesis of AgNPs [45].
HRTEM Analysis
The HRTEM micrographs obtained at 50 and 100 nm magnifications showed polydispersed nanoparticles with different shapes and sizes (Fig. 6a, a1, b, b1, c, c1, d, d1, e, and e1). This could be linked to the nature and quantity of the capping agents found in each plant extract. The micrographs of AgNPs formulated using Garcinia kola showed spherical, triangular, and hexagonal shaped AgNPs (Fig. 6a and a1). The size distribution histogram (Fig. 6a2) confirmed that the sizes of the nanoparticles ranged from 5 to 45 nm (mean size of 19.1 nm). The nanoparticles with the highest frequency were between 10 to 15 nm in sizes similar to those documented by Abdi et al. [13]. The micrographs (Fig. 6b and b1) for Carica papaya AgNPs revealed that the nanoparticles were tetragonal, hexagonal, and spherical with histogram (Fig. 6b2) indicating the sizes of the nanoparticles were in the range of 20 to 120 nm with a mean size of 67.5 nm. The particles of sizes 60 to 70 nm had the highest frequency. In the case of Achillea millefolium AgNPs, the micrographs (Fig. 6c and c1) showed spherical shaped AgNPs. The histogram (Fig. 6c2) disclosed that the average size of nanoparticles was 10.9 nm (range: 4 to 18 nm) with the most frequent ranging from 9 to 10 nm. Furthermore, the HRTEM micrographs of AgNPs synthesized from Perilla frutescens taken at 50 and 100 nm magnifications (Fig. 6d and d1) revealed the presence of tetrahedral, triangular, oblong, and spherical AgNPs. The histogram (Fig. 6d2) indicated that the nanoparticles were ranging from 0 to 50 nm in sizes and with an average size of 26.1 nm. The most frequent nanoparticles were those with sizes ranging from 20 to 25 nm. For Ocimum gratissimum AgNPs (Fig. 6e and e1), the micrographs showed spherical and oblong nanoparticles. The histogram (Fig. 6e2) confirmed that the nanoparticles were ranging from 6 to 33 nm in sizes with a mean size of 15.8 nm and the most frequent particles were ranging from 12 to 14 nm in sizes.
XRD diffractometry
The crystalline nature of AgNPs was determined by XRD analysis. The obtained XRD patterns for the analyzed samples recorded at 2θ are shown in Fig. 7. The XRD pattern of Achillea millefolium AgNPs showed intense peaks at 38.1°, 44.3°, 64.4°, 77.3°, and 81.5°, that correspond to (111), (200), (220), (311), and (222) lattice planes, respectively (Fig. 7a). This XRD data for Achillea millefolium AgNPs match with standard data from the International Centre for Diffraction Data (ICDD) for Ag with code No. 03-065-2871. The XRD pattern for Perilla frutescens AgNPs (Fig. 7b) had major XRD peaks at 38.0°, 44.2°, 64.4°, 77.3°, and 81.4° that correspond with lattice planes at (111), (200), (220), (311), and (222), respectively. This data equally match with the ICDD for Ag with reference code No. 04-001-2617. Fig. 7c is the XRD pattern for Ocimum. gratissimum AgNPs with intense peaks at 38.1°, 44.3°, 64.4°, 77.4°, and 81.5° that corresponds with Ag lattice planes at respectively, (111), (200), (220), (311), and (222). This XRD data matches with standard data of the ICDD for Ag with reference code No. 04-004-6437. The XRD pattern for Carica papaya AgNPs (Fig. 7d) had intense peaks at 38.0°, 44.2°, 64.3°, 77.3°, and 81.4° corresponding with lattice planes of Ag at (111), (200), (220), (311), and (222), respectively. This data agrees with the ICDD for Ag with reference code No. 04-014-0266. For Garcinia kola AgNPs, XRD peaks were observed at 37.9°, 44.1°, 64.2°, 77.0°, and 81.2° (Fig 7e). These peaks correspond to (111), (200), (220), (311), and (222) lattice planes of Ag. The obtained data correlate well with ICDD for Ag (reference code No: 04-003-7118). These XRD patterns all had intense peaks at around 38.0°, 44.0°, 64.0°, 77.0°, and 81.0°, which are assigned with lattice planes of Ag at 111, 200, 220, 311, and 222, respectively (Fig. 7a, b, c, d, and e). These peaks can be indexed as crystalline AgNPs with face-centered cubic (FCC) lattice. The intense diffraction peaks (Bragg reflections) indicate that the x-ray scattering spots are radiating from crystalline materials found in the analyzed samples. These crystalline materials are inorganic compounds, which certainly are Ag nanomaterials [46]. Nonetheless, some of the samples had unassigned peaks, which were denoted (*) (Fig. 7a, b, and c). These unassigned peaks (*) could be coming from other crystalline metalloproteins/bioorganic composites present in the plant extracts used for the biosynthesis of AgNPs [47, 12].
ζ-potential Analysis
The ζ-potential values for all the formulated AgNPs were negative (-13.4, -14.3, -23.0, -16.3 and -22.4) for Garcinia kola, Carica papaya, Achillea millefolium, Perilla frutescens, and Ocimum gratissimum AgNPs, respectively (Fig. 8a, b, c, d, and e). The findings are in agreement with other studies wherein negative values for ζ-potential obtained for AgNPs were biosynthesized using plant extracts [48, 49]. These negative zeta values suggest that the biosynthesized AgNPs are superficially negatively charged and stable demonstrating satisfactory evidence that the AgNPs could have strong surface charges that can hinder agglomeration. Hence, these negatively charged nanoparticles could always be apart from each other in solution due to Coulomb explosion. Moreover, the obtained negative values for ζ-potential affirmed the effectiveness of the bioactive capping agent from plant extracts to stabilize the formulated AgNPs [50, 51].