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. 2016 Apr 7;11(4):e0152728.
doi: 10.1371/journal.pone.0152728. eCollection 2016.

Prevention Effects and Possible Molecular Mechanism of Mulberry Leaf Extract and Its Formulation on Rats With Insulin-Insensitivity

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Free PMC article

Prevention Effects and Possible Molecular Mechanism of Mulberry Leaf Extract and Its Formulation on Rats With Insulin-Insensitivity

Yan Liu et al. PLoS One. .
Free PMC article

Abstract

For centuries, mulberry leaf has been used in traditional Chinese medicine for the treatment of diabetes. This study aims to test the prevention effects of a proprietary mulberry leaf extract (MLE) and a formula consisting of MLE, fenugreek seed extract, and cinnamon cassia extract (MLEF) on insulin resistance development in animals. MLE was refined to contain 5% 1-deoxynojirimycin by weight. MLEF was formulated by mixing MLE with cinnamon cassia extract and fenugreek seed extract at a 6:5:3 ratio (by weight). First, the acute toxicity effects of MLE on ICR mice were examined at 5 g/kg BW dose. Second, two groups of normal rats were administrated with water or 150 mg/kg BW MLE per day for 29 days to evaluate MLE's effect on normal animals. Third, to examine the effects of MLE and MLEF on model animals, sixty SD rats were divided into five groups, namely, (1) normal, (2) model, (3) high-dose MLE (75 mg/kg BW) treatment; (4) low-dose MLE (15 mg/kg BW) treatment; and (5) MLEF (35 mg/kg BW) treatment. On the second week, rats in groups (2)-(5) were switched to high-energy diet for three weeks. Afterward, the rats were injected (ip) with a single dose of 105 mg/kg BW alloxan. After four more days, fasting blood glucose, post-prandial blood glucose, serum insulin, cholesterol, and triglyceride levels were measured. Last, liver lysates from animals were screened with 650 antibodies for changes in the expression or phosphorylation levels of signaling proteins. The results were further validated by Western blot analysis. We found that the maximum tolerance dose of MLE was greater than 5 g/kg in mice. The MLE at a 150 mg/kg BW dose showed no effect on fast blood glucose levels in normal rats. The MLE at a 75 mg/kg BW dose and MLEF at a 35 mg/kg BW dose, significantly (p < 0.05) reduced fast blood glucose levels in rats with impaired glucose and lipid metabolism. In total, 34 proteins with significant changes in expression and phosphorylation levels were identified. The changes of JNK, IRS1, and PDK1 were confirmed by western blot analysis. In conclusion, this study demonstrated the potential protective effects of MLE and MLEF against hyperglycemia induced by high-energy diet and toxic chemicals in rats for the first time. The most likely mechanism is the promotion of IRS1 phosphorylation, which leads to insulin sensitivity restoration.

Conflict of interest statement

Competing Interests: The authors YL and MJL are paid employees of Amway, China. The author YGB is a paid employee of Beijing Pepnoch Co., Ltd. The author Zhong Zhong is a paid employee of Botanic Century (Beijing) Co., Ltd. These commercial affiliations do not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1
Fig 1. Clustering analyses of proteins that are significantly differentially expressed.
N: normal; M: model; MLEF: model treated with MLEF; MLE: model treated with MLE. Black color indicates that the expression level is similar to that in the normal samples. Green color indicates that the expression level is lower compared with that in the normal sample. Red color indicates that the expression level is higher compared with that in the normal sample. (A) Proteins that are involved in the insulin-signaling pathway; (B) Proteins that are not involved in the known insulin-signaling pathway.
Fig 2
Fig 2. Comparison of the expression levels for proteins that are significantly differentially expressed after MLE and MLEF treatment.
Fig 3
Fig 3. Validation of the expression levels of JNK, IRS1, and PDK1 proteins by Western blot analysis.
(A) Relative expression levels of the three proteins in samples from model (M), MLE treatment (MLE), MLEF treatment (MLEF), and normal (N) animals; (B) Western blot analysis of the expression levels of JNK, IRS1 PDK1, and GAPDH in the same set of samples. GAPDH is used as internal control.

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Grant support

The authors YL and MJL are paid employees of Amway, China. The author YGB is a paid employee of Beijing Pepnoch Co., Ltd. The author Zhong Zhong is a paid employee of Botanic Century (Beijing) Co., Ltd. The funder provided support in the form of salaries for authors (YL, YGB, ZZ and MJL), but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.
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