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. 2022 Aug 24;7(35):31233-31245.
doi: 10.1021/acsomega.2c03489. eCollection 2022 Sep 6.

Preparation, Characterization, and In Vitro Evaluation of Inclusion Complexes Formed between S-Allylcysteine and Cyclodextrins

Rino Tachikawa  1 , Hiroki Saito  2 , Hajime Moteki  2 , Mitsutoshi Kimura  2 , Hiroaki Kitagishi  3 , Florencio Arce Jr  4 , Gerard Lee See  4 , Takashi Tanikawa  1 , Yutaka Inoue  1
Affiliations

Preparation, Characterization, and In Vitro Evaluation of Inclusion Complexes Formed between S-Allylcysteine and Cyclodextrins

Rino Tachikawa et al. ACS Omega. .

Abstract

The present study prepared inclusion complexes of S-allylcysteine (SAC) and cyclodextrin (α, β, γ) by the freeze-drying (FD) method and verified the inclusion behavior of the solid dispersion. Also, the study investigated the effect of SAC/CD complex formation on liver tumor cells. Isothermal titration calorimetry (ITC) measurements confirmed the exothermic titration curve for SAC/αCD, suggesting a molar ratio of SAC/αCD = 1/1, but no exothermic/endothermic reaction was obtained for the SAC/βCD and SAC/γCD system. Powder X-ray diffraction (PXRD) results showed that the characteristic diffraction peaks of SAC and CDs disappeared in FD (SAC/αCD) and FD (SAC/γCD), indicated by a halo pattern. On the other hand, diffraction peaks originating from SAC and βCDs were observed in FD (SAC/βCD). Near-infrared (NIR) absorption spectroscopy results showed that CH and OH groups derived from SAC and OH groups derived from αCD and γCD cavity were shifted, suggesting complex formation due to intermolecular interactions occurring in SAC/αCD and SAC/γCD. Stability test results showed that the stability was maintained with FD (SAC/αCD) over FD (SAC/βCD) and FD (SAC/γCD). In 1H-1H of NOESY NMR measurement, FD (SAC/αCD) was confirmed to have a cross peak at the CH group of the alkene of SAC and the proton (H-3, -5, -6) in the αCD cavity. In FD (SAC/γCD), a cross peak was confirmed at the alkyl group on the carbonyl group side of SAC and the proton (H-3) in the cavity of γCD. From the above, it was suggested that the inclusion mode of SAC is different on FD (SAC/CDs). The results of the hepatocyte proliferation inhibition test using HepG2 cells showed that FD (SAC/βCD) inhibited cell proliferation. On the other hand, FD (SAC/αCD) and FD (SAC/γCD) did not show a significant decrease in the number of viable cells. These results suggest that the difference in the inclusion mode may contribute to the stability and cell proliferation inhibition.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
ITC curves of SAC/αCD. The solution (1.4 mL) of CDs (1 mM) dissolved in 0.05 M phosphate buffer were titrated with SAC solution (20 mM) in the same buffer. The solid line in (a) represents the best-fit theoretical curve to determine thermodynamic parameters for this complexation using the ORIGIN software.
Figure 2
Figure 2
ITC curves of SAC/βCD systems.
Figure 3
Figure 3
ITC curves of SAC/γCD systems.
Figure 4
Figure 4
PXRD pattens of SAC intact, SAC/αCD, SAC/βCD, and SAC/γCD systems. (a) SAC intact, (b) αCD intact, (c) PM (SAC/αCD = 1/1), (d) FD (SAC/αCD = 1/1), (e) βCD intact , (f) PM (SAC/βCD = 1/1), (g) FD (SAC/βCD = 1/1), (h) γCD intact, (i) PM (SAC/γCD = 1/1), and (j) FD (SAC/γCD = 1/1).
Figure 5
Figure 5
DSC curves of SAC intact, SAC/αCD, and SAC/βCD SAC/γCD systems. (a) SAC intact, (b) αCD intact, (c) PM (SAC/αCD = 1/1), (d) FD (SAC/αCD = 1/1), (e) βCD intact , (f) PM (SAC/βCD = 1/1), (g) FD (SAC/βCD = 1/1), (h) γCD intact, (i) PM (SAC/γCD = 1/1), and (j) FD (SAC/γCD = 1/1).
Figure 6
Figure 6
Second differentiation NIR absorption spectra of FD SAC/αCD = 1/1 system. (a) 8000–6500, (b) 5600–4600, and (c) 4700–4100 cm–1.
Figure 7
Figure 7
Second differentiation NIR absorption spectra of FD SAC/βCD = 1/1 system. (a) 8000–6500, (b) 5600–4600, and (c) 4700–4100 cm–1.
Figure 8
Figure 8
Second differentiation NIR absorption spectra of FD SAC/γCD = 1/1 system. (a) 8000–6500, (b) 5500–4600, and (c) 4700–4100 cm–1.
Figure 9
Figure 9
Changes in SAC content after storage under vacuum conditions at temperatures of 40 and 80 °C. Each point presents the mean ± SD (n = 3). (a) Under 40 °C conditions. (b) Under 80 °C conditions.
Figure 10
Figure 10
{1H–1H} NOESY NMR spectra of FD (SAC/αCD = 1/1) in D2O. (a) f1 is 0–10 ppm, f2 is 0–10 ppm. (b) f1 is 3.2–3.9 ppm, f2 is 2.5–3.3 ppm. (c) f1 is 3.2–3.9 ppm, and f2 is 4.8–5.8 ppm.
Figure 11
Figure 11
{1H–1H} NOESY NMR spectra of FD (SAC/βCD =1/1) in D2O. (a) f1 is 0–10 ppm, f2 is 0–10 ppm. (b) f1 is 3.2–3.9 ppm, f2 is 2.5–3.3 ppm. (c) f1 is 3.2–3.9 ppm, and f2 is 4.8–5.8 ppm.
Figure 12
Figure 12
{1H–1H} NOESY NMR spectra of FD (SAC/γCD =1/1) in D2O. (a) f1 is 0–10 ppm, f2 is 0–10 ppm. (b) f1 is 3.2–3.9 ppm, f2 is 2.5–3.3 ppm. (c) f1 is 3.2–3.9 ppm, and f2 is 4.8–5.8 ppm.
Scheme 1
Scheme 1. Proposed Structure Images of SAC/αCD Complexes
Scheme 2
Scheme 2. Proposed Structure Images of SAC/βCD Complexes
Scheme 3
Scheme 3. Proposed Structure Images of SAC/γCD Complexes
Figure 13
Figure 13
Inhibitory effects of SAC and/or/α,β,γ-cyclodextrins on the growth of HepG2 cells. Cells were plated at 2 × 105 cell/well with 3% FCS in DMEM, and after a 24 h attachment period, cells were cultured in serum-free medium containing 10–6 M SAC with or without α,β,γ-cyclodextrins (SAC intact, SAC/αCD, αCD intact, SAC/βCD, βCD intact, SAC/γCD, or γCD intact), and the number of nuclei (cell proliferation) were measured 48 h after the addition of SAC/α,β,γ-cyclodextrins. Values are shown as means ± S.E.M. (N = 3, Tukey’s test). *(p < 0.05), **(p < 0.01) shows comparison with SAC intact, +(p < 0.05), ++(p < 0.01) shows comparison with control.
Figure 14
Figure 14
Chemical structures of SAC, CDs. (a) SAC, (b) αCD, (c) βCD, and (d) γCD.
See this image and copyright information in PMC

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