UV visible spectroscopy
The initial indication of the synthesis of zinc oxide nanoparticles (ZnONPs) is change in color of solution. Appearance of yellow color indicates the formation of ZnONPs. UV vis spectroscopy is a useful technique to initially confirm the synthesis of nanoparticles. MCZNPs were prepared using Malvastrum coromandelianum leave extracts as capping and reducing agent. A strong absorption peak was observed in 350-363.5 nm range (Figure 1), corresponding to the band energy of zinc oxide.
X-ray diffraction (XRD)
X-ray diffraction (XRD) analysis determines the crystalline nature, size and physical properties of nanoparticles through interaction of x-rays with atoms of the crystals. The characteristics peaks for ZnONPs were observed for MCZNP (Figure 2). By applying the Debye Scherrer’s equation the average particle size was approximately 23.5 nm and the crystallite was of hexagonal shape. The Bragg’s peaks were present at the angle (31.81, 34.37, 36.23, 47.48, 56.53, 62.84, 67.93), which were in accordance with the crystallographic reflections from entry number 96-230-0451.
Scanning electron microscopy (SEM)
Scanning electron microscopy (SEM) revealed the physical features of the MCZNP. Size of the MCZNP was determined using small amount of MCZNP and was placed on the copper grid coated with carbon. Image generated by SEM of MCZNP are shown in Figure 3, which clarifies that the biosynthesized MCZNP were possessing average size of 32.06 nm and spherical shape.
Energy dispersive x-ray spectroscopy (EDX)
Elemental composition of MCZNP was analyzed by employing EDX in combination with SEM. EDX results confirmed that the samples contain Zn and O and sharp peaks can be seen in Figure 4 which strengthens the view that MCZNPs are free of impurities. Strong indices Zn and O were observed at 1.00 and 0.5 KeV which corresponds to Zn and O as major elements, thus confirms the single-phase purity of MCZNPs.
Antioxidant activities
The antioxidant potential of MCZNP was evaluated at various concentrations (1000, 500, 250, 125, 62.5, 31.25, 15.63 and 7.81 mg/ml) and IC50 values were calculated. The IC50 value for DPPH analysis was 44.3 ± 0.6, 27.6 ± 0.9 for superoxide scavenging activity, 52.3 ± 0.9 was observed for TAC and 47.5 ± 0.9 for TRP. Ascorbic acid was used as a standard and IC50 value was recorded as 17.8 ± 0.1 and 34 ± 0.9 respectively (Table 2).
Table 2 Antioxidant potential of Malvastrum coromandelianum.
Concentrations (µg/mL)
|
1000
|
500
|
250
|
125
|
62.5
|
31.25
|
15.63
|
7.81
|
IC50
|
DPPH (µg/mL)
|
MCZNPs
|
85.8 ± 0.1BC
|
73.2 ± 0.5D
|
67.1 ± 0.2E
|
59.6 ± 0.1C
|
53.9 ± 0.3C
|
47.6 ± 0.3C
|
39.9 ± 0.7B
|
29.5 ± 0.07D
|
44.3 ± 0.6B
|
Ascorbic Acid
|
94.7 ± 0.1A
|
88.2 ± 0.2A
|
81.9 ± 0.05A
|
76 ± 0.2A
|
69.3 ± 0.05A
|
59 ± 0.1A
|
45.6 ± 0.2A
|
39.2 ± 0.1A
|
17.8 ± 0.1E
|
Superoxide Scavenging
(SOS)
|
MCZNPs
|
84.6 ± 0.2C
|
79.3 ± 0.1B
|
72.6 ± 0.06C
|
68.6 ± 0.1B
|
57.8 ± 0.4B
|
53.6 ± 0.4B
|
43.5 ± 0.4A
|
34.1 ± 0.1B
|
27.6 ± 0.9D
|
Ascorbic Acid
|
92.3 ± 0.3A
|
88.1 ± 0.4A
|
77.8 ± 0.5B
|
66.4 ± 0.8B
|
59.7 ± 0.1B
|
48 ± 0.1C
|
38.4 ± 0.1BC
|
28.7 ± 0.1DE
|
34 ± 0.9C
|
TAC (Ascorbic Acid Eq mg/g)
|
87.8 ± 0.8B
|
78.0 ± 0.9BC
|
70.1 ± 0.9D
|
55.6 ± 0.8D
|
51.4 ± 0.8D
|
42.8 ± 0.8D
|
37 ± 0.7C
|
27.4 ± 0.8E
|
52.3 ± 0.9A
|
TRP (Gallic Acid Eq mg/g)
|
84.6 ± 0.7C
|
76.3 ± 0.7C
|
69 ± 0.3DE
|
59.4 ± 0.5C
|
51.7 ± 0.7CD
|
44.4 ± 0.5D
|
36.5 ± 0.3C
|
31.7 ± 0.5C
|
47.5 ± 0.9B
|
Mean ± SE. Superscripts A-E showing highest significance difference (HSD) (p < 0.05). MCZNP: Zinc oxide nanoparticles of Malvastrum coromandelianum
In-vivo analysis
Hematological profiling and acute toxicity analysis
The hematological profiling of normal and MCZNP treated female rats (Table 3) revealed significant results. The white blood cells (WBCs), mean corpuscular value (MCV), platelets (PLT), lymphocytes (LYM), neutrophils (NTP) and mean corpuscular hemoglobin concentration (MCHC) exhibited significance increase as compared to normal rats while values of red blood cells (RBCs) and hemoglobin (HB) showed non-significant increase.
Table 3 Treatment effects of MCNPs on hematological profile.
|
WBCs (103/mm3)
|
RBCs (106/mm3)
|
HB (g/dl)
|
MCV (fl)
|
PLT (103/ml)
|
LYM (103/m3)
|
NTP (103/mm3)
|
MCHC (g/dl)
|
Control
|
6.73 ± 0.10
|
6.06 ± 0.28
|
12.39 ± 0.18
|
58.24 ± 0.43
|
178.67 ± 0.29
|
3.70 ± 0.10
|
28.70 ± 0.59
|
8.90 ± 0.12
|
MCZNPs
(2000 mg/kg)
|
11.34 ± 0.12A
|
7.11 ± 0.29
|
13.76 ± 0.08B
|
64.09 ± 0.15B
|
201.77 ± 0.48A
|
8.57 ± 0.15B
|
33.89 ± 0.56
|
7.34 ± 0.13B
|
MCZNPs
(3000 mg/kg)
|
10.03 ± 0.28B
|
7.43 ± 0.27
|
14.16 ± 0.09A
|
76.25 ± 0.35A
|
105.20 ± 0.13B
|
9.22 ± 0.21A
|
32.80 ± 0.13
|
13.22 ± 0.15A
|
Mean ± SE (n=6). Superscripts A-B showing highest significance difference (HSD) (p < 0.05). MCZNP: Zinc oxide nanoparticles of Malvastrum coromandelianum, WBCs: White blood cells, RBCs: Red blood cells, HB: Hemoglobin, MCV: Mean corpuscular value, PLT: Platelets, LYM: Lymphocytes, NTP: Neutrophils, MCHC: mean corpuscular hemoglobin concentration
Liver and body weight analysis
Table 4 illustrates the % increase in body weight and relative liver weight of the experimental rats increased after treatment with CCl4 alone and also in combination with silymarin and MCZNP.
Table 4 Treatment effects of MCZNPs on body weight and liver weight of rats
Treatment
|
Initial Body Weight (g)
|
Final Body Weight (g)
|
% Increase in body weight
|
Absolute liver weight (g)
|
Relative liver weight (mg/g of body weight)
|
Control
|
159.4 ± 1.0
|
230.1 ± 1.6
|
44.3 ± 1.3
|
7.04 ± 0.01
|
3.06 ± 0.03
|
CCl4 1 ml/kg
|
162.9 ± 1.0
|
201.7 ± 1.3
|
23.8 ± 1.2D
|
10.11 ± 0.03A
|
5.01 ± 0.04A
|
CCl4 1ml/kg+ Sily 200 mg/kg
|
164.6 ± 1.7
|
235.7 ± 1.0
|
43.3 ± 1.8A
|
7.60 ± 0.06C
|
3.23 ± 0.03C
|
CCl4 + MCZNP Low Dose 150 mg/kg
|
158.9 ± 1.1
|
206.3 ± 1.3
|
29.8 ± 1.4C
|
10.05 ± 0.03A
|
4.87 ± 0.05A
|
CCl4 + MCZNP High Dose 300 mg/kg
|
164.3 ± 1.3
|
224.1 ± 1.3
|
36.4 ± 1.2B
|
8.81 ± 0.03B
|
3.93 ± 0.03B
|
MCZNP Low dose 150 mg/kg
|
154.8 ± 1.0
|
224.8 ± 1.3
|
45.2 ± 1.6
|
7.65 ± 0.02
|
3.40 ± 0.03
|
MCZNP High Dose 300 mg/kg
|
161.3 ± 1.1
|
244.8 ± 1.7
|
51.8 ± 1.8
|
7.63 ± 0.03
|
3.12 ± 0.02
|
Mean ± SE (n=6). Superscripts A-D showing highest significance difference (HSD) (p < 0.05). CCl4: Carbon tetrachloride, Sily: Silymarin, MCZNP: Zinc oxide nanoparticles of Malvastrum coromandelianum
Serum markers analysis
Serum analysis of treated and control group rats expressed varied level of hepatic serum markers. An increase in concentration of ALT (u/l) AST u/l), ALP (u/l) and serum proteins (mg/dl) was observed in all the groups. However, highly significant increase can be seen in CCl4 (1 ml/kg) treated rats, while in rest of the groups exhibited significant increase. Albumin (mg/dl) values were nonsignificant while in case of bilirubin significant increase was observed for CCl4 and CCl4 + Low Dose (150 mg/kg) treated groups (Table 5).
Table 5 Treatment effects of MCNPs on hepatic serum markers
Treatment
|
ALT U/L
|
AST U/L
|
ALP U/L
|
Serum Proteins mg/dl
|
Albumin mg/dl
|
Bilrubin mg/dl
|
Control
|
44.78 ± 1.17
|
65.94 ± 1.36
|
64.46 ± 1.55
|
8.43 ± 0.22
|
4.39 ± 0.16
|
0.76 ± 0.02
|
CCl4 1 ml/kg
|
117.88 ± 1.20A
|
137.39 ± 1.21A
|
130.18 ± 1.90A
|
5.88 ± 0.27B
|
2.61 ± 0.13B
|
1.67 ±0.03A
|
CCl4 1ml/kg+ Sily 200 mg/kg
|
60.00 ± 1.12D
|
74.94 ± 1.38C
|
77.04 ± 1.34C
|
7.18 ± 0.18A
|
3.86 ± 0.22A
|
0.80 ± 0.02D
|
CCl4 + MCZNP Low Dose 150 mg/kg
|
99.47 ± 1.23B
|
95.32 ± 1.92B
|
86.79 ± 1.57B
|
6.44 ± 0.16AB
|
3.01 ± 0.12B
|
1.21 ± 0.03B
|
CCl4 + MCZNP High Dose 300 mg/kg
|
69.98 ± 1.06C
|
77.15 ± 1.61C
|
79.58 ± 1.07C
|
7.56 ± 0.18AB
|
3.34 ± 0.13AB
|
0.87 ± 0.01C
|
MCZNP Low dose 150 mg/kg
|
55.50 ± 1.22
|
72.03 ± 1.63
|
77.32 ± 1.03
|
7.97 ± 0.24
|
3.74 ± 0.09
|
0.77 ± 0.02
|
MCZNP High Dose 300 mg/kg
|
47.42 ± 1.14
|
62.95 ± 1.33
|
66.94 ±1.33
|
8.51 ± 0.11
|
3.95 ± 0.29
|
0.73 ± 0.01
|
Mean ± SE (n=6). Superscripts A-D showing highest significance difference (HSD) (p < 0.05). CCl4: Carbon tetrachloride, Sily: Silymarin, MCZNP: Zinc oxide nanoparticles of Malvastrum coromandelianum, ALT: Alanine transaminase, AST: Aspartate aminotransferase, ALP: Alkaline phosphatase
Hepatic biochemical markers
Hepatic biochemical markers represented optimum values in control group. Treatment of CCl4 resulted in elevation of proteins, TBARS, H2O2, and nitrite however, on treatment with silymarin and low and high doses of MCZNPs resulted in normalization of elevated values. However, the values of these biochemical markers vary significantly on treatment only with MCZNP (Table 6).
Table 6 Treatment effects of MCNPs on hepatic biochemical markers
Treatment
|
Protein (µg/mg tissue)
|
TBARS (nM/min/mg protein)
|
H2O2 (nM/min/mg tissue)
|
Nitrite (µM/mg protein)
|
Control
|
13.78 ± 0.33
|
41.23 ± 1.25
|
10.88 ± 0.77
|
46.88 ± 1.26
|
CCl4 1 ml/kg
|
7.53 ± 0.56C
|
93.25 ± 1.53A
|
15.11 ± 0.41A
|
63.98 ± 0.91A
|
CCl4 1ml/kg+ Sily 200 mg/kg
|
10.81 ± 0.19A
|
48.88 ± 1.30D
|
11.20 ± 0.93B
|
50.68 ± 1.21B
|
CCl4 + MCZNP Low Dose 150 mg/kg
|
9.99 ± 0.16B
|
68.71 ± 1.21B
|
14.68 ± 1.06A
|
60.51 ± 1.12A
|
CCl4 + MCZNP High Dose 300 mg/kg
|
10.57 ± 0.12A
|
62.95 ± 0.60C
|
13.55 ± 0.61AB
|
54.18 ± 1.04B
|
MCZNP Low dose 150 mg/kg
|
11.44 ± 0.10
|
50.93 ± 0.98
|
11.58 ± 0.91
|
46.48 ± 1.41
|
MCZNP High Dose 300 mg/kg
|
11.83 ± 0.18
|
44.21 ± 0.85
|
10.66 ± 0.59
|
46.85 ± 0.92
|
Mean ± SE (n=6). Superscripts A-D showing highest significance difference (HSD) (p < 0.05). CCl4: Carbon tetrachloride, Sily: Silymarin, MCZNP: Zinc oxide nanoparticles of Malvastrum coromandelianum, TBARS: Thiobarbituric acid reactive substances, H2O2: Hydrogen peroxide
Antioxidant status in MCNPs treated rats
Hepatic antioxidant enzyme levels were reduced in CCl4 treated group. In positive control group, on treatment with silymarin (200mg/kg) catalase, peroxidase, superoxide dismutase and glutathione levels were restored. Co-administration of MCZNP (150 mg/kg and 300 mg/kg) + CCl4 brought the antioxidant values to normal. Rats treated with MCZNP (high and low doses) alone showed values comparable with control group (Figure 5).
Expression of genetic markers
The mRNA expression of genetic markers regulated under hepatic toxicity and inflammation showed varied values among different groups (Figure 6). High expression was observed in CCl4 treated groups for GRP-78, XBP-1t, XBP-1s, XBP-1u, MCP-1 and CHOP, along with the upregulation of pro-inflammatory cytokines including TNF-α and IL-6. In case of GCLC moderate fold change was observed however, BCL-2 was downregulated (p < 0.05) on CCl4 administration. In positive control group, silymarin (200 mg/kg) restored gene expression of all the genetic markers towards normal. Results depicted that silymarin act as a hepato-protective agent. Fold change analysis showed that low (150 mg/kg) and high (300 mg/kg) doses of MCZNP along with CCl4 revert the toxicity induce by CCl4. While mRNA expression showed that the treatment of MCZNP alone left no toxic effect on rat liver.
Histopathological study
Normal hepatic cell morphology was observed in control group (Figure 7A). CCl4 treatment resulted in liver cell fibrosis (Figure 7B). Neutrophil infiltration, sinusoidal obstruction and hypertrophy, which can be seen in Figure 7C while, on treatment with MCZNPs in combination with CCl4 (Figure 7D and 7E) and only MCZNPs (low and high doses) resulted in attainment of normal morphology of liver cells (Figure 7F and 7G). Level of hepatic injury representing to severity of hepatic injury is shown in Figure 7 H.