Antioxidant Activity:
Table 1: % age Free radical scavenging activity of aqueous extract and extract based silver nanoparticles of S. commune
In-Vitro assessment:
Concentration
|
Aq. Extract of S. commune
|
Extract based Silver NPs
|
BHA
|
10
|
2.14
|
3.36
|
42.65
|
50
|
10.32
|
14.96
|
73.69
|
100
|
21.28
|
33.59
|
98.36
|
In-Vivo assessment:
Animal Groups
|
CAT
|
SOD
|
GPx
|
Group 1
|
19.52±1.02
|
11.56±0.92
|
3.27±0.19
|
Group 2
|
28.89±0.93a
|
7.26±0.42a
|
5.92±0.21a
|
Group 3
|
26.14±1.26ab
|
9.78±0.36ab
|
4.28±0.17ab
|
Group 4
|
22.46±0.88ab
|
10.35±0.75ab
|
4.18±0.12ab
|
aSignificant when compared to Group 1, bSignificant when compared to Group 2; CAT (µ mole of H2O2 decomposed/minute/mg Protein), SOD (U/ mg Protein), GPx (U/mg Protein).
Reduction of Silver ions produces a color change:
The mixing of macrofungal extract with the AgNO3 solution has resulted into a gradual color change in the mixture solution (Fig. 1), which preliminarily indicates the reduction of silver ions and formation of silver nanoparticles (Shankar et al., 2004; Shivshankar et al., 2004). No further color change was observed after 2 hrs, which indicates the completion of reduction of silver ions in the reaction mixture. The further confirmatory characterizations were done through analysis of SEM images, UV-Vis and FT-IR spectra.
UV-Vis spectrum:
The UV-VIS spectrum of the S. commune extract based silver nanoparticles has been shown in Fig. 2. Several studies have established that the plasmonic properties like Surface Plasmon resonance (SPR) in the visible range are correlated with the morphology of the silver nanoparticles and therefore they can provide useful information regarding the silver nanoparticle synthesis, by UV-Vis spectroscopy (Benzamin et al., 2006, Zhang et al., 2009, Cecilia, 2007). Mie's theory predicts that spherical nanoparticles will have a single surface plasmon resonance (SPR) band in their absorption spectra, whereas anisotropic particles may have two or more SPR bands, depending on their form (Joshia et al., 2008). The resulting band in the UV-Vis absorption spectra is due to the phenomenon of SPR, which in turn depends upon the size of the nanoparticles and the refractive index of the solution containing the nanoparticles (Amendola et al., 2010). Several previous studies showed that the SPR peak of silver nanoparticles ranges from 400-500 nm (Dhanlakshmi et al., 2012, Parashar et al., 2008). Borthakur et al. (2017) has studied the macrofungi mediated silver nanoparticle synthesis and reported that the UV-Vis spectrum showed the SPR peak at 400-450 nm. In our previous work (Kumar et al., 2018), we had synthesized silver nanoparticles using Punica granatum leaf extract and found the SPR peak at 420 nm. Athira et al. (2017) had synthesized silver nanoparticles using the macrofungus Pleurotus Florida and reported the absorbance peak at 410 nm. In the present work we had observed the SPR peak at 431 nm, which infers the synthesis of silver nanoparicles in the present sample. Further, in the present work we got a single SPR peak (at 431 nm) hence it may be inferred that the silver nanoparticles synthesized are roughly spherical in shape.
SEM Analysis:
The SEM images (Fig. 3) showed the shape and the dimensions of the synthesized nanoparticles. The nanoparticles were found to have cuboidal and roughly spherical morphology with a size ranging from 80nm to 110 nm, average size was found to be 96.13nm. Mustafa (2019) reported that the silver nanoparticles synthesized using Pleurotus (Oyster mushroom) have spherical shape and a size ranging from 2 to 100 nm. Adeyemi and John (2018) have synthesized silver nanoparticles using Lentinus edodes extract and reported that the nanoparticles had a size range of 50-100nm.
FT-IR spectrum:
The analysis of FT-IR spectrum (Fig. 4) infers the presence of the functional groups from the bioactive chemical constituent compounds present in the macrofungal extract that act as reducing as well as coating or capping agent during the synthesis of silver nanoparticles. The FT-IR spectrum of the present work has been shown in Fig. 4. The different characteristic peaks obtained in the FT-IR spectrum of S. commune extract based silver nanoparticles has been compared with previously established references for the presence of different functional groups (Coates, 2000; Nandiyanto et al., 2019). The peaks obtained in the present FT-IR spectrum belong to the mid-IR region. The spectrum showing the broad spectral peaks at 3224.98 cm-1 corresponds to hydrate (H2O), Hydroxyl (-OH) and Amino groups, which refers to the presence of phenolic compounds and alcohols. The presence of –OH groups is further confirmed by the associated peaks in the range of 1392.61 cm-1 to 1654.92cm-1, 1118.71 to 1219.01 cm-1 and 617.22 to 783.10 cm-1. The peak in the range of 1654.92 and 1597.06 cm-1 indicates the presence of double bonds and aromatic rings, which are found in flavonoid and tannin compounds. The peaks in the range of 1219.01 and 1392.61 cm-1 refers to the presence of proteins, nucleic acids and other carbon related compounds. The peaks at 1118.71 cm-1 region indicates the presence of C-O stretch which may be found in alcoholic compounds or ethers. The peak at 783.10 cm-1 refers to the presence of C-H bond in aromatic ring with C-H, 1-3 (meta) distribution. The peaks at 617.22 and 428.20 cm-1 refers to the presence of disulfide stretch (-S-S-). The peak at 520.78 cm-1 indicates the presence of aliphatic organohalogen compounds.
Antioxidant activity:
The result of comparative in-vitro assessment of antioxidant activity of crude extract and extract based AgNPs (silver nanoparticles) of S. commune has been shown in Table 1. The results revealed that the extract based silver nanoparticles showed comparatively more free radical scavenging activity in comparison to the crude extract. The aqueous extract of S. commune showed 2.14, 10.32 and 21.28 % DPPH radical scavenging activity at 10, 50 and 100 µg concentrations respectively whereas the extract based AgNPs of S. commune showed 4.36, 17.96 and 33.59 % DPPH radical scavenging activity at 10, 50 and 100 µg concentrations respectively. Athira Ragunath (2017) et al., had reported that the extract based AgNPs of macrofungus Pleurotus florida showed increasing DPPH radical scavenging activity with increasing concentration and found to show 96.48% DPPH radical scavenging activity at 200 μg concentration. Dinakar et al. (2008) reported that the AgNPs of I. obliquus (Chaga mushroom) extract showed 76.57 % free radical scavenging activity at 1mM concentration. The results of the present experimental work clearly infer that the extract based AgNPs have more antioxidant potentiality in comparison to the aqueous extract of S. commune.
The in-vivo assessment of antioxidant activity of extract based silver nanoparticles of S. commune involved the measurement of the activity of three key enzymes among other enzymes of the antioxidant defense system of the body viz. CAT, SOD and GPx. CAT is found in almost all the organs of the body and is responsible for breakdown of H2O2 into H2O and O2, thereby protecting the cells from oxidative damage by the Reactive Oxygen Species (ROS) (Iiyukha, 2001). SOD catalyzes the disproportionation or dismutation of Superoxide anion (O2-) into O2 and H2O2 and is found in three forms in mammals SOD1, SOD2 and SOD3, out of which SOD3 is extracellular (Antonyuk et. al., 2009). Superoxide radical is formed in the body as a byproduct of metabolism of oxygen and is responsible for causing oxidative damage if not regulated (Hayyan et.al., 2016). The H2O2 produced as result of SOD activity i.e. dismutation of Superoxide radical is also damaging and hence it is decomposed by the activity of other enzymes like Catalase. The GPx is a family of enzymes which reduces H2O2 into H2O and Lipid hydroperoxides into their corresponding alcohols thereby protecting the body aginst oxidative damage (Muthukumar et. al., 2011). GPx3 is one of the members of this enzyme family abundantly found in blood plasma (Muller et. al., 2007). Previous studies reported that decrease in GPx levels might be responsible for increased life expectancy in mice (Ran et. al., 2007). In fact, under normal physiological conditions there exists a very much delicate balance between rates of formation of H2O2 by diproportionation of Superoxide radical, and, the rate of removal of H2O2 by the activity of GPx and CAT (Kono and Fridovich, 1982).
Previous studies reported that the Triazophos-induced oxidative stress in rat results into increase in the activity of CAT and GPx associated with a decrease in the activity of SOD (Sharma, Sangha and Khera, 2014). The results of the present study showed that in Triazophos-intoxicated rats (Group 2) the CAT and GPx activity significantly (p=0.05) increased and activity of SOD was significantly (p=0.05) decreased in comparison to the control group of rats (Group 1). The rats treated with aqueous extract of S. commune extract (Group 3) showed a significant (p=0.05) decrease in the CAT and GPx activity and a significant (p=0.05) increase in SOD activity in comparison to the Triazophos-intoxicated rats (Group 2). The rats treated with extract based silver nanoparticles also showed a significant (p=0.05) decrease in the CAT and GPx activity and a significant (p=0.05) increase in SOD activity in comparison to the Triazophos-intoxicated rats (Group 2). Further, the decrease in CAT activity in extract based silver nanoparticles treated rats (Group 4) was found to be significantly (p=0.05) more than the aqueous extract treated rats (Group 3) in comparison to the normal control group of rats (Group 1). But, the increase in SOD activity and decrease in GPx activity in extract based silver nanoparticles treated rats (Group 4) and the aqueous extract treated rats (Group 3) were not found to be significantly different. These results revealed that the aqueous extract as well as the extract based silver nanoparticles have significant antioxidant activities which was found in the in-vitro assessment results also.