LCMS analysis of MLHEF suggest following tentative major compounds:
LCMS analysis of M.longifolia hydro ethanolic extract and structure of tentative compounds. LCMS finger printing analysis of MLHEF shows the presence of high amount of sugar moiety i.e D Arabinose (RT 1.08), and flavonoids i.e myricetrin (RT 7.65), quercetin (RT 10.82). 10- Shogaol was also identified at RT 8.30. Two more compound were partially characterized 3-O- 28-O-(R-L-rhamnopyranosyl-(1f3)-β-D-xylopyranosyl-(f4) [R-L-rhamnopyranosyl-(1f3)]-R-L-rham-nopyranosyl-(1f2)-â-D xylopyranosyl) protobassic acid and β-D-glucopyranosyl protobassic acid at RT 9.61 and 11.90 respectively (shown in figure.1 and table.1).
Table 1
LCMS analysis MLHEF suggest following tentative compounds.
S.no
|
RT
|
Tentative Compound
|
Mol. Wt. (M+)
|
References
|
1
|
1.08
|
D Arabinose
|
151.3
|
[24]
|
2
|
7.65
|
Myricetrin
|
319.3
|
[25]
|
3
|
8.30
|
10- Shogaol
|
333.3
|
[26]
|
4
|
9.61
|
3-O- 28-O-(R-L-rhamnopyranosyl-(1f3)-β-D-xylopyranosyl-(f4)[R-L-rhamnopyranosyl-(1f3)]-R-L-rham-nopyranosyl-(1f2)-â-D xylopyranosyl) protobassic acid,
|
1367.9
|
[27]
|
5
|
10.82
|
Quercetin
|
302.6
|
[25]
|
6
|
11.90
|
β-D-glucopyranosyl protobassic acid
|
684.7
|
[27]
|
Table 1
LCMS analysis of M.longifolia hydro ethanolic extract and structure of tentative compounds. LCMS finger printing analysis of MLHEF shows the presence of high amount of sugar moiety i.e D Arabinose (RT 1.08), and flavonoids i.e myricetrin (RT 7.65), quercetin (RT 10.82). 10- Shogaol was also identified at RT 8.30. Two more compound were partially characterized3-O-28-O-(R-L-rhamnopyranosyl-(1f3)-β-D-xylopyranosyl-(f4)[R-L rhamnopyranosyl-(1f3)]-R-L-rham-nopyranosyl-(1f2)-â-D xylopyranosyl) protobassic acid and β-D-glucopyranosyl protobassic acid at RT 9.61 and 11.90 respectively.
MLHEF attenuates physiological changes in diabetes mice.
The MLHEF treatment minimizes the hepatic oxidative stress elevated due to administration of HFD + STZ and is expressed in Table 2. There was significant decrease (p < 0.001) whereby 50% reduction was observed in SOD and CAT level while increase in MDA level in high fat fed group and HFD + STZ group was observed as compared to normal control group. Treatment with MLHEF significantly reduced (p < 0.001) the hepatic oxidative stress in liver.
Table 2
Effect of MLHEF treatment on liver oxidative stress profile altered due to HFD and HFD + STZ. All values are mean ± SD; n = 6; ap≤0.001 compared to control, bp≤0.001 compared to obesity control, cp≤0.001 compared to diabetes control [One-way ANOVA followed by Tukey's Multiple Comparison].
S.no
|
SOD
(U/mg protein)
|
CAT
(nmoles of H2O2 consumed /min/mg protein)
|
MDA
(nmol/g)
|
AST
(IU/L)
|
ALT
(IU/L)
|
Normal Control
|
40.03 ± 1.89
|
45.78 ± 3.01
|
13.11 ± 1.46
|
16.45 ± 1.11
|
26.22 ± 3.21
|
Obesity Control
|
20.11 ± 1.32a
|
25.45 ± 2.33a
|
24.45 ± 2.44a
|
39.65 ± 1.75a
|
53.56 ± 3.16a
|
Diabetic Control
|
24.42 ± 2.01a
|
23.97 ± 1.98a
|
26.89 ± 2.16a
|
45.66 ± 1.36a
|
57.14 ± 2.89a
|
Treated (400 mg/kg)
|
37.45 ± 1.41b,c
|
37.89 ± 2.13b,c
|
16.87 ± 1.84b,c
|
23.47 ± 1.41b,c
|
35.37 ± 2.13b,c
|
Standard
|
41.84 ± 3.10
|
43.86 ± 3.11
|
14.77 ± 3.11
|
20.54 ± 2.66
|
30.42 ± 3.42
|
HFD and HFD + STZ caused oxidative imbalance by altering the oxidising enzymes. In order to examine the chronic HFD associated hepatic injury was determined by the activity of liver enzymes such as AST and ALT. There was twofold elevation (p < 0.01) in AST and ALT level in HFD and HFD + STZ group. There was significant decrease (p < 0.01) in AST and ALT level was observed in MLHEF treatment group (see supplementary material Table 1).
MLHEF administration mitigates GLUT (1–4) expression in liver, adipose tissue, and skeletal muscles.
In liver (Fig. 2), GLUT 2 and GLUT4 protein content was observed to be initially lower in obesity control and diabetic control as compared to normal control. MLHEF (400 mg/kg) treatment reprogramed the GLUT 2 and GLUT 4 protein expression in diabetic group towards the balanced glucotransporter protein expression. On the other hand GLUT 1 and GLUT 3 protein was found to be significantly increased (p < 0.001) in disease control groups (obesity control and diabetic control) as compared to normal control group which was further reduced by the MLHEF treatment (400 mg/kg).
In skeletal muscle tissue (Fig. 2), GLUT 2 and GLUT4 protein expression was shown to be lesser in obesity control and diabetic control as compared to normal control. MLHEF (400 mg/kg) treatment modulated the GLUT 2 and GLUT 4 protein expression in diabetic group towards the balanced gluco transporter protein expression in skeletal muscle. However, GLUT 1 and GLUT 3 protein expression was found to be elevated in disease control groups (obesity control and diabetic control) ascompared to normal control group which was reprogrammed by the MLHEF treatment to diabetic mice.
In adipose tissue (Fig. 2), GLUT 2 and GLUT4 protein content was shown to be reduced in obesity control and diabetic control as compared to normal control. After the treatment with MLHEF (400 mg/kg), the GLUT 2 and GLUT 4 protein expression were upregulated in diabetic animals towards the balanced glucose transporter protein expression. Contrarily it was found that GLUT 1 and GLUT 3 protein was found be significantly downregulated (p < 0.001) in disease control groups (obesity control and diabetic control) as compared to normal control group and was restored by the MLHEF treatment to diabetic group
In this study, the effect on prolonged treatment with MLHEF on Class 1 GLUT transporter (GLUT 1, GLUT2 GLUT 3, and GLUT 4) protein expression in the liver, skeletal muscles and adipose tissue in normal control mice, obesity control, type 2 diabetic control and MLHEF treated group were studied. In liver, skeletal muscles and adipose tissue (Fig. 2), GLUT 2 and GLUT4 protein content was shown to be initially lower in obesity control and diabetic control as compared to normal control. MLHEF (400mg/kg) treatment induced the GLUT 2 and GLUT 4 protein expression in diabetic group towards the balanced glucose transporter protein expression. While GLUT 1 and GLUT 3 protein was found to be increase in disease control group (obesity control and diabetic control) as compared to normal control group which was reduced by the MLHEF treatment (400mg/kg).
MLHEF administration mitigates LDH level in liver, adipose tissue, and skeletal muscles.
Lactate a cytosolic product of glycolytic pathway, is considered as a modulator of energy homeostasis. Figure 3 and Table 3 illustrates the effect of MLHEF treatment on HFD and HFD + STZ induced alteration in LDH activity in different organs (a) liver, (b) white adipose tissue and (c) skeletal muscles, respectively. One-way ANOVA showed that there were significant differences in liver (p < 0.05), skeletal muscle (p < 0.05), and white adipose tissue (p < 0.05) activity among the groups. The HFD and HFD + STZ significantly reduced the respiratory enzyme activities in liver tissues compared to control animals. MLHEF (400 mg/kg) attenuated HFD induced decrease in the activity of LDH activity in liver, skeletal muscle and white adipose tissue as compared to disease control (Obesity control and diabetic control) groups.
MLHEF administration restores mitochondrial complex activity in diabetes mice.
Figure 4 illustrates the effect of MLHEF treatment on HFD and HFD + STZ induced alteration in the mitochondrial complex-I (a), mitochondrial complex-II (b), mitochondrial complex-IV (c), and mitochondrial complex-V (d) activities of liver tissue. One-way ANOVA showed that there were significant difference in complex-I (p<0.05), complex-II (p<0.05), complex-IV(p<0.05), and complex-V (p<0.05) activity among the groups. The HFD and HFD + STZ significantly reduced the respiratory enzyme activities in liver tissues compared to control animals. MLHEF attenuated HFD induced decrease in the activity of complex-I, II, IV, and complex-V in liver tissues as compared to disease control (Obesity control and diabetic control) groups.
Figure 4
Describes the effect of MLHEF treatment on HFD and HFD + STZ induce alteration in the mitochondrial complexes- I(a), II (b), IV (c), and V(d) activities of liver tissue. One way ANOVA analysis indicated the significant difference in complex I (NADH+) (F (4, 24) = 1.97, p < 0.001), complex II (SDH) (F (4, 24) = 18.18, p < 0.001), complex-IV (F (4, 24) = 23.45, p < 0.001), and complex-V (F (4, 24) = 9.8, p < 0.001) activity. The HFD and HFD + STZ significantly decreased the activity of mitochondrial respiratory enzymes as compared to normal control. MLHEF reversed the HFD associated decrease in the activity of mitochondrial complexes in liver tissues as compared to disease control (Obesity control and diabetic control) group mice. All values are mean ± SD; n = 6; ap≤0.001 compared to control, bp≤0.001 compared to obesity control, cp≤0.001 compared to diabetes control [One-way ANOVA followed by Tukey's Multiple Comparison].