Oral administration of hydrolyzed red ginseng extract improves learning and memory capability of scopolamine-treated C57BL/6J mice via upregulation of Nrf2-mediated antioxidant mechanism

Abstract

Background: Korean ginseng (Panax ginseng Meyer) contains a variety of ginsenosides that can be metabolized to a biologically active substance, compound K. Previous research showed that compound K could be enriched in the red ginseng extract (RGE) after hydrolysis by pectinase. The current study investigated whether the enzymatically hydrolyzed red ginseng extract (HRGE) containing a notable level of compound K has cognitive improving and neuroprotective effects.

Methods: A scopolamine-induced hypomnesic mouse model was subjected to behavioral tasks, such as the Y-maze, passive avoidance, and the Morris water maze tests. After sacrificing the mice, the brains were collected, histologically examined (hematoxylin and eosin staining), and the expressions of antioxidant proteins analyzed by western blot.

Results: Behavioral assessment indicated that the oral administration of HRGE at a dosage of 300 mg/kg body weight reversed scopolamine-induced learning and memory deficits. Histological examination demonstrated that the hippocampal damage observed in scopolamine-treated mouse brains was reduced by HRGE administration. In addition, HRGE administration increased the expression of nuclear-factor-E2-related factor 2 and its downstream antioxidant enzymes NAD(P)H:quinone oxidoreductase and heme oxygenase-1 in hippocampal tissue homogenates. An in vitro assay using HT22 mouse hippocampal neuronal cells demonstrated that HRGE treatment attenuated glutamate-induced cytotoxicity by decreasing the intracellular levels of reactive oxygen species.

Conclusion: These findings suggest that HRGE administration can effectively alleviate hippocampus-mediated cognitive impairment, possibly through cytoprotective mechanisms, preventing oxidative-stress-induced neuronal cell death via the upregulation of phase 2 antioxidant molecules.

Keywords: ABTS, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); BW, body weight; CCK-8, cell counting kit-8; Cognition; DCF, dichlorofluorescein; DCFH, 2,7-dichlorodihydrofluorescein; DPPH, 2,2-diphenyl-1-picrylhydrazyl; H&E, hematoxylin and eosin; HO-1, heme oxygenase-1; HRGE, hydrolyzed red ginseng extract; KO, knockout; Korean Red Ginseng; Learning and memory; NQO1, NAD(P):quinone oxidoreductase 1; Neuroprotection; Nrf2, nuclear-factor-E2-related factor 2; PPD, protopanaxadiol; Pectinase-mediated hydrolysis; RGE, red ginseng extract; ROS, reactive oxygen species; WT, wild-type.

Fig. 1

Oral administration of HRGE improved working memory deficits in scopolamine-treated C57BL/6J mice as assessed by the Y-maze test. (A, B) Nrf2-WT or Nrf2-KO mice were orally administered HRGE or RGE at a designated dosage daily and intraperitoneally injected with scopolamine at 1 mg/kg BW before the test. Spontaneous alterations were evaluated in Nrf2-WT (A) or Nrf2-KO mice (B). Values are expressed as mean ± SD (n = 8). Bars not sharing a common letter are significantly different from each other (p < 0.05).

Fig. 2

Oral administration of HRGE improved associative learning and memory deficits in scopolamine-treated mice as assessed by the passive avoidance test. (A, B) The latency time for either Nrf2-WT (A) or Nrf2-KO (B) mice staying in the bright chamber, where no electric shock was given, was measured. Values are expressed as mean ± SD (n = 8). Bars not sharing a common letter are significantly different from each other (p < 0.05). There was no significant difference between the values for training trials.

Fig. 3

Oral administration of HRGE improved spatial learning and memory deficits in scopolamine-treated mice as assessed by the Morris water maze test. The arrival time for each mouse to the platform (A, B) and latency time in the platform quadrant (C, D) were monitored. (A and C) Nrf2-WT mice. (B and D) Nrf2-KO mice. Values are expressed as mean ± SD (n = 8). Bars not sharing a common letter are significantly different from each other (p < 0.05).

Fig. 4

Oral administration of HRGE ameliorated scopolamine-induced hippocampal damage. The whole-brain tissues were obtained after the mice were sacrificed following completion of the behavioral tests, then sectioned to 5-μm thickness, and stained with H&E for microscopic observation. Representative images are shown at 100 × magnification; the CA1 pyramidal cell layer was further magnified.

Fig. 5

Oral administration of HRGE upregulated Nrf2 and its downstream antioxidant enzymes in the hippocampal homogenates. The hippocampal tissues were collected from the decapitated Nrf2-WT mice. The protein expression levels of nuclear Nrf2 and cytoplasmic HO-1 (A, B) and the activity of NQO1 were examined (C). Values are expressed as mean ± SD (n = 6). Bars not sharing a common letter are significantly different from each other (p < 0.05).

Fig. 5

Oral administration of HRGE upregulated Nrf2 and its downstream antioxidant enzymes in the hippocampal homogenates. The hippocampal tissues were collected from the decapitated Nrf2-WT mice. The protein expression levels of nuclear Nrf2 and cytoplasmic HO-1 (A, B) and the activity of NQO1 were examined (C). Values are expressed as mean ± SD (n = 6). Bars not sharing a common letter are significantly different from each other (p < 0.05).

Fig. 6

Treatment with RGE or HRGE protected against glutamate-induced HT22 hippocampal neuronal cell death. HT22 cells were treated with 5 mM glutamate to generate cytotoxic conditions or intracellular ROS production. (A) Glutamate-induced cytotoxicity was reduced by RGE or HRGE. (B‒D) Intracellular ROS levels observed by fluorescence microscopy (B) and determined by the DCF assay (C and D) were increased in glutamate-treated cells, but decreased by concomitant treatment with RGE or HRGE. Values are expressed as mean ± SEM (n = 3). Bars not sharing a common letter are significantly different from each other (p < 0.05).

Fig. 6

Treatment with RGE or HRGE protected against glutamate-induced HT22 hippocampal neuronal cell death. HT22 cells were treated with 5 mM glutamate to generate cytotoxic conditions or intracellular ROS production. (A) Glutamate-induced cytotoxicity was reduced by RGE or HRGE. (B‒D) Intracellular ROS levels observed by fluorescence microscopy (B) and determined by the DCF assay (C and D) were increased in glutamate-treated cells, but decreased by concomitant treatment with RGE or HRGE. Values are expressed as mean ± SEM (n = 3). Bars not sharing a common letter are significantly different from each other (p < 0.05).