L. Chen, W. Yang, C. Gao et al.
Journal of Molecular Liquids xxx (xxxx) xxx
2.2.3. Synthesis of thiodipropionic amide-b-CD dimer (TPACDD)
3,30-Thiodipropionic acid (0.12 g, 0.66 mmol), N, N-
Diisopropylethylamine (DIPEA) (0.17 g, 1.32 mmol), O-(7-
Azabenzotriazol-1-yl)-N, N, N’, N’-tetramethyluronium hexafluo-
rophosphate (HATU) (0.50 g, 1.32 mmol) were dissolved in dry
DMF (15 mL). The resulting solution was stirred at room tempera-
ture for 4 h. Then, ACD (1.00 g, 0.88 mmol) was dissolved in dry
DMF (15 mL) and dripped into the above solution. The mixture
was stirred roughly at room temperature for 48 h. The mixture
was dropped into acetone (200 mL) to precipitate. The precipitate
was collected by centrifugation and washed by acetone
(100 mL ꢀ 3). The resultant was dialyzed (MWCO, 2000) against
distilled water for 48 h and then freeze dried for 24 h. The final
product was TPACDD (0.19 g, 0.079 mmol, 18%). 1H NMR
(600 MHz, D2O, ppm): d 5.00 (dt, J = 8.9, 5.4 Hz, 14H, 1-H of b-
CD), 3.95–3.71 (m, 52H, 3, 5, 6-H of b-CD), 3.60–3.27 (m, 28H, 2,
4-H of b-CD), 2.93–2.18 (m, 14H, 60, 9, 8-H). ESI-MS: m/z calcd.
for [M + H] +: 2409.7803, found: 2409.7884.
micrographs were then obtained with an accelerating potential
of 15 kV under reduced pressure.
2.2.9. Nuclear magnetic resonance (NMR) spectroscopy
All NMR analyses including 1H and 2D ROESY NMR experiments
were conducted on a Bruker Avance III HD spectrometer (600 MHz,
Bruker BioSpin, Switzerland) at 25 °C. The samples were dissolved
in 99.98% D2O or 99.98% CDCl3 and were filtered before use.
2.2.10. Water solubility test
The water solubility of CDs-CBD inclusion complexes was
assessed by preparation of their saturated solution. Briefly, excess
amounts of CDs-CBD inclusion complexes were placed in buffer
solution (KH2PO4/K2HPO4, pH 7.0, 2 mL) and shaken at 25 °C for
2 h, respectively. The mixture was then filtered through 0.45-lm
membrane to obtain a clear solution. The obtained solution was
suitable diluted and measured using ShimadzuÒ UV–Vis spec-
trophotometer (UV-2250) under 274 nm. The water solubility of
CDs-CBD inclusion complexes could be deduced from the standard
curve of CBD.
2.2.4. Preparation of CDs-CBD inclusion complexes
Solid inclusion complexes were prepared by saturated aqueous
solution method. Accurately weighed amounts of DMCD, SACDD
and TPACDD (0.1 mmol) were dissolved in ultrapure water
(20 mL), respectively. Subsequently, CBD (0.2 mmol) was added
to the above solution, respectively. The resulting suspension was
stirred in the dark at room temperature for 72 h. It was then fil-
tered through a 0.45-lm membrane filter and the filtrate was dried
in vacuum freeze dryer to yield CDs-CBD solid inclusion
complexes.
2.2.11. Thermal gravimetric analysis (TGA)
The TGA curves of CDs, CBD, CDs-CBD physical mixtures and
inclusion complexes were performed on a simultaneous thermal
analyzer (NETZSCH STA449F3 Germany). About 2 mg of the sample
was placed in an aluminum crucible and subjected to a tempera-
ture range of 40 ~ 400 °C, under dynamic nitrogen atmosphere
(50 mLꢂminꢁ1) and heating rate of 10 °Cꢂminꢁ1
.
2.2.12. In vitro antioxidant assays
2.2.5. Preparation of CDs-CBD physical mixtures
The DPPH (2, 2-diphenyl-1-picrylhydeazyl) scavenging assay
was used to evaluate the in vitro antioxidant activity of CDs-CBD
inclusion complexes [30,31]. Firstly, the stock solutions of CBD,
CDs and three inclusion complexes (0.5 mM, 1.0 mM, 1.5 mM,
3.0 mM and 5.0 mM, 10.0 mL) were prepared by ethanol and water
The physical mixtures were prepared by mixing the powders in
a 1:1 M ratio of CBD and CDs (DMCD, SACDD and TPACDD) in an
agate mortar for 5 min, respectively.
2.2.6. Phase-solubility diagram
respectively. Then a volume of 50
lL of stock solutions was mixed
The phase-solubility diagram was performed according to the
studies of Higuchi and Connors [29]. Excess amounts of CBD were
added in solutions with increasing concentration of CDs (DMCD,
SACDD and TPACDD), range in 1.0 to 7.2 mM, respectively. The
samples were sealed and were shaken for 72 h at room tempera-
with 150 L of DPPH in ethanol (0.2 mM) in a 96-well plate. After
l
being cultivated at room temperature for 30 min in the dark, they
were subjected to the absorbance determination on Enzyme-
labeled instrument at 517 nm. The free radical scavenging activity
of the sample could be expressed in the percentage of remaining
DPPH (% DPPHrem) which could be calculated from Eq. (1).
ture in the dark. Then, they were filtered on 0.45 lm membranes.
The filtered samples were suitable diluted and analyzed in a Shi-
madzuÒ UV–Vis spectrophotometer (UV-2250) for dissolved con-
centration of CBD at 274 nm. All experiments were performed in
triplicates and the phase-solubility diagram was drawn by plotting
the molar concentration of CBD against the molar concentration of
CDs (DMCD, SACDD and TPACDD) according to the calibration
curve.
AAðtÞ
ACð0Þ
%DPPHrem
¼
ꢀ 100%
ð1Þ
where AC(0) and AA(t) are the initial and the final absorbance of
DPPH, respectively. All tests were run in triplicate.
EC50 represents the concentration of the inhibitory effect of the
antioxidant when the clearance rate is 50%, which is used to eval-
uate the antioxidant capacity of the antioxidant. Under the same
conditions, the lower the EC50 value, the stronger the antioxidant
capacity. All experiments were carried out in triplicate, and the
average value was calculated.
2.2.7. Powder X-ray diffractometry (XRD)
The X-ray powder diffraction patterns were obtained with an
XRD-6000 X-ray Diffractometer (Shimadzu, Japan) using a Ni-
filtered, Cu Ka radiation, a voltage of 40 kV and a 30 mA current.
the physical mixture of CBD and CDs (DMCD, SACDD and TPACDD)
and their inclusion complex were previously dried for 24 h at
110 °C. Each dried powder was measured in the 2h angle range
between 5° and 60° with a scan rate of 5° minꢁ1 and a step size
of 0.02°.
2.2.13. In vitro cytotoxicity assays
The in vitro cytotoxicity of solid inclusion complexes was eval-
uated towards two human cancer cell lines, MCF-7, HT29 and nor-
mal cell line HLF, using cisplatin as a positive reference by MTT (3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide)
2.2.8. Scanning electron microscope (SEM) analysis
assay [32,33]. Cells were suspended in RPMI 1640 (Hyclone Corp.
Utah, USA) supplemented with 10% fetal bovine serum (Hyclone)
at 37 °C in a humidified atmosphere of 5% CO2 in air. Afterward,
cells were seeded into 96-well microculture plates. Culture for
24 h, CBD, DMCD, SACDD, TPACDD and their inclusion complex
were added, respectively. After 48 h exposure to the compounds,
SEM analysis was carried out with a Jeol JSM-840 scanning elec-
tron microscope (Japan). The samples were mounted on metal
stubs with double-sided adhesive tapes. To avoid the issue of elec-
trically charging from the insulating samples, a thin layer of gold
was sputtered on top of the samples prior to the SEM scan. The
3