Effects of ZSS hot water extract on APP23 mice.

Hot water extract of ZSS (Ext- ZSS) was administered to 13-15-month-old and 15-16-month-old APP23 mice at 0.1 and 0.5 mg/shot, respectively, for 1 month. (A) Ext-ZSS at 0.1 mg/shot improved mouse memory, but its effect was not complete. (B) Ext-ZSS at 0.5 mg/shot significantly improved mouse memory to a level similar to that of non-Tg littermates. (C) Aβ pathologies were assessed in mice that received the lower dose. Ext-ZSS at 0.1 mg/shot significantly reduced the levels of Aβ oligomers in the cerebral cortex (CC) and hippocampus (HC). (D) Amyloid deposits in these regions were also significantly decreased with Ext-ZSS. (E) Ext-ZSS significantly restored synaptophysin levels in the mossy fibers of hippocampal CA2/3 regions to a level similar to that of non-Tg littermates. AU, arbitrary unit. Each point and bar represent the mean ± SEM. The numbers of total, male, and female mice analyzed are shown in each figure as n = x: m (male) + f (female).

Effects of ZSS hot water extract on Tau784 mice.

Hot water extract of ZSS (Ext- ZSS) was administered to 14-month-old Tau784 mice at 0.1 and 0.5 mg/shot for 1 month. (A) Ext-ZSS improved mouse memory in a dose-dependent manner; the higher dose achieved a complete recovery to a level similar to that of non-Tg littermates. (B) Ext-ZSS significantly reduced the levels of phosphorylated tau in the hippocampus and tau oligomers in the cerebral cortex in a dose-dependent manner. (C) The levels of synaptophysin in the hippocampal CA2/3 regions were significantly recovered in a dose-dependent manner. Each point and bar represent the mean ± SEM. The numbers of total, male, and female mice analyzed are shown in each figure as n = x: m (male) + f (female).

Comparison of ZSS hot water extract, extraction residue, and non-extracted simple crush powder in Tau784 mice.

Hot water extract (Ext-ZSS), non-extracted simple crush powder (Pwd-ZSS), and extraction residue (Res-ZSS) of ZSS were administered to 8-12- month-old and 8-11-month-old Tau784 mice at 0.1 mg/shot for 1 month. (A) Pwd-ZSS markedly enhanced mouse memory to a level even higher than that of non-Tg littermates, whereas Ext- ZSS showed only a moderate effect. (B) Res-ZSS also showed a moderate effect, similar to that of Ext-ZSS. (C, D) The levels of phosphorylated tau in the hippocampus and tau oligomers in the cerebral cortex were significantly attenuated by all three preparations, with Pwd-ZSS showing the strongest effects. (E) Pwd-ZSS significantly increased the levels of synaptophysin in the hippocampal CA2/3 region to a level similar to or higher than those in non-Tg littermates. Ext-ZSS and Res-ZSS showed only slight effects. (F) Pwd-ZSS significantly enhanced BDNF expression in the hippocampus to a level even higher than that in non-Tg littermates. Ext-ZSS and Res-ZSS showed only slight effects. Each point and bar represent the mean ± SEM. The numbers of total, male, and female mice analyzed are shown in each figure as n = x: m (male) + f (female).

Effects of ZSS simple crush powder on Huα-Syn(A53T) mice.

Non-extracted simple crush powder of ZSS (Pwd-ZSS) was administered to 8-month-old Huα-Syn(A53T) mice at 0.1 mg/shot for 1 month. (A) Pwd-ZSS significantly improved motor function to a level similar to that of non-Tg littermates. (B) Pwd-ZSS significantly reduced the levels of phosphorylated α- synuclein and α-synuclein oligomers in the hippocampus. (C) Pwd-ZSS significantly enhanced BDNF expression in the cerebral cortex (CC) and substantia nigra (SN) to a level even higher than that in non-Tg littermates. (D) Neurogenesis was evaluated by immunofluorescence for BrdU (red) and doublecortin (DCX, green), in which double-positive cells (yellow) were regarded as newly generated neurons. Pwd-ZSS significantly enhanced neurogenesis in the dentate gyrus (DG) and substantia nigra (SN) to a level higher than that in non-Tg littermates. (E) Pwd- ZSS was administered to 6-7-month-old Huα-Syn(A53T) mice at 0.1 mg/shot for 1 month. Mouse memory was significantly improved to a level similar to or slightly less than that of non- Tg littermates. Each point and bar represent the mean ± SEM. The numbers of total, male, and female mice analyzed are shown in each figure as n = x: m (male) + f (female).

Effects of ZSS simple crush powder on normal aged mice.

Non-extracted simple crush powder of ZSS (Pwd-ZSS) was administered to 16-18-month-old (aged) and 8-month-old (young) wild-type mice at 0.1 mg/shot for 1 month. (A) Pwd-ZSS significantly improved the memory of aged mice to that of water-treated young mice. A memory-enhancing effect was also observed in young mice, but it was not significant. Pwd-ZSS significantly increased the levels of synaptophysin in the hippocampal CA2/3 regions (B), BDNF expression in the hippocampus (C), and neurogenesis in the dentate gyrus (D) of aged mice to levels similar to those in water- treated young mice. In (D), blue shows nuclei stained with DAPI. (E) The levels of the cellular senescence markers p16INK4a, p21CIPI/WAF1, and γH2AX in the cerebral cortex of aged mice were significantly decreased by Pwd-ZSS treatment to levels similar to those in water-treated young mice. Each point and bar represent the mean ± SEM. The numbers of total, male, and female mice analyzed are shown in each figure as n = x: m (male) + f (female).

Antioxidant activities of ZSS simple crush powder.

(A) Antioxidant effect of ZSS powder was investigated in the brains of mice used in Figure 5. The levels of the DNA oxidation product 8-OHdG and its autoantibodies were both increased in aged mice, but their levels were significantly reduced by ZSS powder to levels similar to or lower than those in young mice. Each bar represents the mean ± SEM. The numbers of mice analyzed are shown in each figure as n = x. AU, arbitrary unit. (B) The radical-scavenging ability of ZSS powder was measured in a cell- free system, in which the SOD-like activity is expressed as % inhibition against superoxide produced by xanthine oxidase. Compared to Mamaki leaf powder, which is known to have strong antioxidant activity, ZSS powder showed only a weak effect.

Effects of three components of ZSS on Tau784 mice.

A mixture of jujuboside A, jujuboside B, and spinosin was administered to 13-16-month-old Tau784 mice for 1 month. The daily doses were 0.455 μg, 0.2 μg, and 0.7 μg, respectively, which correspond to those in 0.5 mg ZSS hot water extract. This treatment displayed a much weaker effect on mouse memory than that of 0.5 mg ZSS hot water extract (Figure 2A). Each point represents the mean ± SEM. The numbers of total, male, and female mice analyzed are shown in each figure as n = x: m (male) + f (female).

The three major components in 100 g of each ZSS preparation

Effects of ZSS simple crush powder on neurodegenerative diseases and brain aging.

Oral administration of ZSS powder ameliorates pathological phenotypes in mouse models of neurodegenerative diseases, and furthermore, rejuvenates the brain condition of aged mice to a level comparable to that of young mice.