Wound Healing and Antimicrobial Property of Phytofabricated …
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2.2 Synthesis of Silver Nanoparticles
20 ml of 0 0.001 (M) silver nitrate solution was mixed with 1 ml of plant extract. The
mixture was heated to 45 °C for 45 min in water bath and after that, it was kept at room
temperature for 30 min to observe the color change from light yellow to brown. Then
the mixture was incubated at room temperature for 48–72 h for completion of the
reaction (Banerjee and Nath 2015). The change of color of the mixture was from pale
yellow to light brown during the heating process and finally, the color became dark
brown and indication for the synthesis of silver nanoparticles. The conical flask was
incubated at room temperature for 48 h for the complete settling of the nanoparticles.
2.3 Characterization
Prepared silver nanoparticles were characterized by UV–vis spectroscopy at 400–
450 nm after proper incubation. The hydrodynamic size of synthesized nanoparticle
was characterized by DLS. Zeta potential was also observed. Proper size of nanoparticle was characterized by AFM and SEM images. To investigate the surface material
of the nanoparticles, FTIR was done with the dried sample.
2.4 Biocompatibility
The level of biocompatibility was tested on the isolated lymphocytes. The spleen
lymphocytes were obtained using Histopaque (Sigma-Aldrich, St. Louis, MO, USA)
according to the method of Boyum (1976). The extent of nuclear damage was estimated by DAPI staining (Mollick et al. 2014), Comet assay (Sing et al. 1988), and
cell cycle analysis by flow cytometry (Pozarowski and Darzynkiewicz 2004). The
generation of ROS was detected by 2, 7-dichlorofluorescein di-acetate (DCFH-DA)
fluorescence following the methodology of Roy et al. (2008).
2.5 Preparation of Diabetic Animal Model
Twenty-week-old mature Swiss albino male mice were taken. Diabetes was induced
by one-time injection of streptozotocin at 150 mg/kg body weight. 180 mg/dl of
blood glucose level was the indication of diabetes that was developed on the 11th
day of streptozotocin injection. There were six groups of mice and three mice in each
group.
Group 1: diabetic mice + thermal wound (untreated); Group 2: diabetic mice +
thermal wound + SSD treated; Group 3: diabetic mice + thermal wound + AgNPs
477
2.2 Synthesis of Silver Nanoparticles
20 ml of 0 0.001 (M) silver nitrate solution was mixed with 1 ml of plant extract. The
mixture was heated to 45 °C for 45 min in water bath and after that, it was kept at room
temperature for 30 min to observe the color change from light yellow to brown. Then
the mixture was incubated at room temperature for 48–72 h for completion of the
reaction (Banerjee and Nath 2015). The change of color of the mixture was from pale
yellow to light brown during the heating process and finally, the color became dark
brown and indication for the synthesis of silver nanoparticles. The conical flask was
incubated at room temperature for 48 h for the complete settling of the nanoparticles.
2.3 Characterization
Prepared silver nanoparticles were characterized by UV–vis spectroscopy at 400–
450 nm after proper incubation. The hydrodynamic size of synthesized nanoparticle
was characterized by DLS. Zeta potential was also observed. Proper size of nanoparticle was characterized by AFM and SEM images. To investigate the surface material
of the nanoparticles, FTIR was done with the dried sample.
2.4 Biocompatibility
The level of biocompatibility was tested on the isolated lymphocytes. The spleen
lymphocytes were obtained using Histopaque (Sigma-Aldrich, St. Louis, MO, USA)
according to the method of Boyum (1976). The extent of nuclear damage was estimated by DAPI staining (Mollick et al. 2014), Comet assay (Sing et al. 1988), and
cell cycle analysis by flow cytometry (Pozarowski and Darzynkiewicz 2004). The
generation of ROS was detected by 2, 7-dichlorofluorescein di-acetate (DCFH-DA)
fluorescence following the methodology of Roy et al. (2008).
2.5 Preparation of Diabetic Animal Model
Twenty-week-old mature Swiss albino male mice were taken. Diabetes was induced
by one-time injection of streptozotocin at 150 mg/kg body weight. 180 mg/dl of
blood glucose level was the indication of diabetes that was developed on the 11th
day of streptozotocin injection. There were six groups of mice and three mice in each
group.
Group 1: diabetic mice + thermal wound (untreated); Group 2: diabetic mice +
thermal wound + SSD treated; Group 3: diabetic mice + thermal wound + AgNPs