Elaine Hui-Chien Lee et al.
BBB-permeable curcumin analog Ab protection
6
H, OCH 9 2), 6.89 (d, J = 9 Hz, 2H, CH=CH–
3
Ab-induced paralysis assay
CO 9 2), 7.17 (d, J = 9 Hz, 2H, arom-CH=CH 9 2),
.25 (s, 2H, arom), 7.36 (s, 2H, arom), 9.72 (br s, 2H,
[14]
7
Paralysis assays were performed as described previously.
1
3
OH 9 2). C NMR (DMSO-d , 125 MHz): d 195.3, 149.0,
In brief, egg-synchronized population of transgenic
GMC101 strain was prepared by allowing gravid adult
nematodes to lay eggs on the NGM plate containing either
a vehicle or test compound at the desired concentrations
for 2 h at 16 °C. Adult nematodes were then removed and
plates with eggs were allowed to develop into L3 larvae
stage at 16 °C. At 68 h post-egg lay, the test plates were
temperature upshifted to 25 °C to induce transgene expres-
sion. Scoring for paralysis was performed at 2 h intervals
beginning from 24 h post-temperature upshift. Nematodes
were scored as paralysed if they failed to respond to a
touch-provoked movement with platinum wire. At least
three independent experiments were performed for each
test compounds. Approximately 55–100 worms were grown
on each assay plate. Survival curves were generated using
Prism 5 (GraphPad Software Inc., La Jolla, CA, US). The
delays in paralysis were assessed by comparing the median
survival time at which 50% nematodes were paralysed, rep-
resented as median ꢁ95% confident interval, between
nematodes fed with vehicle or compounds. Significant dif-
ferences between groups were assessed using the log-rank
6
1
2
48.2, 135.2, 133.3, 127.6, 125.2, 116.3, 115.0, 56.0,
+
6.3; ESI-MS m/z: 353.29 [M + H] . HPLC (MeOH :
H O = 65 : 35) tR (min): 3.973. Purity: 99.73%. HPLC
2
(
ACN : H O = 50 : 50) tR (min): 3.097. Purity: 99.80%.
2
2
,6-bis (4-hydroxy-3-methoxy benzylidene)
cyclohexanone (C3)
1
Yellow powder, Yield: 73.3%, m.p. 174–176 °C; H NMR
DMSO-d ): d 1.71–1.75 (m, 2H), 2.9 (t, 4H), 3.81 (s,
(
6
6H, OCH 9 2), 6.85 (d, J = 9 Hz, 2H, CH=CH–
3
CO 9 2), 7.04 (d, J = 9 Hz, 2H, arom-CH=CH 9 2),
.12 (s, 2H, arom), 7.57 (s, 2H, arom), 9.57 (s, 2H,
7
1
3
OH 9 2). C NMR (DMSO-d , 125 MHz): d 189.0,
6
1
5
48.3, 147.9, 136.6, 134.0, 127.4, 124.7, 116.0, 115.2,
+
6.1, 28.4, 23.03. ESI-MS m/z: 367.27 [M + H] . HPLC
(
MeOH : H O = 65 : 35) tR (min): 4.870. Purity:
2
98.84%. HPLC (ACN : H O = 50 : 50) t (min): 4.177.
2 R
Purity: 98.84%.
(Mantel-Cox) analysis. A P value <0.05 was considered sta-
tistically significant.
Caenorhabditis elegans strain and culture
conditions
The transgenic strain GMC101 (dvIs100 (unc-54p::A-
0
Parallel artificial membrane permeability
assay for blood–brain barrier
beta-1-42::unc-54 3 -UTR + mtl-2p::GFP). mtl-2p::GFP)
and the food source Escherichia coli (E. coli) strain
OP50 were obtained from the Caenorhabditis Genetics
Center (University of Minnesota, Minnesota, MN,
USA). The GMC101 strain constitutively produces a
full-length human Ab1–42 in bodywall muscle cells that
aggregates in vivo and upshifting L4 or young adults
Porcine brain lipid (PBL) was purchased from Avanti
Polar Lipids (Alabaster, AL, USA). Dodecane, caffeine
and donepezil were sourced from Sigma-Aldrich (St.
Louis, MO, USA) while DMSO was from BioBasic
Canada Inc. The 96-well donor plate microplate (PVDF
membrane, pore size 0.45 lM) and the acceptor micro-
plate were both from Merck Millipore Bioscience (Bed-
[14]
worms from 16 to 25 °C induces paralysis.
The
nematodes were routinely propagated at 16 °C on solid
nematode growth medium (NGM) seeded with spots of
OP50 as the food source.
[
17]
ford, MA, USA). The method by Di et al.
was
followed. Stock solutions of each reference and test com-
pound were prepared in DMSO at 1 mM and then
diluted with phosphate buffered saline (PBS) to obtain
the donor solution with the final concentration of 10 lM.
300 ll of the compounds were added to the donor wells.
The lipid membrane of the PAMPA-BBB model was pre-
pared by dissolving 20 mg of PBL in 1 ml dodecane. The
filter membrane of the acceptor microplate was coated
with 4 ll of the PBL solution and then filled with 300 ll
of PBS. Thereafter, acceptor plate was gently placed into
the donor plate and incubated at room temperature for
18 h. Upon completion of incubation, the plates were
separated and the concentration of the compounds in
both donor and acceptor wells were quantified by the UV
Media preparation
Curcumin and C4 were purchased from Sigma-Aldrich Inc.
(Singapore, Singapore). The curcumin analogues (C1, C2
and C3) were synthesized as described in the previous sec-
tion. Stock solutions of all compounds were made with
100% DMSO. The final concentration of DMSO did not
exceed 1% in the media. All compounds were added
directly into the molten NGM to a final concentration of
100, 10 and 1 lM; NGM with 1% of DMSO served as vehi-
cle control. All media were spotted with 600 ll of OP50
food source.
©
2018 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2018), pp. **–**
3