The synthesis and characterization of NPs and their applications represent a rapidly growing concept and an emerging trend in science and technology [50]. The use of plant materials for the synthesis of NPs could be more advantageous because it does not require elaborate processes [51]. The UV–vis spectroscopy technique can be used to determine the synthesis and stability of AuNPs in an aqueous solution due to their characteristic absorption in the range of 500–600 nm [52, 53]. AuNPs exhibited ruby red color in the aqueous solution due to the excitation of surface plasmon vibrations in the metal NPs which give rise to the surface plasmon resonance (SPR) band [54, 55]. The plasmon resonance (PR) can be pictured as a ‘‘wave’’ of electrons sloshing over the surface of a metal NPs. As a result, an enhanced electromagnetic field at and near the metal NPs surface is set up. The position of the plasmon band (extinction spectrum) is best measured on a conventional UV–vis spectrophotometer and appears as a band with extremely high extinction coefficients [56]. In the present study, when 1 mL of HH aqueous extract was added to 1 mM HAuCl4.3H2O solution at pH 4 the color of the solution start to change from golden yellow to crimson red and then finally to ruby red at an optimized ratio after 24 hrs of incubation at 40ºC. The appearance of the ruddiness color in an aqueous medium is considered the first indication of colloidal AuNPs formation [53, 54]. This color change is due to the reduction of the Au+ into HH-AuNPs by the active molecules of the HH aqueous extract such as phenolic, alkaloids, saponins, amino acids, proteins, etc. The absorption spectrum of the aqueous solution revealed a peak at 576 nm with a maximum absorbance of 1.96 after 24 hrs.
Synthesis of the AuNPs is affected by different factors such as plant extract volume, HAuCl4.3H2O, pH, temperature, time, etc. [57]. An ideal pH is required for synthesis of controlling the shape and size of NPs [58, 59]. The role of pH is significant in changing the size and shape of NPs. Numbers of studies have shown the stability of AuNPs at acidic pH while many achieved stable suspension in the basic region. Furthermore, the synthesis reaction, size, shape, and stability of AuNPs could be controlled by adjusting the initial pH value of the reaction mixture. In the present study, the reaction was performed at different pH ranges from 4 to 12 to identify the effect of pH on the formation of HH-reduced AuNPs. It was detected that the absorbance of the solution increased while changing the initial pH of the solution. With the increase in pH, the SPR band was also blue-shifted to 576 nm at pH 4. The AuNPs were quite stable in an acidic medium; however, a gradual decrease in UV–vis peak intensities showed less stability with an increase in pH of the colloidal solution from 4 to 12. Peak broadening and redshift were noted at pH 10 and 11. UV-vis results suggested that no reaction occurred in the basic region which is in agreement with the previous studies [54]. It was might be due to the deprotonation of hydroxyl and carboxyl groups present in extracts [60]. AuNPs synthesized using banana peel extract were stable at a pH value of 2–5 supporting the results of the present study [61]. A broader range of pH i.e. 2–11 were taken to synthesize AuNPs using oil palm mill effluent and pH 3 was observed optimum to achieve definite shapes particles [62]. Medicinal plants are a rich source of secondary metabolites that act as reducing, capping, and stabilizing agents for NPs. However, the composition of these active secondary metabolites varies from plant to plant depending on the nature, part, type of plant, and method followed for the extraction of these metabolites [63]. In the present study, the SPR peak is blue-shifted from 553 to 576 nm as the added volume of HH extract increased from 0.5 to 1mL; the blue shift of SPR peaks is a sign of the production of small size NPs. The shift towards shorter wavelengths with decreasing NPs size is associated with frequencies of oscillation of different free electrons in the conduction band [64]. The SPR band absorbance increased with an increasing volume of HH extract, which reveal the higher production of AuNPs. This is due to the availability of more reducing agents for the Au ions bioreduction [65]. But the further increase in the volume of HH extract from 1.5 to 2 mL the redshift of SPR was recorded, this redshift is a sign of large size NPs. HH contains active molecules such as carbohydrates, flavones, terpenoids, alkaloids, and proteins that were testified to be responsible for the bio-reduction of Au + to Auo. Proteins and terpenoids are believed to play an important role in AuNPs biosynthesis through the reduction of Au ions, and carbohydrates provide a coating of AuNPs [66, 67]. The HH extract produces more AuNPs than other plant extracts credited to the availability of the larger amount of reducing agents in the extract, such as flavonoid and its antioxidant activity [67]. Varying concentrations of HAuCl4.3H2O (0.25 to 1.5 mM) were prepared while the other factors were kept constant i-e HH extract (1 mL), pH (4), temperature (40ºC), and time (24 hrs). At 0.25 and 0.5 mM concentrations of HAuCl4.3H2O broad peaks were revealed; while at increased HAuCl4.3H2O concentration (1 mM) a sharp peak of 576 nm with maximum absorbance 1.96 was reported but beyond this concentration the peak becomes broad. Thus it can be reported from the present results that the absorption peak intensity increase with an increase in HAuCl4.3H2O salt concentration. All the results of the present study are in good covenant with the results reported in the literature [68]. Temperature is another factor that plays a crucial role in tuning the size and shape of AuNPs. The effect of temperature on the SPR feature of metal NPs is a critical factor in the pure and applied science of the NPs [69, 70]. In the present work, the effect of temperature on AuNPs synthesis was studied at five different temperatures, that is, 20ᵒC, 40ᵒC, 60ᵒC, 80ᵒC, and 100ᵒC, and the UV-vis spectra were recorded after 24 hrs. From the results, it is clear that the rate of AuNPs increase with increasing temperature. Earlier, similar findings related to the increase in the reaction rate of AuNPs synthesis with an increase in reaction temperature have also been reported by [71, 72]. According to the UV-vis spectra of AuNPs synthesized at different temperatures, it is reported that there is a blue shift to 576 nm with a maximum absorbance of 1.96 at 40ᵒC in comparison to the AuNPs synthesized at 20ᵒC. The results suggest that the higher temperature leads to an increase in the activation energy of the molecules and a faster rate of reaction. As a result, there is a decrease in the size of synthesized AuNPs and, hence, monodispersed small NPs are formed without undergoing the phase of particle size growth [73]. However, the present study shows that a further increase in reaction temperature (60ᵒC to 100ᵒC) and an increase in the size of AuNPs were reported as is evident from the sharp and narrow SPR peaks with increased sphericity. These results are consistent with the previous findings discussing the same in context to the increased reaction rate of AuNPs synthesis upon increasing the reaction temperature. The high reaction temperature leads to a rapid nucleation process of metallic NPs involving the enhanced consumption of most of the metal ions with the least secondary reduction of the preformed nuclei [73, 74]. It has been shown that an optimum temperature could help control the rate of AuNPs synthesis and the uniform NPs can be synthesized under optimum pH at different reaction temperatures [75].
The possible role of Phyto-components present in aqueous plant extract responsible for mediating and stabilizing the nanoparticles was depicted using FTIR analysis. A perusal of scientific studies reports FTIR as one of the ideal tools to predict functional moieties. In the present investigation, vibrational stretch occurring at different peaks which corresponds to polyhydroxy, phenol, carboxyl, proteins, lipids, amide, alkynes, alkene etc. Scientific studies on FTIR analysis of plant-mediated NPs report that different functional moieties like hydroxyl, carboxyl, and amide are responsible for the reduction of metal ions to produce NPs [76, 77]. The obtained result of FTIR analysis is by previous findings [78]. Interestingly, in the plant-mediated synthesis of NPs, the Phyto-components also play important role in the stabilization of NPs which is very crucial for rendering its applicative properties. These results also coincide with reports of earlier findings [79, 80].
XRD is used for the phase identification and characterization of the crystal structure of the AuNPs. XRD analysis of HH-AuNPs shows four distinct peaks at 38.02°, 44.29°, 64.37°, and 77.58° which are corresponded to the planes (1 1 1), (2 0 0), (2 2 0), and (3 1 1) respectively. The mean size for HH-AuNPs was calculated using Debye-Sherrer’s equation is 10.72 nm. The “d” and “a” values were calculated by using Debye-Sherrer’s equation. To fulfill Bragg’s Law the incidence theta (θ) must vary with the change in “d” values which showed that as the value of θ increases the “d” values of the atomic layers decrease. Similar results were also reported by [7, 81].
SEM was used to confirm the production and examine the morphology characterization at the nm to µm scale of the obtained AuNPs [82]. In the present study, the SEM data revealed that the AuNPs were spherical with an average particle size of 30 nm. SEM analysis shows uniformly distributed HH-AuNPs that indicate the stabilization of AuNPs by HH extract capping agents. Green synthesis of spherical shape AuNPs with particle size range from 21 to 45 nm was carried out by using Stevia rebadiauna leaf extracts are in good accordance with the results of the present research work [81].
The elemental composition was determined using EDX [83, 84]. The percentage of Ag metal in the present study was found to be appreciable. The EDX analysis showed the percentage relative composition of Au signal of the AuNPs with 48.08%. The other elements served as capping organic agents bound to the surface of the AuNPs [85]. This recognition was made because of the registered energy, which is characteristic of AuNPs. [86] reported the biosynthesis and characterization of AuNPs using extracts of Tamarindus indica L leaves are in good accordance with the results of the present study.
Oxidative stress has been linked to the cause of many deadly diseases such as Neurological disorder, Parkinson disease, mild cognitive impairment, and aging, etc the managing of which is costly [87]. Thus, plant extracts and NPs with antioxidant, antidiabetic, and anticholinesterase properties will be greatly beneficial [88]. Antioxidants are substances that can inhibit or delay the oxidation of a substrate when present in low concentrations. Due to the relationship of oxidative stress to other diseases, we, therefore, investigated the antioxidant capacities of HH extract, and the corresponding HH-AuNPs. Five essays; Ferric reducing antioxidant power, AmmoniuM Molybdate, DPPH, H2O2, and ABTS were carried out. The mechanism with which ferric reducing operates is known as single electron transfer (SET), whereby an antioxidant transfers an electron to the corresponding cation, which would neutralize it [89]. Thus the reducing capacity of AuNPs might be due to their quick electron transferring ability, which makes AuNPs suitable for biosensors [90].
In the present study, strong ferric reducing activities were exhibited by HH-AuNPs (77.2 ± 0.014% with IC50 95.17 ± 0.025µg/mL) as compared to HH extract (55.2 ± 0.035% with 151 ± 0.13 µg/mL IC50) and HAuCl4.3H2O solution (16.92 ± 0.014 with IC50 = 0), while lower scavenging then ascorbic acid (84.4 ± 0.016% with IC50 = 79 ± 0.06 µg/mL). This inhibition is due to the HH extract hydroxyl groups attached to aromatic rings, which will perfectly participate in oxidation during the process. It is well known that phenolics have strong antioxidant activities [91]. However, since the ferric reducing mechanism is by electron transfer, the hydroxyl groups of the HH extract might be interacting with the NPs, thereby limiting the site for the oxidation process. High activities in close ranges were demonstrated for AuNPs in ferric reducing assay which might be due to the size, shape, and the surrounding environment of NPs. Therefore, it is expected that AuNPs behave somewhat differently since the smaller sized particles have a higher surface area. Previously, NPs with smaller-sizes were reported to show enhanced activity in comparison to relatively plant extract [59].
Ammonium molybdate is an ammonium salt composed of ammonium and molybdate ions. It has a role as a poison as it contains a Molybdate. It can be used as a free radical in the antioxidant assay. In the present study, all the four samples of HAuCl4.3H2O solution, HH extract, HH-AuNPs, and ascorbic acid were tested for their Ammonium Molybdate scavenging; HAuCl4.3H2O solution, HH extract, HH-AuNPs, and ascorbic acid showed maximum scavenging potential of 22 ± 0.0014, 62.1 ± 0.0021, 74.3 ± 0.021, and 88.8 ± 0.001% at 160 µg/mL. The calculated IC50 values were found to be 91.48 ± 0.13 for HH extract, 58.5 ± 0.051 for HH-AuNPs, and 48 ± 0.11 µg/mL for ascorbic acid respectively; while the HAuCl4.3H2O solution does not cause 50% scavenging. The scavenging potential of HH-AuNPs is higher as compared to HAuCl4.3H2O solution and HH extract [92].
DPPH is a free radical which changes its color from violet to yellow on reduction by a hydrogen or electron [93]. In the DPPH scavenging assay, the compounds which can reduce DPPH are considered antioxidants. By DPPH radical scavenging assay, it was found that AuNPs were able to react with free oxygen radicals and hence, possessed strong antioxidant activity as compared to HH extract and HAuCl4.3H2O solution (HAuCl4.3H2O solution of 21 ± 0.007, HH extract 55 ± 0.059, HH AuNPs 58.6 ± 0.51, and Ascorbic acid 90 ± 0.0014% % at 160µg/mL). The biological activity of NPs is depending upon the aspect ratio of particles. NPs with a high aspect ratio have been demonstrated to exhibit good antioxidant properties [94]. The result was also confirmed by the finding of [92, 95] who showed that a higher concentration of AuNPs significantly showed high scavenging capacity as compared to Acinetobacter sp. Similar results were also reported by [96, 97].
H2O2 is an unstable inorganic compound that damages the cell membrane in the living organism. It is also known as oxidant or dioxide and can be used as free radicals in antioxidant activity. An anti-oxidant compound donates an electron to H2O2 ions and thus neutralizes it to water [93]. In the present study synthesized HH-AuNPs were able to scavenge 62 ± 0.041% using 160 µg/mL concentrations in comparison to HAuCl4.3H2O solution and HH extract which scavenge 21 ± 0.021 and 56.6 ± 0.0021% respectively. The calculated IC50 values were found to be 136 ± 0.21 for HH extract, 105.66 ± 0.062 for HH-AuNPs, and 51.18 ± 0.3 µg/mL for ascorbic acid. [97] reported that AuNPs can catalyze the rapid decomposition of H2O2. H2O2 scavenging activity shown by AuNPs is almost equal to other embedded 3,6-dihydroxyflavone AuNPs, used to enhance the antioxidant activity (Medhe et al., 2014).
On the other hand, a clear trend can be observed for the ABTS assay. This is probably because of the difference in the mechanism of operation between the assays. ABTS is largely operating on hydrogen atom transfer. The trend in the ABTS results is such that individual AuNPs demonstrated better antioxidant capacity relative to their respective precursors. Recent research reports [19, 91, 98] supported the above submission. AuNPs biosynthesized from Halymenia dilatata also demonstrated higher antioxidant activity than the starting plant extract [99]. Thus biosynthesis of NPs, has been reported for its antioxidant capacity [100]. These antioxidant compounds might get adsorbed onto the active surface of NPs. The surface reaction phenomenon of these biosynthesized NPs (due to adsorbed antioxidant moiety onto the surface) and the high surface area to volume ratio of NPs generate a tendency to interact and scavenge the free radical [101]. NPs donate electrons to free radicals due to which the free radical becomes stable. The enhanced potential of the HH-AuNPs is due to their control size, shape, and type due to which they became more reactive as compared to the plant extract [92].
Dietary antioxidants have been hypothesized to have a protective effect against α-amylase. The human salivary α-amylase digests the starch into small fragments with two or three pieces. Hence, the inhibition of the α-amylase enzyme could control the carbohydrate metabolism which also decreases the amount of glucose absorption. It seems plausible that a sufficient intake of antioxidants plays an important role in protection against type 2 diabetes [102]. In the present study the observed inhibitory percentage of α-amylase by HAuCl4.3H2O solution, HH extract, HH-AuNPs, and standard drug Glucophage are shown. The α-amylase inhibitory activity of HH-AuNPs exhibited the highest inhibitory activity with 56 ± 0.003 µg/mL IC50 when compared to HAuCl4.3H2O solution and HH extract. The positive control Glucophage has revealed the potent α-amylase inhibitory activity with the IC50 value of 37 ± 0.13 µg/mL. This behavior is similar to that reported by [21], therefore, closely related behaviors in the same experimental conditions are obvious. Accordingly, the improved antidiabetic performance of AuNPs over their precursor extracts has been reported by [103, 104]. The IC50 values of α-amylase of HH-AuNPs form also showed improved activity in agreement with previous studies [103]. The inhibiting powers of the nanomaterials may be a function of size and shape. In our results, HH-AuNPs had a size of 10.72 nm. These values are similar to the size of AuNPs in previous investigations [21]. Similarly, [105] affirmed that spherical AuNPs of sizes 20 and 40 nm in diameter induced the west Nile virus better than those of other sizes and shapes. This may be the reason for the improved enzymatic activity of AuNPs. The enzyme kinetics competitively revealed inhibition for HH on α-amylase (Km is increased, whereas Vmax remains the same). These findings indicate that some of the α-amylase inhibitory components in HH extract may be structural analogs of the substrate that compete for binding at the active site of α-amylase, while in the case of HH-AuNPs effect on α-amylase a non-competitive type of inhibition was reported (Km remained constant and Vmax is decreased), this suggests that some of the α-amylase inhibitory components in this HH-AuNPs resulting from HH extract bind only to the enzyme-substrate complex and may alter the active site of the enzyme. α-amylase inhibitors delay the rate of carbohydrate digestion, thereby providing an alternative therapeutic option for modulation of postprandial hyperglycemia [106]. In diabetic patients with a sustained reduction of hyperglycemia is shown to decrease the risk of developing microvascular and macrovascular diseases and their associated complications [107]. The HH extract exhibited higher inhibitory activity towards the α-amylase as opposed to many other hypoglycemic plants reported in previous studies [108]. In comparison, the first time in vitro hypoglycemic assessment of HH-AuNPs indicates higher bioactive properties. The enhanced activity of AuNPs obtained in the α-amylase assessment may be due to their high surface area to volume ratio, thus increasing the surface area phenomenon (promoting the electron transfer reaction) and may increase the pharmacokinetics from a biological view. The effects of oral hypoglycemic drugs depend on several pharmacokinetic factors such as absorption, metabolism, and excretion, and the actions of drugs begin inside the cells, it is believed that AuNPs' small size is easily carried across the cell membrane by transport proteins and may exhibit prolonged effects in bio-systems [109]. Given the α-amylase inhibitory effects of HH-AuNPs, the results obtained in this study are coherent with previous studies [110].
Antioxidants such as vitamin C and vitamin E have been associated with AChE inhibition and play an important role in AD treatment [102] and patients with AD on take high doses of antioxidants will have a slow cognitive deterioration rate [111]. Inhibition of the AChE is considered promising in AD disease treatment and various researches are focused on new inhibitors from the herbal resources [27, 112–114]. To our knowledge, no attention has been given to AuNPs synthesized using the plant for the treatment of AD. The HAuCl4.3H2O solution, HH extract, and HH-AuNPs synthesized using the HH extract were screened for their in vitro AChE inhibitory activity. Their IC50 values are found to be 19.1 ± 0.021 for HH extract and 16.66 ± 0.011 µg/mL for HH-AuNPs while no concentration of HAuCl4.3H2O solution cause 50% inhibition of AChE. Under the same conditions, the IC50 values of HH-AuNPs showed a remarkable increase in activity toward AChE than extract. This finding is noteworthy because AD is associated with AChE deficiency and AuNPs could be potential new AChE inhibitors [85]. The good anti-AChE potential of AuNPs as compared to HH extract is due to the size and shape that may find useful in the AD treatment [115]. According to Line Weaver Burk Plot HH-AuNPs causes an uncompetitive type of inhibition of AChE (both Km and Vmax decrease), while HH extract caused a non-competitive type of inhibition. From the kinetic study, it is reported that AuNPs primarily cause the inhibition by interaction or adsorption with the AChE enzyme. The exact mechanism by which AuNPs inhibit AChE remains unknown. The binding affinity of NPs to AChE might be due to the lipophilicity of the NPs and the hydrophobicity of the enzyme environment in ChE molecules. Another possible mechanism of AChE inhibition might be due to the adsorption of AChE on the surface of NPs resulting in conformational changes, and surface coverage leading to the inactivation of the enzyme as reported by [116]. Inactivation of the enzyme by NPs depends on physicochemical properties like shape, size, curvature, and surface functional groups. [12] proposed that inhibition by NPs is primarily caused by adsorption or interaction with AChE protein and partly by dissolved metal ions.