Samples and reagents
Sugi wood (Cryptomeria japonica) diterpene (Fig. 1): ferruginol (I), 6,7-dehydroferruginol (II), sandaracopimarinal (III), sandaracopimarinol (IV), abietadiene (V), abietatriene (VI) and phyllocladenes (VII-i and -ii; mixture of phyllocladene and isophyllocladene) were isolated and identified according to a previous report [7]. The chemical structure and purity of each compound were confirmed by Gas chromatography (GC) and 1H/13C Nuclear magnetic resonance (NMR). The isolated diterpenoids are more than 95% pure. Ginkgolide A was purchased from Funakoshi Co.; rosmarinic acid was purchased from SIGMA-ALDRICH; carnosic acid and 1,1-Diphenyl-2-picrylhydrazyl (DPPH) were purchased from Tokyo Chemical Industry Co., Ltd. and morin and 2,6-Di-tert-butyl-4-methylphenol (BHT) were purchased from Fujifilm Wako Pure Chemical Corporation. These purchased compounds and reagents are also more than 95% pure.
Caenorhabditis elegans and management
C.elegans CL4176 strains were provided by Caenorhabditis Genetics Center (University of Minnesota, Minneapolis, MN) (Fig. 2). Aβ synthetic gene with the temperature dependency is incorporated in CL4176 strain, toxic Aβ1-42 is expressed in a muscle tissue-specific manner by raising breeding temperature from 16 to 25 °C and it becomes the paralysis [8]. The C.elegans were bred and preserved on a Nematode Growth Medium (NGM) storage plate at 16 °C with Escherichia coli OP50 as feed, and moved to new storage plates once in 4 days. E. coli OP50 that was inoculated into 8 mL of Luria–Bertani (LB) media and cultured at 25 °C overnight was used as bacterial cell suspension according to the usual manner. For the storage plate for C. elegans, 10 μL of E. coli OP50 suspension was dropped onto an NGM plate, spread with a spreader and cultured at 25 °C for one night before use.
Aβ toxicity reduction test (C. elegans paralysis test)
Preparation of a test plate, 10 μL of E. coli OP50 suspension and 10 μL of sample solution (0.5 mg/mL dimethyl sulfoxide solution, as molarity of I:1.7 μM, II:1.8 μM, III:1.8 μM, IV:1.7 μM, V:1.8 μM, VI:1.8 μM, VII:1.8 μM, ginkgolide A:1.2 μM, rosmarinic acid:1.4 μM, carnosic acid:1.5 μM, morin:1.7 μM) were dropped onto an NGM plate, and spread with a spreader. Only DMSO was used for a control plate instead of the sample solution. Twenty five L3 larvae of synchronized C. elegans CL4176 that was grown at 16 °C were taken to each of two plates and moved to an incubator of 25 °C to have Aβ genes expressed. Observation was started 20 h after raising temperature to 25 °C and the number of paralyzed individuals was counted every two hours. Aβ toxicity reduction effects of each sample were assessed by statistically analyzing data obtained from the test plate and control plate using the Kaplan–Meier method (log-rank test).
Radical scavenging activity test
DPPH was dissolved in ethanol at the concentration of 0.2 mM. Each diterpenoid sample was dissolved in ethanol at a concentration of 10 mM. BHT was used as a positive control. First, 0.2 mL of sample solution, 1.0 mL of DPPH solution, 0.2 mL of 0.1 M Tris–HCL (pH 7.4) buffer, and 0.6 mL of ethanol were mixed in a screw tube. Then, the screw tubes were shaken, and allowed to react at room temperature under dark conditions. After 30 min, the absorbance was measured at 517 nm by UV mini 1240 (SHIMADZU, Japan). Based on the data obtained by each concentration, concentration for 50% inhibition concentration (IC50) was calculated.
GC
GC was performed on a 7890B GC System (Agilent Technologies, Santa Clara, CA, USA) equipped with an HP-5 column (30 m × 0.25 mm i.d., 0.25 μm film thickness, Agilent Technologies). The oven temperature started at 60 °C and increased at 3.0 °C/min to at 240 °C. The injector and the detector temperatures were both 250 °C.
NMR
NMR spectra (1H: 400 MHz, 13C: 100 MHz) were recorded on a JEOL AL400 FT-NMR spectrometer. Each purified diterpenoid (50 mg) was dissolved in 0.7 mL chloroform-d. The experimentally measured spectra were compared with data from the previous report [7].