Antidiabetic and Immunoregulatory Activities of Extract of Phyllanthus emblica L. in NOD with Spontaneous and Cyclophosphamide-Accelerated Diabetic Mice

Abstract

Oil-Gan, also known as emblica, is the fruit of the genus Phyllanthus emblica L. The fruits are high in nutrients and display excellent health care functions and development values. The primary aim of this study was to investigate the activities of ethyl acetate extract from Phyllanthus emblica L. (EPE) on type 1 diabetes mellitus (T1D) and immunoregulatory activities in non-obese diabetes (NOD) mice with spontaneous and cyclophosphamide (Cyp)-accelerated diabetes. EPE was vehicle-administered to spontaneous NOD (S-NOD) mice or Cyp-accelerated NOD (Cyp-NOD) mice once daily at a dose of 400 mg/kg body weight for 15 or 4 weeks, respectively. At the end, blood samples were collected for biological analyses, organ tissues were dissected for analyses of histology and immunofluorescence (IF) staining (including expressions of Bcl and Bax), the expression levels of targeted genes by Western blotting and forkhead box P3 (Foxp3), and helper T lymphocyte 1 (Th1)/Th2/Th17/Treg regulatory T cell (Treg) cell distribution by flow cytometry. Our results showed that EPE-treated NOD mice or Cyp-accelerated NOD mice display a decrease in levels of blood glucose and HbA1c, but an increase in blood insulin levels. EPE treatment decreased blood levels of IFN-γ and tumor necrosis α (TNF-α) by Th1 cells, and reduced interleukin (IL)-1β and IL-6 by Th17 cells, but increased IL-4, IL-10, and transforming growth factor-β1 (TGF-β1) by Th2 cells in both of the two mice models by enzyme-linked immunosorbent assay (ELISA) analysis. Flow cytometric data showed that EPE-treated Cyp-NOD mice had decreased the CD4⁺ subsets T cell distribution of CD4⁺IL-17 and CD4⁺ interferon gamma (IFN-γ), but increased the CD4⁺ subsets T cell distribution of CD4⁺IL-4 and CD4⁺Foxp3. Furthermore, EPE-treated Cyp-NOD mice had decreased the percentage per 10,000 cells of CD4⁺IL-17 and CD4⁺IFNγ, and increased CD4⁺IL-4 and CD4⁺Foxp3 compared with the Cyp-NOD Con group (p < 0.001, p < 0.05, p < 0.05, and p < 0.05, respectively). For target gene expression levels in the pancreas, EPE-treated mice had reduced expression levels of inflammatory cytokines, including IFN-γ and TNF-α by Th1 cells, but increased expression levels of IL-4, IL-10, and TGF-1β by Th2 cells in both two mice models. Histological examination of the pancreas revealed that EPE-treated mice had not only increased pancreatic insulin-expressing β cells (brown), and but also enhanced the percentage of Bcl-2 (green)/Bax (red) by IF staining analyses of islets compared with the S-NOD Con and the Cyp-NOD Con mice, implying that EPE displayed the protective effects of pancreas β cells. EPE-treated mice showed an increase in the average immunoreactive system (IRS) score on insulin within the pancreas, and an enhancement in the numbers of the pancreatic islets. EPE displayed an improvement in the pancreas IRS scores and a decrease in proinflammatory cytokines. Moreover, EPE exerted blood-glucose-lowering effects by regulating IL-17 expressions. Collectively, these results implied that EPE inhibits the development of autoimmune diabetes by regulating cytokine expression. Our results demonstrated that EPE has a therapeutic potential in the preventive effects of T1D and immunoregulation as a supplementary.

Keywords: Phyllanthus emblica L.; cyclophosphamide; diabetes; immunoregulatory; interleukin 17 (IL-17); non-obese diabetes.

Figure 1

(A) The fruits of Phyllanthus emblica L. (B) Preparation of ethyl acetate extract from Phyllanthus emblica L. (EPE). The fruits of Phyllanthus emblica were extracted with methanol three times at room temperature to concentration under vacuum, and then the crude methanolic extract was obtained. The crude methanolic extract was subjected three times to the of suspension in H₂O and partition with EtOAc, respectively, and followed by concentration under reduced pressure, and then the H₂O fraction and the EtOAc fraction were obtained (B).

Figure 1

(A) The fruits of Phyllanthus emblica L. (B) Preparation of ethyl acetate extract from Phyllanthus emblica L. (EPE). The fruits of Phyllanthus emblica were extracted with methanol three times at room temperature to concentration under vacuum, and then the crude methanolic extract was obtained. The crude methanolic extract was subjected three times to the of suspension in H₂O and partition with EtOAc, respectively, and followed by concentration under reduced pressure, and then the H₂O fraction and the EtOAc fraction were obtained (B).

Figure 2

Effects of ethyl acetate from Phyllanthus emblica L. (EPE) on (A) body weight, (B) blood glucose, (C) HbA1_C, (D) insulin, (E) adiponectin, (F) leptin levels, (G) blood cytokine levels-1: IL-4, IL-10, and IFN-γ, and (H) blood cytokine levels-2: IL-1β, IL-6, TGF-1β, and TNF-α levels in spontaneous non-obese diabetes (S-NOD) mice. * p < 0.05, or *** p < 0.001 compared to the S-NOD plus vehicle (S-NOD Con) group. All values are means ± SE (n = 7 per group). EPE: 400 mg/kg body weight.

Figure 2

Effects of ethyl acetate from Phyllanthus emblica L. (EPE) on (A) body weight, (B) blood glucose, (C) HbA1_C, (D) insulin, (E) adiponectin, (F) leptin levels, (G) blood cytokine levels-1: IL-4, IL-10, and IFN-γ, and (H) blood cytokine levels-2: IL-1β, IL-6, TGF-1β, and TNF-α levels in spontaneous non-obese diabetes (S-NOD) mice. * p < 0.05, or *** p < 0.001 compared to the S-NOD plus vehicle (S-NOD Con) group. All values are means ± SE (n = 7 per group). EPE: 400 mg/kg body weight.

Figure 2

Effects of ethyl acetate from Phyllanthus emblica L. (EPE) on (A) body weight, (B) blood glucose, (C) HbA1_C, (D) insulin, (E) adiponectin, (F) leptin levels, (G) blood cytokine levels-1: IL-4, IL-10, and IFN-γ, and (H) blood cytokine levels-2: IL-1β, IL-6, TGF-1β, and TNF-α levels in spontaneous non-obese diabetes (S-NOD) mice. * p < 0.05, or *** p < 0.001 compared to the S-NOD plus vehicle (S-NOD Con) group. All values are means ± SE (n = 7 per group). EPE: 400 mg/kg body weight.

Figure 2

Effects of ethyl acetate from Phyllanthus emblica L. (EPE) on (A) body weight, (B) blood glucose, (C) HbA1_C, (D) insulin, (E) adiponectin, (F) leptin levels, (G) blood cytokine levels-1: IL-4, IL-10, and IFN-γ, and (H) blood cytokine levels-2: IL-1β, IL-6, TGF-1β, and TNF-α levels in spontaneous non-obese diabetes (S-NOD) mice. * p < 0.05, or *** p < 0.001 compared to the S-NOD plus vehicle (S-NOD Con) group. All values are means ± SE (n = 7 per group). EPE: 400 mg/kg body weight.

Figure 3

Flow cytometric data of ethyl acetate from Phyllanthus emblica L. (EPE) on numbers of (A) IL-17, (B) IL-4, (C) IFNγ, (D) Foxp3 subsets of CD4⁺ T cell distribution, and (E) four subset CD4⁺ T cell distribution per 10,000 cells (%) in spontaneous non-obese diabetes (S-NOD) mice. * p < 0.05 or ** p < 0.01 compared to the S-NOD plus vehicle (S-NOD Con) group. All values are means ± SE (n = 7 per group). EPE: 400 mg/kg body weight.

Figure 3

Flow cytometric data of ethyl acetate from Phyllanthus emblica L. (EPE) on numbers of (A) IL-17, (B) IL-4, (C) IFNγ, (D) Foxp3 subsets of CD4⁺ T cell distribution, and (E) four subset CD4⁺ T cell distribution per 10,000 cells (%) in spontaneous non-obese diabetes (S-NOD) mice. * p < 0.05 or ** p < 0.01 compared to the S-NOD plus vehicle (S-NOD Con) group. All values are means ± SE (n = 7 per group). EPE: 400 mg/kg body weight.

Figure 4

Target gene expression levels (including IFN-γ, TNF-α, IL-4, IL-10, or TGF-β1) in the pancreas following treatment of ethyl acetate extract from Phyllanthus emblica L. (EPE) in spontaneous non-obese diabetes (S-NOD) mice by Western blotting. (A) Representative image; (B) target gene expression quantification to β-actin. Protein was separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) detected by Western blotting. *** p < 0.001 compared to the S-NOD plus vehicle (S-NOD Con) group. All values are means ± SE (n = 7 per group). EPE: 400 mg/kg body weight.

Figure 4

Target gene expression levels (including IFN-γ, TNF-α, IL-4, IL-10, or TGF-β1) in the pancreas following treatment of ethyl acetate extract from Phyllanthus emblica L. (EPE) in spontaneous non-obese diabetes (S-NOD) mice by Western blotting. (A) Representative image; (B) target gene expression quantification to β-actin. Protein was separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) detected by Western blotting. *** p < 0.001 compared to the S-NOD plus vehicle (S-NOD Con) group. All values are means ± SE (n = 7 per group). EPE: 400 mg/kg body weight.

Figure 5

Histological examination of ethyl acetate extract from Phyllanthus emblica L. (EPE) on: (A) representative pathogenesis photographs of 400× immunohistochemical (IHC) staining of pancreatic insulin-expressing β cells (brown) and glucagon-expressing (green) α cells; (B) the immunoreactive system (IRS) score of the pancreas according to both the intensity and quantity of the dyeing signal for makers (including glucagon and insulin), and the numbers of the pancreatic islets within the pancreas; and (C–E): immunofluorescence staining effects on the expression levels of Bcl-2 (green) and Bax (red) in spontaneous non-obese diabetes (S-NOD) mice (C) at magnification of 100×. Left: The expression levels of Bcl-2 and Bax of the Cyp-NOD Con group. Right: The expression levels of Bcl-2 and Bax of the Cyp-NOD +EPE group; (D) at magnification of 200×. (E) Quantification of effects of EPE on expression levels of Bcl-2 (green) and Bax (red). The percentage (%) of Bcl-2/Bax was calculated using three slides per group under nine visions to count cell numbers using Image J software. The green fluorescence/red fluorescence under one vision was presented as Bcl-2/Bax per cell, and the numbers of Bcl-2/Bax per 100 cells were calculated as % of Bcl-2/Bax. All values are means ± SE (n = 7 per group). *** p < 0.001 compared to the S-NOD plus vehicle (S-NOD Con) group. EPE: 400 mg/kg body weight.

Figure 5

Histological examination of ethyl acetate extract from Phyllanthus emblica L. (EPE) on: (A) representative pathogenesis photographs of 400× immunohistochemical (IHC) staining of pancreatic insulin-expressing β cells (brown) and glucagon-expressing (green) α cells; (B) the immunoreactive system (IRS) score of the pancreas according to both the intensity and quantity of the dyeing signal for makers (including glucagon and insulin), and the numbers of the pancreatic islets within the pancreas; and (C–E): immunofluorescence staining effects on the expression levels of Bcl-2 (green) and Bax (red) in spontaneous non-obese diabetes (S-NOD) mice (C) at magnification of 100×. Left: The expression levels of Bcl-2 and Bax of the Cyp-NOD Con group. Right: The expression levels of Bcl-2 and Bax of the Cyp-NOD +EPE group; (D) at magnification of 200×. (E) Quantification of effects of EPE on expression levels of Bcl-2 (green) and Bax (red). The percentage (%) of Bcl-2/Bax was calculated using three slides per group under nine visions to count cell numbers using Image J software. The green fluorescence/red fluorescence under one vision was presented as Bcl-2/Bax per cell, and the numbers of Bcl-2/Bax per 100 cells were calculated as % of Bcl-2/Bax. All values are means ± SE (n = 7 per group). *** p < 0.001 compared to the S-NOD plus vehicle (S-NOD Con) group. EPE: 400 mg/kg body weight.

Figure 6

Effects of ethyl acetate from Phyllanthus emblica L. (EPE) on (A) body weight, (B) blood glucose, (C) HbA1_C, (D) insulin, (E) adiponectin, (F) leptin levels, (G) blood cytokine levels-1: IL-4, IL-10, and IFN-γ, and (H) blood cytokine levels-2: IL-1β, IL-6, and TGF-β1 levels in cyclophosphamide (Cyp)-accelerated non-obese diabetes (Cyp-NOD) mice. All values are means ± SE (n = 12). * p <0.05 and *** p < 0.001 compared with the Cyp induction plus vehicle (distilled water) (Cyp-NOD Con) group. EPE: 400 mg/kg body weight.

Figure 6

Effects of ethyl acetate from Phyllanthus emblica L. (EPE) on (A) body weight, (B) blood glucose, (C) HbA1_C, (D) insulin, (E) adiponectin, (F) leptin levels, (G) blood cytokine levels-1: IL-4, IL-10, and IFN-γ, and (H) blood cytokine levels-2: IL-1β, IL-6, and TGF-β1 levels in cyclophosphamide (Cyp)-accelerated non-obese diabetes (Cyp-NOD) mice. All values are means ± SE (n = 12). * p <0.05 and *** p < 0.001 compared with the Cyp induction plus vehicle (distilled water) (Cyp-NOD Con) group. EPE: 400 mg/kg body weight.

Figure 6

Effects of ethyl acetate from Phyllanthus emblica L. (EPE) on (A) body weight, (B) blood glucose, (C) HbA1_C, (D) insulin, (E) adiponectin, (F) leptin levels, (G) blood cytokine levels-1: IL-4, IL-10, and IFN-γ, and (H) blood cytokine levels-2: IL-1β, IL-6, and TGF-β1 levels in cyclophosphamide (Cyp)-accelerated non-obese diabetes (Cyp-NOD) mice. All values are means ± SE (n = 12). * p <0.05 and *** p < 0.001 compared with the Cyp induction plus vehicle (distilled water) (Cyp-NOD Con) group. EPE: 400 mg/kg body weight.

Figure 6

Effects of ethyl acetate from Phyllanthus emblica L. (EPE) on (A) body weight, (B) blood glucose, (C) HbA1_C, (D) insulin, (E) adiponectin, (F) leptin levels, (G) blood cytokine levels-1: IL-4, IL-10, and IFN-γ, and (H) blood cytokine levels-2: IL-1β, IL-6, and TGF-β1 levels in cyclophosphamide (Cyp)-accelerated non-obese diabetes (Cyp-NOD) mice. All values are means ± SE (n = 12). * p <0.05 and *** p < 0.001 compared with the Cyp induction plus vehicle (distilled water) (Cyp-NOD Con) group. EPE: 400 mg/kg body weight.

Figure 7

Flow cytometric data of ethyl acetate from Phyllanthus emblica L. (EPE) on numbers of (A) IL-17, (B) IL-4, (C) IFNγ, (D) Foxp3 subsets of CD4⁺ T cell distribution, and (E) four subset CD4⁺ T cell distribution per 10,000 cells (%) in cyclophosphamide (Cyp)-accelerated non-obese diabetes (Cyp-NOD) mice. * p < 0.005, ** p < 0.001, or *** p < 0.0001 compared to the Cyp-NOD mice plus vehicle (distilled water) (Cyp-NOD Con) group. All values are means ± SE (n = 12 per group). EPE: 400 mg/kg body weight.

Figure 7

Flow cytometric data of ethyl acetate from Phyllanthus emblica L. (EPE) on numbers of (A) IL-17, (B) IL-4, (C) IFNγ, (D) Foxp3 subsets of CD4⁺ T cell distribution, and (E) four subset CD4⁺ T cell distribution per 10,000 cells (%) in cyclophosphamide (Cyp)-accelerated non-obese diabetes (Cyp-NOD) mice. * p < 0.005, ** p < 0.001, or *** p < 0.0001 compared to the Cyp-NOD mice plus vehicle (distilled water) (Cyp-NOD Con) group. All values are means ± SE (n = 12 per group). EPE: 400 mg/kg body weight.

Figure 8

The target gene IFN-γ, TNF-α, IL-4, IL-10, or TGF-β1 expression levels in the pancreas in Cyp-accelerated NOD (Cyp-NOD) mice following treatment with ethyl acetate extract from Phyllanthus emblica L. (Cyp-NOD+EPE) (EPE: 400 mg/kg body weight) by Western blotting analysis. (A) Representative image. (B) Target gene expression quantification to β-actin. Protein was separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and detected by Western blotting. *** p < 0.0001 compared to the Cyp-NOD mice plus vehicle (distilled water) (Cyp-NOD Con) group. All values are means ± SE (n = 12 per group). EPE: 400 mg/kg body weight.

Figure 9

Histological examination of extract of ethyl acetate from P. emblica (EPE) on: (A) representative pathogenesis photographs of 400× immunohistochemical (IHC) staining of pancreatic insulin-expressing β cells (brown) and glucagon-expressing (green) α cells; (B) the immunoreactive system (IRS) score of the pancreas according to both the intensity and quantity of the dyeing signal for makers (including glucagon and insulin) and the numbers of the pancreatic islets within the pancreas; (C–E): immunofluorescence staining effects on expression levels of Bcl-2 (green) and Bax (red) in cyclophosphamide (Cyp)-accelerated non-obese diabetes (Cyp-NOD) mice (C) at magnification of 100x. Left: The expression levels of Bcl-2 and Bax of the Cyp-NOD Con group. Right: The expression levels of Bcl-2 and Bax of the Cyp-NOD+EPE group (D) at magnification of 200× and (E) quantification of effects of EPE on expression levels of Bcl-2 (green) and Bax (red). The percentage (%) of Bcl-2/Bax was calculated using the method of each group. Three slides under nine visions were observed and the cell numbers were counted by Image J software. The green fluorescence/red fluorescence under one vision was presented as Bcl-2/Bax per cell, and the numbers of Bcl-2/Bax per 100 cells were calculated as % of Bcl-2/Bax. All values are means ± SE (n = 12 per group). EPE: 400 mg/kg body weight. ** p < 0.01, *** p < 0.001.

Figure 9

Histological examination of extract of ethyl acetate from P. emblica (EPE) on: (A) representative pathogenesis photographs of 400× immunohistochemical (IHC) staining of pancreatic insulin-expressing β cells (brown) and glucagon-expressing (green) α cells; (B) the immunoreactive system (IRS) score of the pancreas according to both the intensity and quantity of the dyeing signal for makers (including glucagon and insulin) and the numbers of the pancreatic islets within the pancreas; (C–E): immunofluorescence staining effects on expression levels of Bcl-2 (green) and Bax (red) in cyclophosphamide (Cyp)-accelerated non-obese diabetes (Cyp-NOD) mice (C) at magnification of 100x. Left: The expression levels of Bcl-2 and Bax of the Cyp-NOD Con group. Right: The expression levels of Bcl-2 and Bax of the Cyp-NOD+EPE group (D) at magnification of 200× and (E) quantification of effects of EPE on expression levels of Bcl-2 (green) and Bax (red). The percentage (%) of Bcl-2/Bax was calculated using the method of each group. Three slides under nine visions were observed and the cell numbers were counted by Image J software. The green fluorescence/red fluorescence under one vision was presented as Bcl-2/Bax per cell, and the numbers of Bcl-2/Bax per 100 cells were calculated as % of Bcl-2/Bax. All values are means ± SE (n = 12 per group). EPE: 400 mg/kg body weight. ** p < 0.01, *** p < 0.001.

Figure 10

(A) Preparation of seven fractions of ethyl acetate extract from Phyllanthus emblica L. (EtOAc soluble fractions). The Fruits of Phyllanthus emblica were extracted with methanol, and then evaporated and suspended in H₂O and partitioned with EtOAc. The EtOAc-soluble fraction was then subjected to column chromatography on silica gel using a gradient solvent system of n-hexane and EtOAc as a mobile phase and seven fractions were obtained. Seven fractions of EPE are described as EA. EA included 2–10% EA fraction 1 (EA-1), 10–20% EA fraction 2 (EA-2), 20–30% EA fraction 3 (EA-3), 30–50% EA fraction 4 (EA-4), 50–70% EA fraction 5 (EA-5), 70–100% EA fraction 6 (EA-6), and 100% EA fraction 7 (EA-7). (B–D) Effects of seven fractions of EPE (EA) on expression levels of membrane GLUT4 and phospho-Akt/total-Akt in C2C12 myoblast cells by Western blotting analyses. C2C12 skeletal myoblast cells were treated with seven fractions and equal amounts of lysates were resolved by SDS-PAGE and blotted for GLUT4, total-Akt, and phospho-Akt (Ser⁴⁷³). (B) Representative blots of seven fractions (EA) in C2C12 myoblast cells. (C) Quantification of the expression levels of membrane GLUT4 and (D) the ratio of phospho-Akt to total-Akt. All values are means ± S.E. * p < 0.05, *** p < 0.001 compared with the control group. (E) HPLC chromatogram of the polyphenol standards and EA-6 at 280 nm. Determination of phenolic compounds: the peaks indicate the following: 1. Gallic acid (4.3 min); 2. Protocatechuic acid (6.9 min); 3. Chlorogenic acid (9.1 min); 4. Catechin (9.9 min); 5. Protocatechualdehyde (12.9 min); 6. Vanillic acid (13.3 min); 7. Caffeic acid (13.7 min); 8. Epicatechin (14.8 min); 9. Syingic acid (15.5 min); 10. ρ-Coumaric acid (19.1 min); 11. Ferulic acid (21.9 min); 12. Rutin (27.4 min); 13. Narigin (28.3 min); 14. Quercetin (37.2 min); 15. Hesperetin (38.8 min).

Figure 10

(A) Preparation of seven fractions of ethyl acetate extract from Phyllanthus emblica L. (EtOAc soluble fractions). The Fruits of Phyllanthus emblica were extracted with methanol, and then evaporated and suspended in H₂O and partitioned with EtOAc. The EtOAc-soluble fraction was then subjected to column chromatography on silica gel using a gradient solvent system of n-hexane and EtOAc as a mobile phase and seven fractions were obtained. Seven fractions of EPE are described as EA. EA included 2–10% EA fraction 1 (EA-1), 10–20% EA fraction 2 (EA-2), 20–30% EA fraction 3 (EA-3), 30–50% EA fraction 4 (EA-4), 50–70% EA fraction 5 (EA-5), 70–100% EA fraction 6 (EA-6), and 100% EA fraction 7 (EA-7). (B–D) Effects of seven fractions of EPE (EA) on expression levels of membrane GLUT4 and phospho-Akt/total-Akt in C2C12 myoblast cells by Western blotting analyses. C2C12 skeletal myoblast cells were treated with seven fractions and equal amounts of lysates were resolved by SDS-PAGE and blotted for GLUT4, total-Akt, and phospho-Akt (Ser⁴⁷³). (B) Representative blots of seven fractions (EA) in C2C12 myoblast cells. (C) Quantification of the expression levels of membrane GLUT4 and (D) the ratio of phospho-Akt to total-Akt. All values are means ± S.E. * p < 0.05, *** p < 0.001 compared with the control group. (E) HPLC chromatogram of the polyphenol standards and EA-6 at 280 nm. Determination of phenolic compounds: the peaks indicate the following: 1. Gallic acid (4.3 min); 2. Protocatechuic acid (6.9 min); 3. Chlorogenic acid (9.1 min); 4. Catechin (9.9 min); 5. Protocatechualdehyde (12.9 min); 6. Vanillic acid (13.3 min); 7. Caffeic acid (13.7 min); 8. Epicatechin (14.8 min); 9. Syingic acid (15.5 min); 10. ρ-Coumaric acid (19.1 min); 11. Ferulic acid (21.9 min); 12. Rutin (27.4 min); 13. Narigin (28.3 min); 14. Quercetin (37.2 min); 15. Hesperetin (38.8 min).

Figure 10

(A) Preparation of seven fractions of ethyl acetate extract from Phyllanthus emblica L. (EtOAc soluble fractions). The Fruits of Phyllanthus emblica were extracted with methanol, and then evaporated and suspended in H₂O and partitioned with EtOAc. The EtOAc-soluble fraction was then subjected to column chromatography on silica gel using a gradient solvent system of n-hexane and EtOAc as a mobile phase and seven fractions were obtained. Seven fractions of EPE are described as EA. EA included 2–10% EA fraction 1 (EA-1), 10–20% EA fraction 2 (EA-2), 20–30% EA fraction 3 (EA-3), 30–50% EA fraction 4 (EA-4), 50–70% EA fraction 5 (EA-5), 70–100% EA fraction 6 (EA-6), and 100% EA fraction 7 (EA-7). (B–D) Effects of seven fractions of EPE (EA) on expression levels of membrane GLUT4 and phospho-Akt/total-Akt in C2C12 myoblast cells by Western blotting analyses. C2C12 skeletal myoblast cells were treated with seven fractions and equal amounts of lysates were resolved by SDS-PAGE and blotted for GLUT4, total-Akt, and phospho-Akt (Ser⁴⁷³). (B) Representative blots of seven fractions (EA) in C2C12 myoblast cells. (C) Quantification of the expression levels of membrane GLUT4 and (D) the ratio of phospho-Akt to total-Akt. All values are means ± S.E. * p < 0.05, *** p < 0.001 compared with the control group. (E) HPLC chromatogram of the polyphenol standards and EA-6 at 280 nm. Determination of phenolic compounds: the peaks indicate the following: 1. Gallic acid (4.3 min); 2. Protocatechuic acid (6.9 min); 3. Chlorogenic acid (9.1 min); 4. Catechin (9.9 min); 5. Protocatechualdehyde (12.9 min); 6. Vanillic acid (13.3 min); 7. Caffeic acid (13.7 min); 8. Epicatechin (14.8 min); 9. Syingic acid (15.5 min); 10. ρ-Coumaric acid (19.1 min); 11. Ferulic acid (21.9 min); 12. Rutin (27.4 min); 13. Narigin (28.3 min); 14. Quercetin (37.2 min); 15. Hesperetin (38.8 min).

Figure 11

The graphical abstract of the effects of ethyl acetate extract from Phyllanthus emblica L. in NOD with spontaneous and cyclophosphamide-accelerated diabetic mice.