**4. Results and analysis**

#### **4.1 UV-visible absorbance spectra**

The surface plasmon resonance, which is the interaction between free electrons surrounded by metal nanostructures and radiation, was used to validate the formation and stability of Ag and Au NPs. Fruit extracts reduce aqueous silver and gold ions. From **Figure 1** shows the well-dispersed formation of Ag and Au NPs, resulted in the SPR peaks were observed in the visible region in the ranges of 350–600 nm and 500–700 nm, respectively. Due to the excitation of SPR vibrations, *P. granatum* synthesized Ag and Au NPs (**Figure 1a** and **b**) and *Citrus reticulata* Ag and Au NPs (**Figure 1c** and **d**) produced respectively. The ionization of phenolic groups in fruit extract results the formation of nanostructures. For a few months, these metal nanoparticles remain stable.

*Cytotoxicity Studies of Fruit-Extracted Metal Nanostructures DOI: http://dx.doi.org/10.5772/intechopen.106140*

**Figure 1.**

*Absorption spectrum of silver and gold NSs prepared by (a) Punica granatum and (b) Citrus reticulata extracts.*

#### **4.2 TEM images**

The TEM images in **Figure 2a** and **b** shows that Ag nanostructures are in spherical shape and range in size from 30 to 70 nm (average size 62 nm). Ag NPs have crystalline FCC structure in their SAED (selected area electron diffraction) patterns. The circular planes corresponding to (1 1 1), (2 0 0), (2 2 0), and (3 1 1) match the XRD pattern very well. With a spherical dimension of 26 nm and edge length of a triangle 52 nm, Au NPs have spherical and triangular shape. Crystallinity of Au NPs with SAED patterns are shown in the planes (1 1 1), (2 0 0), (2 2 0), and (3 1 1). Similarly, the TEM images of Ag and Au NPs by citrus reticulata are shown in **Figure 2c** and **d**. The Ag NPs had a diameter of 56 nm and were spherical. The circular rings in the SAED pattern revealed that the produced Ag NPs were of crystalline structure. **Figure 2d** shows the presence of triangles in addition to spherical nanoparticles. Au NPs had a mean spherical particle size of 21 nm and a triangle edge size of 48 nm. The crystalline structure of Au NPs is revealed by the SAED pattern, which was indexed to (1 1 1), (2 0 0), (2 0 2) and (3 1 1) reflections.

#### **Figure 2.**

*TEM images and diffraction patterns of Ag and Au NSs (a) & (b) Punica granatum and (c) & (d) Citrus reticulata extract synthesized.*

**Figure 3.**

*XRD of Ag and Au NPs (a) & (b) Punica granatum and (c) & (d) Citrus reticulata extract synthesized.*

#### **4.3 XRD**

**Figure 3a–d** reveals the XRD of *Punica granatum* and *Citrus reticulata* synthesized Ag and Au NSs. It shows different diffraction peaks in the range 2θ = 20°–80° at 38.5°, 44.3°, 64.2° and 77.4° corresponds to (1 1 1), (2 0 0), (2 0 2), (3 1 1) planes with FCC, respectively.

#### **4.4 Luminescence emission**

In **Figure 4a** and **c** showed that emission of europium with Ag NPs excited at 350 nm. The Ag concentrations varied from 5 to 300 μl at 15 μl of Eu. The emission at 577 nm, 590 nm (magnetic), and 614 nm (electric) are contributing to <sup>5</sup> D0 → <sup>7</sup> F0, 5 D0 → <sup>7</sup> F1, 5 D0 → <sup>7</sup> F2 transitions respectively. These emission bands were enhanced but emission wavelengths were remains unaltered. With concentration increment of Ag, intensities increased and decreased with further increment of Ag. Similarly, **Figure 4b** and **d** shows the emission of Samarium with Au. The emissions at 645 nm(4 G5/2 → <sup>6</sup> H9/2), 566 and 602 nm (4 G5/2 → <sup>6</sup> H5/2 and 4 G5/2 → <sup>6</sup> H7/2) correspond to electric and magnetic dipole transitions respectively. In the inset of **Figure 4b** and **d**, we show the effect of silver concentration on different transitions.

The Ag NPs' concentration dependence on the emission enhancement for Eu(TTFA)3 is shown in the inset of **Figure 4a**, where the normalized intensity is the intensity ratio of Eu(TTFA)3 solution containing Ag NPs to pure Eu(TTFA)3 solution for the 5 D0 → <sup>7</sup> F2 transition. The luminescence enhancement gets strongly affected by the Ag NPs concentration, the maximum enhancement factor was 23 at 70 μl Ag concentration. For <sup>5</sup> D0 → <sup>7</sup> F0 and 5 D0 → <sup>7</sup> F1, the enhancement factor was observed as 3. The luminescence enhancement is greater for electric dipole transitions than for magnetic dipole transitions, according to this. The hyper-sensitive transition is influenced by changes in the refractive index and ligand fields around rare-earth ions caused by interactions with metal NPs [44].

#### *Cytotoxicity Studies of Fruit-Extracted Metal Nanostructures DOI: http://dx.doi.org/10.5772/intechopen.106140*

The emission of rare-earth ions near NSs are affected by the near-field environment, which induces enhancement or quenching relay on the distance between nanoparticles and rare-earth ions [45]. Rare-earth ions transfer non-radiative energy to metal particles through very low distances between NPs and molecules. Because of plasmonic resonances in metal NPs, magnetic field enhancement occurs only until a certain distance [46]. More Eu ions can be closer to NSs and the distance between Eu ions and Ag NPs can be reduced by increment in number of Ag NSs. With more NPs or reabsorption of SPR, the emission intensity is quenched by energy transmit between europium and NSs. The electric dipole transition (5D0 7F2) and magnetic dipole transitions (5D0 7F0 and 5D0 7F1), on the other hand, had distinct concentration dependence on luminescence intensities. The enhancement is considered to be caused to a hard coupling between SPR and probes. **Figure 1** shows that the absorption spectra of Ag NPs are in the wavelength range of 350–600 nm, which corresponds to the absorption of rare-earth ions. The enhancement resulted from a sensitive balance between surface Plasmon resonance reabsorption and local electromagnetic (EM) field enhancement.

**Figure 4e** and **g** presents the emission of 20 μl europium with Au NSs and excited at 350 nm. The emission bands at 614 nm is 5 D0 → <sup>7</sup> F2 electric dipole and at 577 nm ( 5 D0 → <sup>7</sup> F0), and 590 nm (5 D0 → <sup>7</sup> F1) are magnetic dipole transitions. The dependency of emission of europium at 10, 20, 30 μl with gold at 5 to 300 μl is shown in the inset of

**97**

#### **Figure 4.**

*((a) Punica granatum and (c) citrus reticulata) Photoluminescence of 15 μl Eu(TTFA)3 with Ag NPs concentration: (a) 5, (b) 10, (c) 30, (d) 50, (e) 70, (f) 100, (g) 200 μl and (h) 300 μl. Inset figures: dependency of Ag with various dipole transitions and Ag concentration with emission at 10, 15, 30 μl of europium concentrations. ((b) P. granatum and (d) Citrus reticulata) Photoluminescence of 200 μl Sm(TTFA)3 with Ag (a) 5, (b) 10, (c) 20, (d) 30, (e) 50, (f) 70, (g) 100 and (h) 200 μl concentrations. Inset figures: dependency of Ag with various dipole transitions and Ag concentration with emission at 180, 200, 220 μl of samarium concentrations. ((e) Punica granatum and (g) Citrus reticulata) Photoluminescence of 20 μl Eu(TTFA)3 with Ag NPs concentration: (a) 5, (b) 10, (c) 30, (d) 50, (e) 70, (f) 100, (g) 200 μl and (h) 300 μl. Inset figures: dependency of Au with various dipole transitions and Au concentration with emission at 10, 20, 30 μl of europium concentrations. ((f) Punica granatum and (h) Citrus reticulata) Photoluminescence of 220 μl Sm(TTFA)3 with Au (a) 5, (b) 10, (c) 20, (d) 30, (e) 50, (f) 70, (g) 100 and (h) 200 μl concentrations. Inset figures: dependency of Au with various dipole transitions and Au concentration with emission at 200, 220, 240 μl of samarium concentrations.*

**Figure 4e** and **g**. Similarly, **Figure 4f** and **h** displays the emission of samarium with Au NTs. The band at 645 nm (4 G5/2 → <sup>6</sup> H9/2) is an electric dipole and 566 nm (4 G5/2 → <sup>6</sup> H5/2) and 602 nm (4 G5/2 → <sup>6</sup> H7/2) are magnetic dipole transitions. In **Figure 4e** and **g** inset, emission of samarium 10, 20, 30 μl with Au at 5 to 300 μl is shown. The emission is quenched exponentially for Eu3+ at 10 and 30 μl and Sm3+ at 100 and 120 μl. The quenching is owing to the re-absorption of nanoparticles.

#### **4.5 In vitro cytotoxicity**

Green Ag and Au NTs were tested in MTT assays to see how they affected the growth of cancer cell lines. This is the first study to test the cytotoxicity of Ag NPs and Au NTs generated from *Punica granatum* and *Citrus reticulata* against cancer cells. **Figure 5a–d** shows the cell viability of Ag and Au nanoparticles, as well as their
