Long term administration of loquat leaves and their major component, ursolic acid, attenuated endogenous amyloid-β burden and memory impairment

Abstract

Loquat (Eriobotrya japonica) leaves contain many bioactive components such as ursolic acid (UA) and amygdalin. We investigated the effects of loquat leaf powder and methanol extract in human neuroglioma H4 cells stably expressing the Swedish-type APP695 (APP_NL-H4 cells) and C57BL/6 J mice. Surprisingly, the extract greatly enhanced cellular amyloid-beta peptide (Aβ) 42 productions in APP_NL-H4 cells. Administration of leaf powder increased Aβ42 levels after 3 months and decreased levels after 12 months compared to control mice. Leaf powder had no effect on working memory after 3 months, but improved working memory after 12 months. Administration of UA decreased Aβ42 and P-tau levels and improved working memory after 12 months, similar to the administration of leave powder for 12 months. Amygdalin enhanced cellular Aβ42 production in APP_NL-H4 cells, which was the same as the extract. Three-month administration of amygdalin increased Aβ42 levels slightly but did not significantly increase them, which is similar to the trend observed with the administration of leaf powder for 3 months. UA was likely the main compound contained in loquat leaves responsible for the decrease in intracerebral Aβ42 and P-tau levels. Also, amygdalin might be one of the compounds responsible for the transiently increased intracerebral Aβ42 levels.

Figure 1

Effects of loquat leaves on Aβ42 level and mice behavior. Aβ42 production in APP_NL-H4 cells treated with loquat leaf methanol extract for 48 h (A). Mice fed a powdered diet containing 5% loquat leaves for 1, 3, 6, and 12 months. Aβ42 levels in the mice cortex (B). Data are presented as the mean ± SEM, n = 6 per group. Working memory (Alternation) (C) in Y-maze test. Motor performance on the rotarod. Mice were assessed for locomotion time (D) and number of falls (E) during a period of 600 s. Data are presented as the mean ± SEM, n = 10 per group. Statistical analysis was performed using a student’s t-test (*p < 0.05; **p < 0.01; ***p < 0.001 vs. Control).

Figure 2

Effects of loquat leaves on APP cleavage, tau phosphorylation, and CREB-BDNF pathway. Mice fed a powdered diet containing 5% loquat leaves for 12 months. The protein level was determined by western blot analysis (A). Protein levels of sAPPα (B), sAPPβ (C), Total tau (D), P-tau (E), P-CREB (F), and BDNF (G) in the hippocampus. Data are presented as the mean ± SEM, n = 5 per group. Statistical analysis was performed using a student’s t-test (*p < 0.05; **p < 0.01 vs. Control).

Figure 3

Aβ42 levels and mice behavior following administration of UA for 3 and 12 months. Mice fed with powdered diet containing 0.3125% UA for 3 and 12 months. Aβ42 production in the mice cortex (A). Data are presented as the mean ± SEM, n = 6 per group. Working memory (Alternation) (B) in Y-maze test. Motor performance on the rotarod. Mice were assessed for locomotion time (C) and number of falls (D) during a period of 600 s. Data are presented as the mean ± SEM, n = 10 per group. Statistical analysis was performed using a student’s t-test (**p < 0.01; ***p < 0.001 vs. Control). Aβ42 production in the APP_NL-H4 cells treated with UA for 48 h (E). Data are presented as the mean ± SEM, n = 5 per group. Statistical analysis was performed using one-way ANOVA followed by post-hoc Newman-Keuls test.

Figure 4

Effects of UA on APP cleavage, tau phosphorylation, and CREB-BDNF pathway. Mice fed a powdered diet containing 0.3125% loquat leaves for 12 months. The protein level determined by western blot analysis (A). Protein levels of sAPPα (B), sAPPβ (C), Total tau (D), P-tau (E), P-CREB (F), and BDNF (G) in the hippocampus. Data are presented as the mean ± SEM, n = 5 per group. Statistical analysis was performed using a student’s t-test (*p < 0.05 vs. Control).

Figure 5

Effects of amygdalin on Aβ42 level and mice behavior. Aβ42 production in APP_NL-H4 cells treated with amygdalin for 48 h (A). Data are presented as the mean ± SEM, n = 5 per group. Statistical analysis was performed using one-way ANOVA followed by post-hoc Newman-Keuls test (*p < 0.001 vs. Control). Mice fed a powdered diet containing 0.0695% amygdalin for 3 months. Aβ42 levels in the mice cortex (B). Working memory (Alternation) (C) in Y-maze test. Data are presented as the mean ± SEM, n = 5 per group. Statistical analysis was performed using a student’s t-test.