**2.3 Mechanism and factors affecting the biogenic synthesis of metallic nanoparticles**

Detailed literature report on the mechanism of biogenic synthesis using plant phytochemicals is still lacking although there exists extensive discussion on the roles of chemical functional groups present in plant phytochemicals. From a typical experience, proposed mechanism may proceed through a four-step reaction [16, 39, 40]. The first step which begins with the ionization of the metal in an aqueous solution and trapping of the metal ion on the surface of the phytochemical (dispersion medium) involves electrostatic interaction between the metal ions and the surface charge of phytochemicals. In the second step, attracted ions at the surface of the dispersion medium are reduced to form metal nuclei (nucleation) through electron transfer. In the case of an extract rich in polyphenol or other biomolecules, a metal-polyphenol framework is formed in the process. The continuous reduction and nucleation of ions result in the growth and accumulation of stabilized NPs. In the final stage, accumulated NPs are capped by plant phytochemicals which leads to the growth termination of NPs [24, 36, 41, 42]. Beyond the mechanism, researchers are also concerned about the size, shape, distribution of NPs, and time required in the synthesis. Hence, several factors that affect these properties have been identified and discussed. Factors such as the concentration and nature of plant extract, metal precursor concentration,

temperature, time, and pH play significant roles in the properties (shape, size, and crystallinity) of the synthesized PM-NPs [42–44].

The potential of any plant species for bio-reduction of metal ion for synthesis depends on the contents of its active biomolecules and their concentrations in the plant extract. Plant extracts with high concentrations of biomolecules like proteins, phenolics, and flavonoids are known to greatly enhanced PM-NPs formation. Literature report shows that polyphenols and flavonoids are one of the most frequently reported phytochemicals responsible for plant-mediated metallic nanoparticle synthesis [33]. *Hibiscus sabdariffa* is a tea plant rich in polyphenols, anthocyanine compounds, and flavonoids including hibiscic acid, ascorbic acid luteolin, gallic acid, chlorogenic acid, caffeic acid, protocatechuic acid, eugenol, quercetin, delphinidin-3-sambubioside, delphinidin-3-glucoside, cyanidin-3-sambubioside and cyanidin-3-glucoside [45]. Soto-Roblesa et al. studied the effect of the different concentrations (1%, 4%, and 8%) of *H. sabdariffa* extract on the biogenic synthesis of ZnO NPs and their application in the photocatalytic degradation of methylene blue dye. The result showed that the formation of smaller (5–12 nm) ZnO-NPs of uniform shapes and photocatalytic degradation of methylene blue was achieved with the highest (8%) concentration of *H. sabdariffa* extract compared to the 1%, and 4% concentration [41, 46].

The ratio of the concentration of the metal substrate to plant extract plays a key role in biogenic synthesis. Generally, a concentration range of 1–10 mM of the metal substrate is employed in the synthesis [47]. Within this range, a lower concentration of metal precursor takes longer reaction time but is beneficial for the formation of monodispersed spherical shape and non-aggregated nanoparticles during the nucleation and growth process. The use of a high concentration of the metal precursor has the advantage to speed up the reaction rate but often leads to polydisperse and amorphous NPs due to the aggregation of a large number of nuclei [10]. This is supported by a study conducted by Vitta et al. on the synthesis of iron nanoparticles from an aqueous extract of *Eucalyptus robusta* Sm using different concentrations of iron salt concentrations; 1 mM, 5 mM, and 0.1 M (NH4)2 FeSO4.6H2O (Mohr's Salt). The authors showed that the size distribution of FeNPs formed was dependent on the concentration of the metal substrate used, as the 1 mM concentration of Fe precursor gave average size distribution of 0.8 nm smaller than the size obtained with 5 mM and 0.1 M concentrations. Interestingly, this agrees with the report of the particle size distribution for Cu-NPs biosynthesized from *A. indica* leaf extract by Nagar and Devra [48]. Nagar and Devra, attributed an increase in particle size from 48.01 nm to 78.51 nm due to increased concentration of CuCl2 substrate from 6 <sup>10</sup><sup>3</sup> mol/L to 7.5 <sup>10</sup><sup>3</sup> mol/L, respectively [27, 48].

The effect of temperature is noticeable mainly on the nucleation and NPs growth time, and the size distribution of PM-NPs. Most PM-NPs are formed between 30 and 90°C (working temperature) depending on the metal substrate used and their electrochemical potentials [49]. The working temperature is important for the excitation of electrons and interaction with phytochemicals for the bio-reduction of metals. In an experiment to study the effect of different temperatures (70, 75, 80, 85, and 90°C) on the size of the biosynthesized silver nanoparticle, Liu et al. [50] explained that high working temperatures have a positive effect on the nucleation kinetics constant k1 and growth kinetics constant k2. Nucleation rate constant k1 was found to increases slightly when temperature was raised from 70 to 80°C, while it rises sharply when the temperature exceeds 80–90°C . To further the effect of temperature on the nucleation and growth of NPs, Jayachandran et al. [51] showed temperature dependence in the

*Phyto-Metallic Nanoparticles: Biosynthesis, Mechanism,Therapeutics, and Cytotoxicity DOI: http://dx.doi.org/10.5772/intechopen.112382*

biosynthesis of zinc oxide nanoparticles using *Cayratia pedata* leaf extract for the immobilization of glucose oxidase enzymes. From the selected working temperature of 55, 65, and 75°C. It was noted that ZnO NPs were formed with a working temperature of 65°C while no synthesis occurred at 55 and 75°C respectively.

The role of pH maintained for reaction synthesis is also a key factor to be considered for controlled synthesis. Different literature studies attributed the effect of pH on the stability, morphology, optical and bioactivity of the synthesized PM-NPs [39, 52, 53]. A comparative stability and biological activity (cytotoxicity and antimicrobial) study of AgNPs synthesized with citrate, and green tea by Bélteky et al. [52] reveals that both NPs are more stable in alkaline and neutral media than in acidic medium with higher stability for green tea AgNPs. Additionally, synthesis in alkaline and neutral pH tend to produce smaller size particles than in acidic pH [9]. Also, the effects of pH in synthesis reflects on the optical properties of the synthesized NPs and their therapeutic applications. The optical properties of ZnO NPs were shown to depend on the pH. This was demonstrated by the report of Thipe et al. [34] showing different colors of ZnO-NPs synthesized from orange fruit peel extract for antibacterial activities. In the study, pH values of 4.0, 6.0, and 9.0 had ivory color, pH values of 7.0 and 8.0 had burnt black color and white color was noticed at pH values of 10 and 11. With Pt NPs synthesis via orange peel extract, Karim et al. [54] illustrated different size distribution and crystallinity properties for pH 3, 5, 9, 11, and 13. The report shows a reduction in sizes (2 nm – 1.8 nm), agglomeration, and uniform spherical shapes of NPs as pH increase from pH 3 through pH 11. pH 11 was noted with the smallest particle size (1.8 nm) and uniformity (which could be standard for the biosynthesis of Pt-NPs) while at pH 13 particles became agglomerated.

### **3. Therapeutic applications of phyto-metallic nanoparticles**

New innovative technologies to improve the development of novel therapeutics and treatment of disease conditions using combined synergies between synthetic and natural products have been of interest to many nanomedicine experts. Extracts from plant has a long history of usage as traditional herbal medicine, as prototype in the formulation of synthetic drugs and in the detoxification of heavy metal accumulation in the body [55, 56]. The pharmacological potentials of many plants are closely associated to the major phytochemicals they contain. For instance, plants rich in polyphenols are often indicated as therapeutic agents for complications resulting from oxidative stress. A systematic formulation of these therapeutic agents as nano drugs for use in biomedicine is gaining acceptance due to the rising preference for natural product-based therapies and their versatilities [32]. The bioinorganic network provided by PM-NPs and their biocompatibility make them suitable as a drug over conventional speciality drugs with many side effects. Hence, their roles in therapeutic applications such as in bactericidal, anticancer, antioxidants, antidiabetic, and photothermal treatment (PTT) are briefly discussed below.

#### **3.1 Bactericidal effects**

The antibacterial potentials of various PM-NPs have been extensively discussed by different researchers given the resistance of bacterial to the first generation and conventional antibiotic drugs. Most reports show that the activities of the PM-NPs are attributed to the binding and interaction of PM-NPs on surfaces of bacterial cell membranes. The interaction inhibits synthesis of new cell membrane thus preventing cell growth. Also, the binding of PM-NPs could disrupt the energy transduction process, and cause damage to the cell membrane through the generation of reactive oxygen species leading to the eventual death of bacterial cells [57–59]. The efficacies of the bactericidal effect of PM-NPs have been tested both as a singular drug and in combination treatment with conventional antibiotic drugs [9]. Among metals and metal compounds, Ag ions and Ag-based compounds are the most pernicious on microorganisms showing strong antibacterial properties [60]. Hence, PM- AgNPs are the most studied for the inhibition of a broad spectrum of both gram-negative (G-ve) and gram-positive (G + ve) bacteria species including *Pseudomonas aeruginosa*, *Staphylococcus aureus*, *Bacillus cereus, Salmonella enterica*, *Escherichia coli* and *Serratia marcescens* [60]. This was recently demonstrated by Balachandar et al., [61] for antibacterial activities of AgNPs mediated by *Glochidion candolleanum* (GC) leaf against *Bacillus subtilis, Listeria monocytogens* and *S. aureus* (G+ve)*; E. coli, P. aeruginosa, and S. enterica* (Gve). The study shows a general inhibition for all bacteria based on the concentration of GC-AgNPs used. The highest maximum inhibition zone (12.2 mm diameter) due to GC-AgNPs was observed against S. enterica and for P. aeruginosa *(*11.8 mm diameter), *L. monocytogens* (11.0 mm), *S. aureus* (10*.9* mm) and 10.8 mm for both *B. subtilis and E. coli* in the descending order [61]*.* A similar result was presented by Nishanthi et al., [9], which showed a synergistic antibacterial activity of rind extract of *Garcinia mangostana* biosynthesized Au, Ag, and Pt NPs in combination with commercial antibiotics (penicillin G (2.0 μg), methicillin (5.0 μg), vancomycin (30 μg), gentamycin (50 μg), streptomycin (10 μg), ciprofloxacin (5.0 μg), azithromycin (30 μg) and clotrimoxazole (25 μg) and tested against *Staphylococcus* sp., and *Bacillus* sp. (G+ve) and *Pseudomonas* sp. and *Klebsiella* sp. (G+ve). The authors found that AgNPs, displayed relatively higher antibacterial activity when compared to AuNP and PtNPs against all the tested pathogens [9]. In addition to Ag, Au, and Pt NPs, biosynthesized ZnO-NPs, and FeNPs using extracts of orange fruit peel, and *E. robusta* Sm, respectively have been reported [27, 62]. ZnO/Zn (OH)2 NPs are known to exhibit photocatalysis when illuminated with UV light producing reactive oxygen species (ROS), such as superoxide anion (O2 •–) or hydroxyl radical (OH• ) which may cause disruption of the electron transport chain and or oxidative stress in the microbial cell membrane [59]. ZnO-NPs were shown to exhibit strong antibacterial activities towards *E. coli* and *S. aureus* at a concentration of 0.025 mg/mL without UV radiation after 8 h of incubation [62]. Vitta et al. [27] recorded no significant differences (p > 0.05) for *E. Coli* with Fe-NPs (positive control), while for *S. aureus, P. aeruginosa and B. subtilis*, statistically significant differences (p < 0.05) were found as there was an increase in the values of inhibition zone as the size of the nanoparticles diminished [62]. Also reported is, the biogenic synthesis and antimicrobial evaluation of Cu-NPs and Mg-NPs from leaf extracts of strawberry and *Viola betonicifolia (L.)* respectively. Bayat et al. [63] noted that the Cu-NPs like their Ag-NPs counterpart displayed a bactericidal effect on *P. aeruginosa* at an effective concentration (EC50) of 2.2 mg/mL. Interestingly, the authors indicated a higher antibacterial activity of Ag-NPs over Cu-NPs due to a greater minimum bactericidal concentration (MBC) of Cu-NPs (5 mg/mL) than for MBC of Ag-NPs (0.01 mg/mL). According to Lu et al. [31], synthesized *Viola betonicifolia*-MnO2 NPs displayed higher antimicrobial and reductions in colony forming unit for *K. pneumoniae* (4.14 0.03 log10 reduction),

*Phyto-Metallic Nanoparticles: Biosynthesis, Mechanism,Therapeutics, and Cytotoxicity DOI: http://dx.doi.org/10.5772/intechopen.112382*

and *S. aureus* (4.65 0.07 log10 reduction) respectively, than the value obtained from commercially available Mn-NPs [31].

Among other metals that have received considerable attention for the biosynthesis of PM-NPs as antimicrobial agents are titanium (TiO2-NPs), palladium (Pd-NPs) nickel (NiO-NPs), selenium (Se-NPs), and tin (SnO2-NPs) [30, 64–67]. Amanulla and Sundaram [64] reported that at dose concentrations from 6.75 to 50 mg/mL, TiO2-NPs synthesized from orange peel extract showed bactericidal effects on *S. aureus, E. coli, and P. aeruginosa*. In a similar report, Pd–NPs (3 nm) obtained via *Garcinia pedunculata roxb* leaf extract was observed to elicit antimicrobial activity against *Cronobacter sakazakii* AMD 04 at dose concentration from 0.39 mM and 0.52 mM [66]. From the report of Srihasam et al. [65], biogenic synthesis and antimicrobial activities of NiO-NPs from the leaf extract of stevia plant were implicated in significant bactericidal effects on *E. coli, Streptococcus pneumoniae*, *and B. subtilis* at a concentration of 200 μg/mL. Furthermore, Alvi et al. [30] demonstrated that Se-NP synthesized from *Citrus paradisi and Citrus limon* for antimicrobial activities against *E. coli, M. luteus, B. subtilis*, *and K. pneumoniae* show significant activities against all the bacterial pathogens when compared with the standard antibiotic Ciprofloxacin. SnO2- NPs are also promising PM-NPs that have been found effective against microbes. SnO2-NPs from fruit extract of *Averrhoa bilimbi* showed satisfying inhibitory activities against *S. aureus* and, *K. aerogenes* while from *Saraca indica* flowers were effective in inhibiting the growth of *E. coli* [67, 68].

#### **3.2 Anticancer effects**

The applications of metallic nanoparticles in the treatment of cancer cells from different organs such as breast, cervical, colon, ovarian, and lung cancers have attracted more interest in biomedicine. In this regard, MNPs act as nanocarriers due to the available large surface area for the attachment of a large number of vectors for targeted delivery at designated sites [69]. This method has been reported to improve the efficacy of anti-cancer drugs over traditional drugs which are sometimes identified and removed from the systemic circulation by the liver and the spleen [16, 36]. Usually, studies on anticancer potentials of nanoparticles require both in vitro and in vivo (animal and clinical) studies. In vitro, studies are mostly reported using conventional MTT and apoptosis assay to determine cell viability via cytoprotection, and extrinsic cell death respectively. In the experiment, a decrease in cell metabolic activities is implicated in the induced secretion of reactive oxygen species by cells due to biogenic NPs which gradually leads to the death of cellular components [70]. The values of half maximal inhibitory concentration IC50 are used to determine the cell behaviors when treated with PM-NPs. The in vivo experiment is a follow-up and confirmation of in vitro, although limited studies of in vivo antitumor studies are reported [71]. Because MNPs are functionalised and stabilized for selective delivery at specific sites, biodistribution and degradation of the MNPs may take much circulation time in systemic circulation resulting in metal accumulation and particle-induced toxicity of mammalian cells and tissues. Different biodegradable materials that help with bio-clearance such as plant phytochemicals, polysaccharides such as dextran and chitosan, polyvinyl alcohol (PVA), phosphorylcholine-based copolymers and so forth have been suggested for coating of MNPs [15, 72, 73].

Addressing the drawback of drug residence time and biodistribution MNPs as a potential anticancer agent in mammalian cells, Akinfenwa et al. [36] showed that phytochemicals from *Aspalathus linearis* plant from PM-NPs could serve to reduce, capped, and increase bio-clearance of biosynthesized AgNPs and AuNPs. From the results of the study, the authors highlighted that AgNPs are more efficacious showing antiproliferation effects on human hepatocellular carcinoma (HepG2), and neuroblastoma (SH-SY5Y) cells in vitro than AuNPs. In a complementary report by Alharbi and Alsubhi [74], in vitro anticancer activity of AgNPs prepared using fruit extract of *Azadirachta indica* on pneumocyte lung tumor (A549) cells show that all treatments involving biosynthesized AgNPs only, and AgNPs in combination with cisplatin were more toxic to A549 cells than with the extracts and cisplatin only. A remarkable combination of both in vitro and in vivo anticancer experiments of biogenic AgNPs was demonstrated by Kabir et al. [70]. The authors concluded that Ag/AgCl-NPs synthesized from *Geodorum densiflorum* rhizome extracts inhibit human cancer cell proliferation in vitro and also for Ehrlich ascites carcinoma (EAC) cell growth in vivo. From the in vitro experiment, the authors found that samples treated with *G. densiflorum*-Ag/AgCl-NPs induced apoptosis in glioblastoma stem cells (GSCs), pancreatic cancer (BxPC-3) and breast cancer (MCF-7) cells. While in vivo, there was inhibition of up to 60 and 95% of EAC-mice cell growth at the doses of 2 and 4 mg/kg/ day after intraperitoneal treatment respectively. Noteworthy is the overall increase in mice life span by 75% compared to EAC-bearing control mice which gives credence to the efficacy of PM-NPs. A prototype drug for chemotherapeutic treatment of cancer is Cisplatin, a platinum coordination complex found with most antitumor properties although with some side effects such as neurotoxicity, ototoxicity and renal impairment. Formulation of the complex analogue for cancer therapy through biosynthesis is a welcome idea to overcome its side effects. In the study of the anticancer effects of PtNPs synthesized from black cumin seed extract, Aygun et al. [75] found that PtNPs were efficient against the proliferation of MDA-MB-231 breast and HeLa cervical cancer lines (IC50: 36.86 μg/mL and 19.83 μg/mL, respectively). The result agrees with a previous result by Chuang et al. [76] of biogenic PtNPs from peppermint leave extract which showed a decrease in the viability of HTC 116 colon cancer cells at an IC50 value of 20 μg/mL. Similarly, dose-dependent anticancer activities of biogenic Au NPs (inhibition of MCF-7 cells by 70.2%), CuO NPs (decrease breast cancer MDA-MB-231 viability and increase ROS at IC50: 20 μg/mL), MnONPs (cytotoxic against MCF-7 cells at 120 μg/mL concentration), ZnO NPs (toxicity on both A549 and MOLT4 cells and a size reduction in A549 tumor), NiO NPs (toxicity against HepG2 cancer cells, IC50: 37.84 μg/mL) and Se NPs (decrease in cell viability at a concentration of 2–6 μg Se/mL) have been described as promising anticancer agents through different studies [31, 77–82].

It is believed that combining two metals in biosynthesized as bimetallic NPs (BMNPs) often synergize their effects with more biological potentials than their monometallic counterpart. As anticancer agents, biogenic BMNPs have been reported with improved activities over their respective single metals [5]. Several reports supported this hypothesis. Tamimi et al. [83] report showed that chemically synthesized bimetallic Ag-Cu NPs showed significant toxicity on MCF-7 cells at 10 μg/ mL concentration than the separate AgNPs (20 μg/mL), and Cu-NP (showed no toxic effects). Hence, the hypothesis should be true for biogenic BM-NPs. Fahmy et al. [28] in their report on green synthesis of Pt-Pd NPs using *Peganum harmala* L. seed alkaloids revealed that bimetallic Pt–Pd NPs exhibited significant cytotoxic activities against A549 (IC50; 8.8 μg/mL) and, MCF-7 (IC50; 3.6 μg/mL) cells when compared to the individual metals, and carboplatin standard (IC50; 23 and 9.5 μg/mL,). In the study, relatively lower anticancer activities of each mono MNPs of Pt NPs and PdNPs for A549 and MCF-7 were noted at IC50; 10.9 μg/mL and 6.7 μg/mL, and IC50; 31 μg/

mL and 10.8 μg/mL, respectively [28]. A similar study by Athinarayanan et al. [84] revealed that bimetallic Pt-Cu NPs synthesized with catechin extract of green tea induced cell death in human cervical cancer dells (SiHa) cells with increasing the dose and time with IC50 value of the Pt-Cu NPs ranged from 32 to 35 g/mL for 24 and 48 h. All these reports of BMNPs from plants portend higher efficacy in therapeutics within the limit of acceptable toxicity.

#### **3.3 Antioxidants potentials**

Over-expression of free radicals is regarded as a lead factor to a variety of diseases including cancer, hyperglycemia, hyperlipidemia, inflammation, and so forth via apoptosis signaling pathways and oxidative stress on cellular components. Free radicals such as reactive nitrogen species (RNS; nitrogen dioxide, NO2), reactive sulfur species (RSS; hydrogen sulphide, H2S), reactive chlorine species (RCS; hypochlorous acid, HOCl), and most especially reactive oxygen species (ROS; superoxide, (O2 •–), are pro-oxidants which induce cellular oxidative stress [85]. Natural antioxidants produced by cells help to check on the excess production of ROS by scavenging activity. However, when the ROS level becomes higher than the natural antioxidant activities of cells, the use of natural antioxidants from plants or food rich in antioxidants to mitigate oxidative stress becomes necessary. Interestingly, compounds of the phenolic and flavonoid classes especially vitamins A, C, and E, quercetin, and rutin have been shown to attenuate ROS-induced oxidative stress with different radicalbased and metal-related assays [86, 87]. Methods used to evaluate free radical scavenging includes 1,1-diphenyl-2-picryl-hydrzyl (DPPH) radical, 2,2-azino-bis-3 ethylbenzothiazoline-6-sulfonic acid (ABTS) radical, ferric reducing antioxidant power (FRAP), nitric oxide (NO), superoxide (O2 •–), hydroxyl (OH• ) cupric reducing antioxidant capacity (CUPRAC), Trolox equivalent antioxidant capacity (TEAC) assays etc. [88]. It is proposed that during the interaction, the metal ions in PM-NPs scavenge free radicals by electron transfer and proton loss thus inhibiting oxidative DNA damage. Hence, the therapeutic potentials of PM-NPs are largely associated with the phenolics, and antioxidant properties of the phyto-reducing agent and the metal ions used during synthesis. According to Vera et al. [89], the antioxidant activities of plant phytochemicals are a strong indication of the synthesis efficiency of MNPs and their biomedical applications. Antioxidant-functionalized NPs are reported to show comparative advantages over antioxidants due to their high permeability and stability during membrane trafficking. Different PM-NPs have demonstrated nanoantioxidant activities which serve as an alternative route to the use of conventional synthetic antioxidant therapy. For example, *Rhazya stricta* plant (Apocynaceae), used in folk medicine in the Middle East and Indian subcontinent is reported with antioxidant activities. When functionalized as AgNPs, exhibited superior two-folds antioxidant activity measured at 75.16% 0.04 over the plant extract (43.12% 2.1) [90]. It is also documented that the antioxidant activity of biosynthesized MnO2 NPs using leaves extract of *Viola betonicifolia* on linoleic acid peroxidation was higher (84.94 0.77%) than for the leaves extract and a little less than that of ascorbic acid (90.57 1.21%) standard [31]. When the inhibition of DPPH radicals of FeNPs from an aqueous extract of *E. robusta* leaves was compared to the extract alone, Vitta et al. [27] showed that FeNPs were significantly more potent (p < 0.01) than the extract alone, showing IC50 values of 81.63 11.75 μg/mL and 423.14 73.27 μg/mL respectively [27]. Overall, the improved antioxidant activities of PM-NPs have opened

research interest in phyto-nano therapy for the repair of damaged cellular macromolecules (like proteins, lipids, DNA) and regulation of cellular functions from degenerative and pathological ailments such as aging, cancer, diabetes, and neurodegenerative diseases.

#### **3.4 Antidiabetic application**

Type 2 diabetes is a result of hepatic dysfunction and insufficient insulin secretion from pancreatic β-cells for glucose uptake accounts for over 90% the worldwide diabetic patients [91]. Being a global disease, a lot of efforts on developing therapeutic agents and advocacies to reduce the increasing number of undiagnosed and diabetes are being explored. Synthetic drugs such as sulfonylureas, metformin, acarbose are among current antidiabetic drugs. In addition to these synthetic drugs, natural products from plants general and plant-derived phytochemicals such as chalconaringenin 20 -*O*-β-*D*-glucopyranoside and aureusidin 6-*O*-β-*D*-glucopyranoside, obtained from flower extract of *H. arenarium* have been reported [55]. Despite the several *in vivo, in vitro,* and clinical studies, till date, a complete cure for treatment of diabetes is yet to be achieved. Recently, several nanomedicine research are focusing on nanoscale drugs by combining the synergies between potential antidiabetic phytochemicals and metallic nanoparticles. In this regard, Ul Haq et al. [92] showed that plant mediated silver nanoparticles from *Taverniera couneifolia* (*TC*-AgNPs) significantly improved Alloxan-induced diabetic Wistar rats. The effects of *TC*-AgNPs on the lipid, liver, and kidney profiles of treated rats (10 mg/kg body weight) were observed with lowered blood glucose levels, and improvements in the lipid, liver, and renal profiles after treatment with *TC*-AgNPs. A similar therapeutic application of ZnO NPs synthesized from *Amygdalus scoparia* stem bark extract showed significant antidiabetic potentials on treated rat (30 mg/kg). As reported, a higher level of insulin and lower AST, ALT, lower blood glucose, higher levels of IR, GluT2, and GCK expression and lower TNFα expression were recorded when compared with the STZ induced diabetic rats [93]. These reports together give insight into the biomedical and therapeutic application of phyto-metallic nanoparticles.

#### **3.5 Photothermal and photocatalytic effects**

The most important property of zerovalent metallic NPs is their electronic excitation and light scattering characters under incident laser beam. The absorption and scattering of light give rise to intense colors and interesting optical properties. These properties are used in theranostic nano probe and therapy in biomedicals such as in biosensing, tissue imaging, molecular imaging, cancer therapy etc. Photothermal therapy is less invasive using therapeutic agents like noble metals and near infrared radiation (NIR). Specifically, AuNPs are known to exhibit excellent photothermal transduction in the interconversion of heat-to-light by absorbing light in the near infrared region (NIR; 700–1400 nm). For example, NIR laser radiations delivery by AuNPs could induce stimulations in cells for necrosis, healing of open wound, in pain relief therapy etc. This property of gold and the paramagnetic property of iron metals and irradiation by NIR have been used for active targeting cancer cells, drug carrier and delivery, and removal of cancer cells [94]. The synergy between the NIR and PM-NPs (as therapeutic agents) has been explored by different researchers. Ali et al. [95] and Wang et al. [96] studies showed an enhanced cytotoxicity on lung (A549), and breast cancer (MCF-7) cell lines when treated with NIR irradiated synthesized *Cordia* *Phyto-Metallic Nanoparticles: Biosynthesis, Mechanism,Therapeutics, and Cytotoxicity DOI: http://dx.doi.org/10.5772/intechopen.112382*

*myxa L.* leaf extract-PtNPs and *Memecylon edule* leaf extract-graphene oxide nanoparticles (GO-NPs) respectively. The effects were induced apoptosis in MCF-7/ A549 cells due to photothermal transduction (conversion of low energy into heat*)* by the respective PtNPs and GO-NPs.

In photocatalysis experiment, the decolorization of dyes; methyl orange, methylene blue, Eosin Y, etc. by UV light in the presence of metal catalyst which result in degradation products are usually reported. During UV irradiation of samples, the photocatalysts cause electron excitation from the Valence to the Conduction band where the material becomes chemically responsive indicated by absorption peak at λ wavelength. The difference between the valence and the conduction band is calculated as Bandgap (Eq. 1). Phytochemical-inspired metallic nanoparticles could improve the photocatalytic degradation of dyes for treatment of wastewater through reduction of conduction electrons for UV absorption spectral determination. This effect was implied by Gupta and Chundawat [97] for the photocatalytic deterioration of methyl orange by fungus mediated PtNPs for potential application in wastewater remediation.

$$\mathbf{E\_g} = \mathbf{h} \,\mathbf{c\_e} \mathbf{V} / \lambda \,\text{or}\,\mathbf{E\_g} = \mathbf{1240\_e} \mathbf{V} / \lambda \tag{1}$$

H = 6.626 � <sup>10</sup>�<sup>34</sup> JS, c = 3 � <sup>10</sup><sup>8</sup> ms�<sup>1</sup> , λ = absorption wavelength.

### **4. Cellular uptake and toxicity of PM-NPs**

Cellular uptake of PM-NPs accounts for the dose of internalized NPs and their interactions in physiological media. The uptake of NPs has been investigated by different methods; quantitatively using inductively coupled plasma-mass spectrometry (ICP-MS), inductively coupled plasma-optical emission spectroscopy (ICP-OES), and inductively coupled plasma-atomic emission spectroscopy (ICP-AES). The qualitative method uses transmission electron microscopic (TEM), energy dispersive X-ray (EDX) analysis, colourimetry, and fluorescence imaging. Cellular uptake experiment is important to understand the stability of NPs and their toxicities on both normal and cancer cells. Most uptake studies discussed uptake efficiency and toxicity. Still, the exact mechanisms of cellular uptake for PM-NPs in different cells have not been well characterized [98]. It is however clear that in the process of uptake NPs used as drug carriers permeate biological barriers of the bilayer cell membrane (consisting of hydrophobic and hydrophilic layers) through a passive diffusion pathway to distribute the NPs within cells. Most studies show that permeation and assimilation of NPs depend on several factors such as surface modification/interactions between the NPs and the cells, size, shape and concentrations of NPs [99]. NPs have surface charges (positive, negative, or neutral) that attract ions of opposite charges in the cytosol for cellular uptake. Different reports have shown that positively charged NPs show better cellular uptake than neutral and negatively charged NPs [2]. This is supported by a report of high cellular uptake of AuNP synthesized from *A. linearis* by SH-SY5Y and HepG2 cells. The authors attributed the uptake of AuNPs at a concentration of 5.368 μg/mL and 3.625 μg/mL by SH-SY5Y and HepG2 cells respectively to high acidity and positive surface charges from *A. linearis* [36, 100]. Also, the nano (small) size factor of PM-NPs plays an important role in the cellular internalization of NPs. Because microorganisms and cell organelles such as DNA have nano-size ranges, they can cross the plasma membrane faster and with uniform distribution for intracellular

particle trafficking. Different reports revealed that smaller-size PM-NPs (≤ 40 nm) easily permeate cells than sizes greater than 50 nm. Amaliyah et al. demonstrated that the antibacterial activity of biogenic AgNP from *Piper retrofractum* Vahl fruit extract was facilitated by the entry of the small sizes (1–5 nm) AgNPs through *E. Coli* and *S. Aureus* [98, 101].

Nanotoxicological assessment of NPs has become essential before application in therapy to determine their toxicity safe level. Toxicological studies are mostly conducted in vitro using standard toxicological assays, such as the MTT assay, colony forming efficiency (CFE), lactose dehydrogenase (LDH) assays and so forth in immortalized cell lines. MTT assay measures cell viability via mitochondrial enzymatic activities, CFE checks the ability of a single cell to form a colony by survival from toxic media, and LDH assay measures the damage to the cell membrane. Every organism is made up of natural defense mechanisms which protect it from toxic exogeneous particles. Despite this, exposure to MNPs beyond a threshold could overcome the body's defense to cause hazards [102]. Toxicity by MNPs may occur during preparation or on therapeutic administration leading to cytotoxicity and oxidative DNA damage. Like cellular uptake, the toxicological behavior of NPs is dependent on their surface activities and morphological properties (size, shape, and agglomeration). It is well-known that spherical, smaller sizes and non-agglomerated NPs are more compatible and less toxic to cell membranes. Again, the toxicity of NPs is dependent on exposure concentration and the amount of the NPs that reached the targeted cells [15]. PM-NPs are considered non toxic due to the synergy that exist between metals and bioorganic networks of the reducing phytochemicals. However, PM-NPs have safe level if delivered to specific cancer cells to elicit DNA damage and cell multiplication [103]. Cellular toxicity of PM-NPs is beneficial when it displays specificity on targeted cells. The therapeutic potential of PM-NPs is lost where it is toxic against normal cells. Toxicity on cells is generally established through in vivo and in vitro experiments. Few published reports have also shown the toxic effects of PM-NPs on normal cells and these reports are briefly discussed. For example, Majoumouo et al. [25] reported in vitro cytotoxic effects of biosynthesized *Terminalia mantaly* extracts gold NPs (TM-AuNPs) investigated on cancer (Caco-2, MCF-7 and HepG2) and noncancer (KMST-6) cell lines. According to the study,*T. mantaly* extracts showed some cytotoxicity towards the cancer cells while the TM-AuNPs exhibited more cytotoxicity on all the cells. The effects of PM-NPs concentrations on toxicity were studied in vivo on EAC cells using *G. densiflorum* -AgNP. At a higher concentration of 4 mg/kg/day, the biosynthesized inhibited growth of mice tumor cells up to 95% greater than 60% recorded on a dose of 2 mg/kg/day [70].
