Memory-Enhancing Effect of a Phytosome Containing the Combined Extract of Mulberry Fruit and Ginger in an Animal Model of Ischemic Stroke with Metabolic Syndrome

Abstract

The prevalence of dementia following cerebral ischemia in metabolic syndrome (MetS) condition is increasing, and most of the cases are often severe. Unfortunately, no effective strategy for treating this condition is available. Based on the positive modulation effect of a polyphenol-rich substance on dementia and the improvement in bioavailability and stability of polyphenols induced by the phytosome technique together with the use of the synergistic concept, we hypothesized that a phytosome containing the combined extract of mulberry fruit and ginger (PMG) should mitigate dementia and memory impairment following ischemic stroke in MetS. MetS was induced in male Wistar rats weighing 180-200 g by exposure to a 16-week feeding period of high-carbohydrate high-fat (HCHF) diet. MetS rats were orally given PMG at doses of 50, 100, and 200 mg·kg^-1 BW 21 days before and 21 days after the occlusion of the right middle cerebral artery (Rt. MCAO). Then, their spatial memory was determined and the possible underlying mechanisms explored via the alterations of acetylcholinesterase (AChE), neuron density, malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), interleukin-6 (IL-6), and signal transduction via extracellular signal-regulated kinase (ERK) pathway in both the cerebral cortex and the hippocampus. It was found that PMG significantly enhanced memory. It also decreased AChE, IL-6, and MDA but increased SOD, CAT, GSH-Px, neuron density, and phosphorylation of ERK. These data suggested the cognitive enhancing effect of PMG. The possible underlying mechanisms might occur partly via the improvement of cholinergic function via the ERK pathway together with the decrease in neurodegeneration induced by the reduction of oxidative stress and inflammation. However, a subchronic toxicity study is also required to assure the safety of PMG consumption before moving forward to a clinical trial study.

Figure 1

Schematic diagram showing all experimental procedures. (a) Experimental protocol of PMG treatment and the determination of various parameters. (b) Right MCAO induction and schedule for PMG treatment. IL-6: interleukin-6; P-Erk: phosphorylated-extracellular signal-regulated protein kinase; Erk: extracellular signal-regulated protein kinase; PMG50, PMG100, and PMG200: the phytosomes containing the combined extract of mulberry fruit and ginger at doses of 50, 100, and 200 mg·kg⁻¹ BW, respectively.

Figure 2

Effect of PMG on escape latency time under cognitive deficit induced by Rt. MCAO. Data are presented as mean ± SEM (n = 6/group). ^ap value < 0.05, ^aap value < 0.01, and ^aaap value < 0.001; compared between naïve control which received a normal diet and vehicle and MetS+MCAO rats which received HCHF, MCAO, and vehicle. ^bp value < 0.05, ^bbp value < 0.01, and ^bbbp value < 0.001; compared between sham rats which received HCHF, sham operation, and vehicle and MetS+MCAO rats which received HCHF, MCAO, and vehicle. ^∗p value < 0.05 and ^∗∗p value < 0.01; compared to MetS+MCAO rats which received HCHF, MCAO, and vehicle. ND: normal diet; HCHF: high-carbohydrate high-fat diet; MetS: metabolic syndrome; MCAO: middle cerebral artery occlusion; Vitamin C: vitamin C at a dose of 250 mg·kg⁻¹ BW; Donepezil: donepezil at a dose of 3 mg·kg⁻¹ BW; PMG50, PMG100, and PMG200: the phytosomes containing the combined extract of mulberry fruit and ginger at doses of 50, 100, and 200 mg·kg⁻¹ BW, respectively.

Figure 3

Effect of PMG on retention time under cognitive deficit induced by Rt. MCAO. Data are presented as mean ± SEM (n = 6/group). ^aaap value < 0.001; compared between naïve control which received a normal diet and vehicle and MetS+MCAO rats which received HCHF, MCAO, and vehicle. ^bbbp value < 0.001; compared between sham rats which received HCHF, sham operation, and vehicle and MetS+MCAO rats which received HCHF, MCAO, and vehicle. ^∗p value < 0.05 and ^∗∗∗p value < 0.001; compared to MetS+MCAO rats which received HCHF, MCAO, and vehicle. ND: normal diet; HCHF: high-carbohydrate high-fat diet; MetS: metabolic syndrome; MCAO: middle cerebral artery occlusion; Vitamin C: vitamin C at a dose of 250 mg·kg⁻¹ BW; Donepezil: donepezil at a dose of 3 mg·kg⁻¹ BW; PMG50, PMG100, and PMG200: the phytosomes containing the combined extract of mulberry fruit and ginger at doses of 50, 100, and 200 mg·kg⁻¹ BW, respectively.

Figure 4

Effect of PMG on locomotor activities: (a) the number of cross lines and (b) the number of center square entries. Data are presented as mean ± SEM (n = 6/group). ND: normal diet; HCHF: high-carbohydrate high-fat diet; MCAO: middle cerebral artery occlusion; Vitamin C: vitamin C at dose of 250 mg·kg⁻¹ BW; Donepezil: donepezil at a dose of 3 mg·kg⁻¹ BW; PMG50, PMG100, and PMG200: the phytosomes containing the combined extract of mulberry fruit and ginger at doses of 50, 100, and 200 mg·kg⁻¹ BW, respectively.

Figure 5

Effect of PMG on exploratory activities: (a) rearing, (b) licking, and (c) grooming behaviors. Data are presented as mean ± SEM (n = 6/group). ND: normal diet; HCHF: high-carbohydrate high-fat diet; MCAO: middle cerebral artery occlusion; Vitamin C: vitamin C at a dose of 250 mg·kg⁻¹ BW; Donepezil: donepezil at a dose of 3 mg·kg⁻¹ BW; PMG50, PMG100, and PMG200: the phytosomes containing the combined extract of mulberry fruit and ginger at doses of 50, 100, and 200 mg·kg⁻¹ BW, respectively.

Figure 6

Effect of PMG on neuron density in the medial prefrontal cortex. (a) Light microscopy of coronal sections in the medial prefrontal cortex which were stained with cresyl violet at 40x magnification. (b) Density of survival neurons in the medial prefrontal cortex. Data are presented as mean ± SEM (n = 6/group). ^aaap value < 0.001; compared between naïve control which received a normal diet and vehicle and MetS+MCAO rats which received HCHF, MCAO, and vehicle. ^bbbp value < 0.001; compared between sham rats which received HCHF, sham operation, and vehicle and MetS+MCAO rats which received HCHF, MCAO, and vehicle. ^∗p value < 0.05 and ^∗∗p value < 0.01; compared to MetS+MCAO rats which received HCHF, MCAO, and vehicle. ND: normal diet; HCHF: high-carbohydrate high-fat diet; MetS: metabolic syndrome; MCAO: middle cerebral artery occlusion; Vitamin C: vitamin C at a dose of 250 mg·kg⁻¹ BW; Donepezil: donepezil at a dose of 3 mg·kg⁻¹ BW; PMG50, PMG100, and PMG200: the phytosomes containing the combined extract of mulberry fruit and ginger at doses of 50, 100, and 200 mg·kg⁻¹ BW, respectively.

Figure 7

Effect of PMG on neuron density in various subregions of the hippocampus. (a) Light microscopy of coronal sections in CA1, CA2, CA3, and dentate gyrus of the hippocampus which were stained with cresyl violet at 40x magnification. (b) Density of survival neurons in CA1, CA2, CA3, and dentate gyrus of the hippocampus. Data are presented as mean ± SEM (n = 6/group). ^aaap value < 0.001; compared between naïve control which received a normal diet and vehicle and MetS+MCAO rats which received HCHF, MCAO, and vehicle. ^bbbp value < 0.001; compared between sham rats which received HCHF, sham operation, and vehicle and MetS+MCAO rats which received HCHF, MCAO, and vehicle. ^∗p value < 0.05, ^∗∗p value < 0.01, and ^∗∗∗p value < 0.001; compared to MetS+MCAO rats which received HCHF, MCAO, and vehicle. ND: normal diet; HCHF: high-carbohydrate high-fat diet; MetS: metabolic syndrome; MCAO: middle cerebral artery occlusion; Vitamin C: vitamin C at a dose of 250 mg·kg⁻¹ BW; Donepezil: donepezil at a dose of 3 mg·kg⁻¹ BW; PMG50, PMG100, and PMG200: the phytosomes containing the combined extract of mulberry fruit and ginger at doses of 50, 100, and 200 mg·kg⁻¹ BW, respectively.

Figure 8

Effect of PMG on the expression of total p-Erk/Erk in the prefrontal cortex was detected by Western blotting. (a) Representative Western blot showing the levels of total p-Erk/Erk. (b) Relative density of total p-Erk/Erk. Data are presented as mean ± SEM (n = 6/group). ^aaap value < 0.001; compared between naïve control which received a normal diet and vehicle and MetS+MCAO rats which received HCHF, MCAO, and vehicle. ^bbbp value < 0.001; compared between sham rats which received HCHF, sham operation, and vehicle and MetS+MCAO rats which received HCHF, MCAO, and vehicle. ^∗p value < 0.05, ^∗∗p value < 0.01, and ^∗∗∗p value < 0.001; compared to MetS+MCAO rats which received HCHF, MCAO, and vehicle. ND: normal diet; HCHF: high-carbohydrate high-fat diet; MetS: metabolic syndrome; MCAO: middle cerebral artery occlusion; Vitamin C: vitamin C at a dose of 250 mg·kg⁻¹ BW; Donepezil: donepezil at a dose of 3 mg·kg⁻¹ BW; PMG50, PMG100, and PMG200: the phytosomes containing the combined extract of mulberry fruit and ginger at doses of 50, 100, and 200 mg·kg⁻¹ BW, respectively.

Figure 9

Effect of PMG on the expression of total p-Erk/Erk in the hippocampus was detected by Western blotting. (a) Representative Western blot showing the levels of total p-Erk/Erk. (b) Relative density of total p-Erk/Erk. Data are presented as mean ± SEM (n = 6/group). ^aaap value < 0.001; compared between naïve control which received a normal diet and vehicle and MetS+MCAO rats which received HCHF, MCAO, and vehicle. ^bbbp value < 0.001; compared between sham rats which received HCHF, sham operation, and vehicle and MetS+MCAO rats which received HCHF, MCAO, and vehicle. ^∗∗p value < 0.01 and ^∗∗∗p value < 0.001; compared to MetS+MCAO rats which received HCHF, MCAO, and vehicle. ND: normal diet; HCHF: high-carbohydrate high-fat diet; MetS: metabolic syndrome; MCAO: middle cerebral artery occlusion; Vitamin C: vitamin C at a dose of 250 mg·kg⁻¹ BW; Donepezil: donepezil at a dose of 3 mg·kg⁻¹ BW; PMG50, PMG100, and PMG200: the phytosomes containing the combined extract of mulberry fruit and ginger at doses of 50, 100, and 200 mg·kg⁻¹, respectively.

Figure 10

Effect of PMG on the expression of IL-6 in the prefrontal cortex was detected by Western blotting. (a) Representative Western blot showing the levels of total IL-6. (b) Relative density of total IL-6. Data are presented as mean ± SEM (n = 6/group). ^aaap value < 0.001; compared between naïve control which received a normal diet and vehicle and MetS+MCAO rats which received HCHF, MCAO, and vehicle. ^bbbp value < 0.001; compared between sham rats which received HCHF, sham operation, and vehicle and MetS+MCAO rats which received HCHF, MCAO, and vehicle. ^∗∗p value < 0.01; compared to MetS+MCAO rats which received HCHF, MCAO, and vehicle. ND: normal diet; HCHF: high-carbohydrate high-fat diet; MetS: metabolic syndrome; MCAO: middle cerebral artery occlusion; Vitamin C: vitamin C at a dose of 250 mg·kg⁻¹ BW; Donepezil: donepezil at a dose of 3 mg·kg⁻¹ BW; PMG50, PMG100, and PMG200: the phytosomes containing the combined extract of mulberry fruit and ginger at doses of 50, 100, and 200 mg·kg⁻¹ BW, respectively.

Figure 11

Effect of PMG on the expression of IL-6 in the hippocampus was detected by Western blotting. (a) Representative Western blot showing the levels of total IL-6. (b) Relative density of total IL-6. Data are presented as mean ± SEM (n = 6/group). ^aaap value < 0.001; compared between naïve control which received a normal diet and vehicle and MetS+MCAO rats which received HCHF, MCAO, and vehicle. ^bbbp value < 0.001; compared between sham rats which received HCHF, sham operation, and vehicle and MetS+MCAO rats which received HCHF, MCAO, and vehicle. ^∗p value < 0.05; compared to MetS+MCAO rats which received HCHF, MCAO, and vehicle. ND: normal diet; HCHF: high-carbohydrate high-fat diet; MetS: metabolic syndrome; MCAO: middle cerebral artery occlusion; Vitamin C: vitamin C at a dose of 250 mg·kg⁻¹ BW; Donepezil: donepezil at a dose of 3 mg·kg⁻¹ BW; PMG50, PMG100, and PMG200: the phytosomes containing the combined extract of mulberry fruit and ginger at doses of 50, 100, and 200 mg·kg⁻¹ BW, respectively.

Figure 12

Schematic diagram demonstrating the neuroprotective effect of PMG against cognitive impairment following ischemic stroke in the animal model of ischemic stroke in MetS condition. ERK: extracellular signal-regulated kinase; IL-6: interleukin-6; MDA: malondialdehyde; SOD: superoxide dismutase; CAT: catalase; GSH-Px: glutathione peroxidase; MetS: metabolic syndrome.