3.1. Characterizations of biosynthesized Au NPs
Biogenic reduction of Au3+ ions to Au NPs over the plant extract could be monitored visibly by color change and spectroscopically by UV-Vis technique. The instant the Mentha pulegium extract was added into the aqueous solution of HAuCl4, color of the solution started changing from yellow to dark red (Fig. 1). Surface Plasmon resonance phenomenon confirmed the formation of Au NPs. As the reaction progressed, intensity of the color increased. In addition to visible tracking, a UV-Vis spectrum of the reaction solution was also recorded to detect the progression of the reaction. Fig. 1 displays the characteristic change as the initial peak due to Au(III) at ~520 nm disappeared as the reaction went to completion to Au (0).
The as synthesized Au NPs were characterized with analytical techniques like Fourier Transformed Infrared Spectroscopy (FT-IR), Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), Energy Dispersive X-ray spectroscopy (EDX) and Powder X-ray diffraction (XRD).
Fig. 2 represents the FT-IR spectra of Mentha pulegium extract itself (a) and the biomolecule modified Au NPs (b). The extract shows strong signals at 3402 cm−1, 2872 cm−1 and 1679 cm−1 which are attributed to phenolic O-H, C-H and C=O bonds respectively. The FT-IR profile of the flower extract almost overlaps with the Au NP spectrum. This clearly indicates the successful immobilization of flower phytochemicals over Au NPs. The results obtained from the FT-IR spectra confirmed that nanoparticles are coated with biomolecules, especially with the amino acid residues of the proteins. The amino acid residues of proteins have a strong ability to bind with metal by coating their surface and preventing them from aggregation and are responsible for reduction and stabilizing. Thus, from the above discussion, it can be concluded that biomass (protein, phenolic, and alcoholic compounds) perform the function of stabilizing and reducing agents for Au NPs [15].
Structural morphology of the biosynthesized Au NPs was investigated by SEM and HR-TEM techniqes. The SEM image, presented in Fig. 3, depicts lump like agglomerations which might be due to high concentration during sampling. However, in a closer view it shows polyhedron and spherical globules adhered homogeneously. Due to tiny size the particles have high tendency towards association. The HR-TEM images display a better structural insight with a clear view of the particle shape and size of Au NPs. As can be seen from Fig. 4, most of the nanoparticles are of homogeneous pherical and octahedral shapes. Average diameter of the particles are within 30-45 nm. Such shape variance could be explained based on Ostwald ripening theory where a large number of small active particles are associated resulting in the growth of larger nanoparticles of anonymous shape and size [32]. There is an agreement between the SEM image and HR-TEM images of the biosynthesized Au NPs.
Energy Dispersive X-ray Spectroscopy (EDX) confirmed the existence of pure Au particles in the specimen (Fig. 5). The presence of C and O elements justifies the phytochemical functionalizations over Au NPs. As the sample preparation was done on a Cu grid, it appears in the profile by default.
In order to investigate the crystalline nature of Au NPs, XRD analysis was performed. As shown in Fig. 6, the characteristic peak position, width and height in the pattern resembles the face centered cubic (fcc) gold. The standard cubic phase of gold, (JCPDS No.65-2870) contains (1 1 1), (2 0 0), (2 2 0) and (3 1 1) planes at 2θ values of 38.2, 44.55, 63.85 and 76.85 respectively which is found to be in close agreement with our material. The other small insignificant peaks can be attributed to phytochemicals present in the flower extract.
3.2. Catalytic performance of the biogenic Au NPs
Following the meticulous physicochemical characterizations of the biosynthesized catalyst, it was the turn to explore its catalytic activity. We investigated the catalyst in the reduction of nitroarenes using NaBH4 as hydride source. At the outset, optimization of reaction conditions was intended and thereby different reaction conditions like catalyst load, solvent and temperature were assorted over a probe reaction, the reduction of nitrobenzene. The experimental outcomes are shown in table 1. The reaction was failed in the absence of any catalyst which signifies alone NaBH4 is not sufficient to carry out the nitro reduction (entry 1, Table 1). 2.0 mmol NaBH4 was required for complete reduction in presence of the catalyst. While studying the solvent effect over the model reaction, the best result was achieved in MeOH/H2O mixture (2 : 1) among the different tested solvents like EtOH, MeOH, H2O, DMF and CH3CN. (entries 2-6, Table 1). 0.1 mol% of catalyst load was found to produce the best result (entry 8, table 1). Again, keeping the other conditions intact, it was observed that the reaction worked the best at 60 oC.
Table 1. Optimization of the reduction of nitrobenzene with NaBH4 in the presence of Au NPs under various conditions.a
Entry
|
Au (mol%)
|
Solvent
|
NaBH4 (mmol)
|
T (°C)
|
Time (h)
|
Yield (%)b
|
1
|
-
|
EtOH
|
2
|
60
|
24
|
0
|
2
|
0.1
|
EtOH
|
2
|
60
|
1
|
60
|
3
|
0.1
|
MeOH
|
2
|
60
|
1
|
70
|
4
|
0.1
|
H2O
|
2
|
60
|
2
|
50
|
5
|
0.1
|
DMF
|
2
|
60
|
1
|
55
|
6
|
0.1
|
CH3CN
|
2
|
60
|
1
|
40
|
7
|
0.1
|
H2O-MeOH (1:1)
|
2
|
60
|
1
|
80
|
8
|
0.1
|
H2O-MeOH (2:1)
|
2
|
60
|
1
|
98
|
9
|
0.05
|
H2O-MeOH (2:1)
|
2
|
60
|
1
|
80
|
10
|
0.1
|
H2O-MeOH (2:1)
|
1.5
|
60
|
1
|
88
|
11
|
0.1
|
H2O-MeOH (2:1)
|
2.5
|
60
|
1
|
98
|
12
|
0.1
|
H2O-MeOH (2:1)
|
2
|
50
|
2
|
88
|
13
|
0.1
|
H2O-MeOH (2:1)
|
2
|
25
|
2
|
50
|
14
|
0.1
|
H2O-MeOH (2:1)
|
2
|
70
|
1
|
98
|
aReaction conditions: nitrobenzene (1.0 mmol), solvent (3.0 mL), open air; bIsolated yield.
After having the standard conditions in hand for the reduction of nitro compounds, it was the turn to establish the scope and generality of those conditions. Diverse ranges of nitroarenes were reduced to corresponding amines in presence of the Au NP over NaBH4. The results have been depicted in Table 2. Both electron attracting and electron withdrawing groups are found to be equally compatible under the reaction conditions producing excellent yields within 1-2 h time range. Turnover frequencies (TOF) are being calculated in all the cases which are moderate to high as presented in table 2.
Table 2. Au NPs catalyzed reduction of aromatic nitroarenes.a
Entry
|
RC6H4NO2
|
Time (h)
|
Yield (%)b
|
TOF (h-1)c
|
1
|
H
|
1
|
98
|
980
|
2
|
4-OH
|
1
|
96
|
960
|
3
|
2-OH
|
1.5
|
95
|
633
|
4
|
4-NH2
|
1.5
|
96
|
640
|
5
|
4-CH3
|
1
|
96
|
960
|
6
|
4-OCH3
|
1
|
92
|
920
|
7
|
4-CN
|
1
|
92
|
420
|
8
|
2-NH2
|
2
|
90
|
450
|
9
|
4-CHO
|
2
|
85
|
420
|
10
|
4-Cl
|
2
|
90
|
450
|
aReaction conditions: Catalyst (0.1 mol%), nitroarene (1.0 mmol), NaBH4 (2.0 mmol), MeOH:H2O (1:2, 3.0 mL), 60 ºC; bIsolated yields; cTOF, turnover frequencies (TOF = (Yield/Time)/Amount of catalyst (mol).
3.3 Study of reusability
In spite of the excellent catalytic efficiency of the biogenic Au NPs, it is very essential to assess the reusability in view of sustainable catalysis. Therefore, after completion of the fresh batch of probe reaction, the catalyst was retrieved by centrifugation, washed thoroughly with MeOH/H2O mixture, dried and reused in successive cycles. The result has been presented in Fig. 7. Interestingly, the catalyst could be reused up to 12 cycles with almost consistent reactivity.
We compared the outcomes of our developed protocol with the hitherto reported articles in the catalytic reduction of nitrobenzene in order to show the distinctiveness. They have been documented in Table 3. It clearly demonstrates that our method is superior in most over the others.
Table 3. A comparison our protocol with some reported methods for the reduction of nitrobenzene.
Entry
|
Catalyst
|
Conditions
|
Time (h)
|
Yield (%)
|
Refs.
|
1
|
Fe3O4 Ni MNPs
|
Glycerol, KOH, 80 ºC
|
3
|
94
|
56
|
2
|
[Pt]@SiC6
|
AcOEt, H2, RT
|
3
|
95
|
57
|
3
|
Nickel-iron mixed oxide
|
N2H4.H2O, propan-2-ol, Reflux
|
1.5
|
96
|
58
|
4
|
Rh
|
N2H4, EtOH, 80 °C
|
2.5
|
99
|
59
|
5
|
Pd Cu/graphene
|
NaBH4, EtOH:H2O (1:2), 50 °C
|
1.5
|
95
|
60
|
6
|
Fe–phenanthroline/C
|
N2H4.H2O, THF, 100 °C
|
10
|
99
|
61
|
8
|
Amberlite-Au NPs
|
NaBH4, MeOH:H2O
(1:1), 40 ºC
|
0.33
|
85
|
62
|
8
|
Au NPs
|
NaBH4, MeOH:H2O
(1:2), 60 ºC
|
1
|
98
|
This Work
|
3.4 Anti-human colon cancer potentials of gold nanoparticles
In our study, the treated cells with several concentrations of the present HAuCl4, Mentha pulegium extract, and AuNPs were examined by MTT test for 48h regarding the cytotoxicity properties on normal (HUVEC), colorectal adenocarcinoma (HT-29), colorectal carcinoma (HCT 116), ileocecal colorectal adenocarcinoma (HCT-8 [HRT-18]), and Burkitt's lymphoma (Ramos.2G6.4C10) cell lines (Fig. 8; Table 4). The absorbance rate was determined at 570 nm, which indicated extraordinary viability on normal cell line (HUVEC) even up to 1000μg/mL for HAuCl4, Mentha pulegium extract, and AuNPs.
In the case of colorectal adenocarcinoma, colorectal carcinoma, ileocecal colorectal adenocarcinoma, and Burkitt's lymphoma cell lines, the viability of them reduced dose-dependently in the presence of HAuCl4, Mentha pulegium extract, and AuNPs. The IC50 of Mentha pulegium and AuNPs against HT-29 cell line were 421 and 218 µg/mL, respectively; against Human HCT 116 cell line were 380 and 193 µg/mL, respectively; against HCT-8 [HRT-18] cell line was 445 and 269 µg/mL, respectively; and against Ramos.2G6.4C10 cell line were 457 and 285 µg/mL, respectively. The best result of cytotoxicity property of gold nanoparticles against the above cell lines was seen in the case of the HCT 116 cell line.
The anticancer of gold nanoparticles was found to be highly dependent on a range of factors related to their physical characteristics, such as surface coating, shape, and size. About the size, it has been reported that gold nanoparticles with small size can transfer of cell membrane of tumor cells and remove them. In the larger size, the above ability significantly is confined [63]. As can be observed in Figures 3 and 4 of our study, gold nanoparticles had uniform spherical morphology in range sizes of 30-45 nm. The size of gold nanoparticles in lower than 50 nm is very suitable for the killing of tumor cell lines in vivo and in vitro [63].
About the anticancer properties of gold nanoparticles, they have used for the treatment of several cancers including human lung cancer, mammary carcinoma, uterus cancer, lung epithelial cancer, Lewis lung carcinoma, colon cancer, and human glioma [64].
Table 4. The IC50 of HAuCl4, Mentha pulegium, and AuNPs in the cytotoxicity test.
|
HAuCl4 (µg/mL)
|
M. pulegium (µg/mL)
|
AuNPs (µg/mL)
|
IC50 against HT-29
|
-
|
421
|
218
|
IC50 against HCT 116
|
-
|
380
|
193
|
IC50 against HCT-8 [HRT-18]
|
-
|
445
|
269
|
IC50 against Ramos.2G6.4C10
|
-
|
457
|
285
|