Mulberry (Morus alba) Twig and Leaf Extracts Ameliorate Obesity-Related Metabolic Disorders via Gut Microbiota Modulation in High-Fat Diet-Fed Mice

Abstract

Mulberry (Morus alba) twigs and leaves, rich in flavonoids, polyphenols, polysaccharides, and alkaloids with multi-target regulatory properties on glucose/lipid metabolism, were evaluated for their anti-obesity effects using methanol-extracted twigs (MTE) and aqueous-extracted leaves (MLE) in high-fat diet (HFD)-induced obese mice. Both extracts significantly ameliorated obesity-related metabolic dysregulation, as evidenced by attenuated body weight gain, visceral fat accumulation, serum lipid profiles, homeostatic model assessment of insulin resistance (HOMA-IR), and hepatic inflammation compared to HFD controls (p < 0.05). Concurrently, MTE and MLE enhanced systemic antioxidant capacity and elevated high-density lipoprotein cholesterol (HDL-C) levels. Notably, high-dose MTE (MTEH, 1000 mg/kg) markedly reduced perirenal adiposity while increasing brown adipose tissue mass (p < 0.05). Mechanistic investigations revealed that MTEH reshaped gut microbiota composition by suppressing Firmicutes and Enterococcus, while enriching beneficial Faecalibaculum and Bifidobacterium spp. (p < 0.05). Furthermore, cecal short-chain fatty acid (SCFA) profiling demonstrated MTEH and MLEH-mediated metabolic reprogramming, characterized by increased propionic acid and decreased butyric acid, suggesting microbiota-dependent modulation of host energy metabolism. These findings collectively highlight the potential of mulberry extracts as multi-targeted nutraceuticals for obesity intervention via gut microbiota-SCFA axis regulation.

Keywords: glycolipid metabolism; gut microbiology; mulberry extract; obesity.

Figure 1

Design of the experiment. Mulberry twig extract high dose, 1000 mg/kg group (MTEH); mulberry twig extract low dose, 500 mg/kg group (MTEL); mulberry leaf extract high dose, 1000 mg/kg group (MLEH); mulberry leaf extract high dose, 500 mg/kg group (MLEL); orlistat 200 mg/kg group (OC); blank control group (CON, normal diet + equal amount of distilled water); and high-fat diet feeding group (HFD, high-fat diet + equal amount of distilled water).

Figure 2

Effects of MTE and MLE interventions on fat accumulation in obese mice (n = 8/group). (A) Final body weight; (B) Feed intake; (C) Weights of inguinal fat; (D) Perirenal fat; (E) Epididymal fat; (F) Brown fat. Data were expressed as mean ± SD and analyzed by ordinary one-way ANOVA compared to the HFD group (model group). Different letters represent different significances (n = 8). Different letters above bars denote significant differences (p < 0.05, one-way ANOVA with Tukey’s post hoc test).

Figure 3

Effects of MTE and MLE on blood glucose and lipid levels in obese mice (n = 6/group). Serum (A) Cholesterol; (B) Triglycerides; (C) HDL Cholesterol; (D) LDL Cholesterol; (E) Insulin Resistance index; (F) Fasting blood glucose level. Data were expressed as mean ± SD and analyzed by ordinary one-way ANOVA compared to the HFD group (model group). Different letters represent different significances (n = 8). Different letters above bars denote significant differences (p < 0.05, one-way ANOVA with Tukey’s post hoc test).

Figure 4

Effect of MTE and MLE on serum hormone levels in obese mice (n = 6/group). Serum levels of (A) Leptin; (B) Insulin; (C) Adiponectin. Data were expressed as mean ± SD and analyzed by ordinary one-way ANOVA compared to the HFD group (model group). Different letters represent different significances (n = 8). Different letters above bars denote significant differences (p < 0.05, one-way ANOVA with Tukey’s post hoc test).

Figure 5

Effects of MTE and MLE on liver function and metabolic indexes of obese mice (n = 6/group). (A) Serum alanine aminotransferase; (B) Serum glutamate aminotransferase; (C) Hepatic cholesterol; (D) Hepatic weight; (E) Hepatic organ index; (F) Hepatic triglyceride levels. Data were expressed as mean ± SD and analyzed by ordinary one-way ANOVA compared to the HFD group (model group). Different letters represent different significances (n = 8). Different letters above bars denote significant differences (p < 0.05, one-way ANOVA with Tukey’s post hoc test).

Figure 6

Effects of MTE and MLE on liver inflammatory factors and oxidative stress indices in obese mice (n = 6/group). (A) Interleukin-8 (IL-8); (B) Interleukin-6 (IL-6); (C) Interleukin-17 (IL-17); (D) Tumor Necrosis Factor-α (TNF-α); (E) Malondialdehyde (MDA); (F) Total Superoxide Dismutase (T-SOD); (G) Glutathione Peroxidase (GSH-Px); (H) Total antioxidant capacity (T-AOC). Data were expressed as mean ± SD and analyzed by ordinary one-way ANOVA compared to the HFD group (model group). Different letters represent different significances (n = 8). Different letters above bars denote significant differences (p < 0.05, one-way ANOVA with Tukey’s post hoc test).

Figure 7

Effect of MTE and MLE interventions on liver lipid deposition profile. Representative images of liver morphology, H&E staining of liver tissue in CON, MTEL, MTEH, MLEH, MLEL, and HFD groups (original magnification ×20, original magnification ×40).

Figure 8

Effect of MTE and MLE interventions on liver lipid deposition profiles (n = 6/group). Data are presented as the mean ± SD (n = 6). Different letters above bars denote significant differences (p < 0.05, one-way ANOVA with Tukey’s post hoc test).

Figure 9

Effect of MTE and MLE on the diversity of intestinal flora in obese mice (n = 6/group). (A) Venn diagram; (B) PCoA analysis; (C) Alpha-diversity analysis (Chao1, Shannon and Simpson indices). ***, p < 0.001 vs. control group (one-way ANOVA with Tukey’s multiple comparisons test).

Figure 10

Effect of MTE and MLE on the composition of intestinal flora at the portal level in obese mice (n = 6/group). (A) Relative abundance of TOP 10 bacteria at the gate level for each sample and treatment group; (B) relative abundance and Firmicutes/Bacteroidota (F/B) ratio of bacteria differing at the gate level; data are presented as mean ± SD deviation, and different letters indicate statistical differences between the two data sets (p < 0.05).

Figure 11

Effect of MTE and MLE on gut flora composition at the genus level in obese mice (n = 6/group). (A) Relative abundance of genus-level TOP 10 bacteria; (B) linear discriminant effect size analysis (LefSe) to identify representative bacterial taxa at different taxonomic levels for each treatment group; (C) relative abundance of genus-level differential bacteria; data are presented as mean ± SD deviation, and different letters indicate statistical differences between the two data sets (p < 0.05).

Figure 12

Correlation analysis between genus-level gut microbes and short-chain fatty acids. Each grid represents the Spearman correlation coefficient between rows and columns. A red color indicates positive correlation and a blue color indicates negative correlation. Significance: * 0.01 < p < 0.05; ** p < 0.01.

Figure 13

Mechanisms of MTE and MLE in Attenuating Lipid Deposition in Obese Mice. MTE, mulberry twig extract; MLE, mulberry leaf extract;red arrows indicate an increase, blue arrows indicate a decrease.