Preparation, characterization, and anticancer effects of an inclusion complex of coixol with β-cyclodextrin polymers

Figures & data

Figure 1. Ultraviolet absorption spectra of coixol (A) and CDP (B).

Figure 2. (A) HPLC figures of the coixol-CDP inclusion compound; (B) HPLC figure of the sample of CDP; (C) standard curve of coixol.

Figure 3. Standard curve of coixol.

Table 1. The accuracy test of coixol.

Concentration	Peak area (Au)					Average	RSD(%)
(μg/mL)	1	2	3	4	5
10	1435488	1416845	1422154	1410214	1409874	1418915	0.74
50	7212358	7254684	7120246	7315487	7246583	7229872	0.99
150	21587895	21854682	21459872	21789543	21548963	21648191	0.77

Table 2. The stability test of coixol.

Concentration	Peak area (Au)					medium	RSD(%)
(μg/mL)	2 h	6 h	12 h	18 h	24 h
10	1426589	1435215	1421547	1412154	1425749	1424251	0.59
50	7254682	7254684	7354854	7154684	7354897	7274760	1.15
150	21554895	21645648	21745542	21548963	21689789	21636967	0.39

Table 3. The average recovery test of coixol.

Groups	moriginal/μg	madded/μg	Mfound/μg	Recovery (%)	Average recovery (%)	RSD(%)
Low	13.5	5.5	18.91	99.52	99.92 ± 0.73	0.73
	13.5	5.5	19.24	101.26
	13.5	5.5	18.86	99.26
Medium	13.5	15.5	29.11	100.38
	13.5	15.5	28.74	99.28
	13.5	15.5	28.97	99.89
High	13.5	25.5	39.31	100.79
	13.5	25.5	38.76	99.38
	13.5	25.5	38.82	99.54

Table 4. The yield and inclusion rate of coixol-CDP compounds.

Molar ratios of coixol and CDP	Average inclusion rate (%)	Average yield rate (%)	Comprehensive score
A (8:1)	88.77 ± 0.61	67.67 ± 0.76	84.55 ± 0.68
B (8:2)	86.55 ± 0.94	84.09 ± 0.46	86.06 ± 0.82
C (8:3)	82.61 ± 0.91	78.85 ± 0.83	81.85 ± 0.85

Table 5. Data of light stability experiments.

Inspection condition	Appearance	Rehydration	Inclusion rate (%)	Content (%)	Drying or moisture absorption rate (%)
Illumination	White powder	good	86.36 ± 0.48	5.63 ± 0.22
High temperature	White powder	good	85.97 ± 0.71	5.61 ± 0.31	1.72 ± 0.83 weight loss
High humidity	Surface wetting	good	85.43 ± 0.94	5.56 ± 0.28	26.18 ± 2.65 moisture absorption

Figure 4. Scanning electron micrographs of different samples. (A) β-CD; (B) CDP; (C) coixol; (D) physical mixture of CDP and coixol; (E) coixol-CDP inclusion compound.

Figure 5. FT-IR figures of different samples. (A) β-CD; (B) CDP; (C) coixol; (D) physical mixture of CDP and coixol; (E) coixol-CDP inclusion compound.

Figure 6. DSC figures of different samples. (A) β-CD; (B) CDP; (C) coixol; (D) physical mixture of CDP and coixol; (E) coixol-CDP inclusion compound.

Figure 7. XRD figures of different samples. (A) β-CD; (B) CDP; (C) coixol; (D) physical mixture of CDP and coixol; (E) coixol-CDP inclusion compound.

Figure 8. Inhibitory effects of coixol and the coixol-CDP inclusion compound on the A549 cells (x ± s, n = 3). Note: the coixol-CDP inclusion compound compares with coixol (**p < 0.01).

Table 6. The inhibition effects of the coixol-CDP inclusion compound on A549 cells (x ± s, n = 3).

Concentration (mg/L)	Growth-inhibiting rate (%)
	24	48	72
0	0	0	0
4.129	0.85 ± 1.23 (9.88 ± 0.34)	10.68 ± 1.41 (19.21 ± 1.19)	15.86 ± 2.01 (27.75 ± 2.76)
8.259	0.88 ± 1.42 (18.15 ± 2.66)	17.58 ± 2.50 (28.22 ± 1.02)	25.36 ± 3.85 (61.07 ± 2.13)
16.518	8.80 ± 1.39 (38.90 ± 1.39)	34.58 ± 3.13 (48.38 ± 4.04)	51.36 ± 4.76 (78.48 ± 1.39)
33.035	19.04 ± 0.82 (48.05 ± 4.05)	46.61 ± 3.80 (70.38 ± 4.40)	73.63 ± 3.31 (90.71 ± 1.11)

Figure 9. Hoechst33258 Fluorescent staining assay for morphological changes of the A549 cells induced by coixol and the coixol-CDP inclusion compound (×200). (A, B, C, D) the concentrations of coixol are 0, 8.259, 16.518, and 33.035 mg/L in sequence. (A′, B′, C′, D′) the concentrations of coixol-CDP inclusion compound are 0, 8.259, 16.518, and 33.035 mg/L in sequence.

Figure 10. Flow cytometry results. (A, B, C, D) The concentrations of coixol are 0, 8.259, 16.518, and 33.035 mg/L in sequence; (A′, B′, C′, D′) the concentrations of the coixol-CDP inclusion compound are 0, 8.259, 16.518, and 33.035 mg/L in sequence. (E) The apoptotic effects of coixol and the coixol-CDP inclusion compound on the A549 cells (x ± s, n = 3). Note: the coixol-CDP inclusion compound compares with coixol (** p < 0.01).

Figure 11. Cell cycle examination on the A549 cells. (A, B, C, D) The concentrations of coixol are 0, 8.259, 16.518, and 33.035 mg/L in sequence; (A′, B′, C′, D′) the concentrations of the coixol-CDP inclusion compound are 0, 8.259, 16.518, and 33.035 mg/L in sequence. (E) The cycle effect of coixol and its compounds on A549 cell (x ± s, n = 3). (*, p < 0.05 vs. corresponding coixol; **, p < 0.01 vs. corresponding coixol).

Figure 12. The protein levels of cleaved-Caspase-3, Caspase-8, Caspase-9, Bax, and Bcl-2 detected by Western blots (x ± s, n = 3). (*, p < 0.05 vs. corresponding coixol).

Data availability statement

The datasets used and/or analyzed are available from the corresponding author upon reasonable request.