Screening assay for the identification of candidate compounds used in Kampo medicine. (A) Screening of candidate compounds to determine which have chemical chaperone activity. The relative intensity of α-LA aggregation was analyzed by measuring optimal density at 488 nm to detect turbidity after incubation at 37°C for 7 hours. Mean ± SD, n = 3. (B) Screening of candidate compounds that could inhibit amyloid β aggregation. The relative intensity of amyloid β aggregation using ThT fluorescence intensity measured at an excitation wavelength of 450 nm and emission wavelength of 480 nm after incubating at 37°C for 6 hours. Mean ± SD, n = 3. Candidate compounds were as follows: #5: alkannin, #15: barbaloin, #29: coptisine chloride, #65: (Z)-ligustilide, #69: luteolin, #91: shikonin. A list of all the compounds tested is shown in Supplementary Fig. 1.

Alkannin inhibits the aggregation of amyloid β. (A) Chemical structure of alkannin. (B) A time-course analysis of the effect of alkannin on ThT fluorescence intensity was measured at 0, 6, 12, 24, and 48 hours after incubating Aβ_1-42 (10 µM) and alkannin (10 µM). Aβ_1-42 alone dramatically increased ThT fluorescence intensity whereas Aβ_1-42 + alkannin did not. Alkannin alone did not affect ThT fluorescence intensity. Mean ± SD, n = 3. (C) Alkannin inhibited preformed amyloid β aggregates. Amyloid β was incubated for 6 hours to form amyloid β aggregates in the absence of alkannin, after which the amyloid β aggregates were incubated with alkannin for the additional times and ThT fluorescence was measured. *P < 0.05, ***P < 0.001 versus cont (DMSO). Tukey-Kramer. Mean ± SD, n = 5. (D) Alkannin inhibited preformed amyloid β aggregates. Aβ was incubated for 24 hours to form amyloid β aggregates in the absence of alkannin, after which the amyloid β aggregates were incubated with alkannin for the additional times. ThT fluorescence was measured after incubation. *P < 0.05, **P < 0.01 versus control (DMSO). Tukey-Kramer. Mean ± SD, n = 5. (E) A time-course analysis of the effect of alkannin and shikonin on ThT fluorescence. The intensity was measured at 0, 6, 12, and 24 hours after incubating Aβ_1-42 (10 µM) and alkannin (10 µM) or shikonin (10 µM). Sikonin similarly attenuated ThT fluorescence intensity compared with alkannin. Mean ± SD, n = 4.

Alkannin attenuates the β-sheet structure and fibril formation of amyloid β. Aβ_1-42 (10 μM) was incubated with or without alkannin (10 μM) for 24 hours at 37°C and CD spectra were analyzed. CD spectra of amyloid β and alkannin at (A) 0 hour and (B) 24 hours after incubation. Alkannin was found to attenuate the typical CD spectra signal of β-sheet structure formation of amyloid β. Mean ± SD, n = 4. (C) Alkannin attenuated amyloid β fibril formation. Scanning electron micrograph analysis of Aβ_1-42 (10 μM) alone or Aβ_1-42 + alkannin (10 μM) after incubation at 37°C for 48 hours.

Alkannin attenuated formation of toxic conformer of amyloid β. (A) Amyloid β_1-42 was incubated for 8 hours with or without alkannin and analyzed toxic conformer of amyloid β using specific antibody 11A1. We observed inhibition of formation of the toxic conformer of amyloid by alkannin. On the other hand, similar signal was detected when we used anti-Aβ1-17 antibody (6E10) antibody. (B) Densitometric analysis of the amyloid β dot blot analysis. Mean ± SD, n = 5. Tukey-Kramer *p < 0.05 versus control 8 hours.

Alkannin protects amyloid β-induced neuronal cell death. PC12 cells were treated with amyloid β (5 µM) and alkannin (0.1, 0.3, and 1 µM) or shikonin (0.1, 0.3, and 1 µM). The percentage of cell death was measured by LDH assay after 48 hours of incubation. Alkannin was found to attenuate amyloid β-induced neuronal cell death. Mean ± SD, n = 5. Dunnett’s test compared with amyloid β-treatment alone was done. Aβ versus Aβ + alkannin (P=0.14). Aβ versus Aβ + shikonin (P = 0.11).

Alkannin ameliorates AD and amyloid β aggregation in C. elegans. (A) Alkannin ameliorates chemotaxis index of AD in C. elegans. Treatment of CL2355 (AD model of C. elegans) with alkannin increased CI. CL2122 (normal control of C. elegans) was incubated at 25°C. CL2355 was incubated at 16°C initially; however, 36 hours after hatching, the temperature was increased to 25°C. Concurrently, CL2355 was treated with alkannin (0.3, 1, 3 µM). At 72 hours after hatching, all the worms were treated with 5-fluoro-2’-deoxyuridine (0.6 mM) for sterilization. The chemotaxis assay was performed 144 hours after hatching. CL2355 showed a reduced CI, which indicated AD, while treatment with alkannin showed a concentration-dependent increase in CI, which reached significance at 3 µM. Mean ± SD, n = 6. *P < 0.05. Dunnett’s test compared with AD model worms was performed.