An optimized molecular inclusion complex of diferuloylmethane: enhanced physical properties and biological activity

Figures & data

Table 1 Results of orthogonal design (n = 3)

Experiment number	Factor			Mean solubility (mmol/L)
	A	B	C
1	1:1	20	0.5	1.08
2	1:1	40	1	1.49
3	1:1	60	1.5	1.76
4	1:2	20	1.5	0.85
5	1:2	40	0.5	1.55
6	1:2	60	1	0.83
7	1:3	20	1	0.76
8	1:3	40	1.5	0.4
9	1:3	60	0.5	0.28
k1	1.44	0.90	0.77	–
k2	1.08	1.15	0.87	–
k3	0.48	0.96	1.36	–
Δk	0.96	0.25	0.59	–

Notes: “A” represents the molar ratio of diferuloylmethane to hydroxypropyl-β-cyclodextrin; “B” represents the optimal preparation temperature; “C” represents the optimal kneading time; k_i is the average scores of each factor at its different level i (here, i equals 1, 2, and 3, respectively).

Figure 1 Ultraviolet spectra of diferuloylmethane (A) and hydroxypropyl-β-cyclodextrin (B).

Figure 2 Optical photomicrographs of (A) diferuloylmethane, (B) hydroxypropyl-β-cyclodextrin, (C) physical mixture, and (D) a molecular inclusion complex consisting of diferuloylmethane and hydroxypropyl-β-cyclodextrin.

Figure 3 Schematic structure models of a molecular inclusion complex consisting of diferuloylmethane (DFH) and hydroxypropyl-β-cyclodextrin (HBCD). (A) Initial view and (B) rotated view.

Notes: DFH is represented as a space-filling model, while HBCD is represented as a stick model. The DFH and HBCD molecules consist of different atoms of oxygen (red), carbon (gray), and hydrogen (white).

Table 2 Mathematical models of mean cumulative release rate versus time

Model	MICDH		Free DFM (12–120 hours)
	0.1 mol/L HCl	pH 6.8 PBS	0.1 mol/L HCl	pH 6.8 PBS
Zero-order kinetic	Q = 0.3842t + 31.471	Q = 0.5512t + 26.117	Q = 0.1954t + 4.0409	Q = 0.197t + 4.0818
	r = 0.9480	r = 0.9692	r = 0.9818	r = 0.9786
First-order kinetic	ln(1 − Q) = −0.0082t − 0.360	ln(1 − Q) = −0.014t − 0.244	ln(1 − Q) = −0.0023t − 0.0348	ln(1 − Q) = −0.0024t − 0.0346
	r = 0.9832	r = 0.9947	r = 0.9850	r = 0.9799
Higuchi^Citation17	Q = 4.7755t^1/2 + 21.537	Q = 0.1459t^1/2 − 1.7299	Q = 2.8491t^1/2 − 4.9889	Q = 2.8418t^1/2 − 4.7928
	r = 0.9822	r = 0.9956	r = 0.9857	r = 0.9718
Retrer-Peppas^Citation33	lnQ = 0.2521lnt + 3.064	lnQ = 0.3555lnt + 2.735	lnQ = 0.7487lnt − 0.2637	lnQ = 0.6918lnt − 0.0217
	r = 0.9798	r = 0.9921	r = 0.9742	r = 0.9673
Weibull^Citation17	lnln[1/(1 − Q)] = 0.3438	lnln[1/(1 − Q)] = 0.4972	lnln[1/1 − Q] = 0.8016	lnln[1/1 − Q] = 0.7441
	lnt − 1.4846	lnt − 1.9177	lnt − 4.9844	lnt − 4.7398
	r = 0.9825	r = 0.9872	r = 0.9767	r = 0.9682

Note: Q is cumulative DFM release at time t.

Abbreviations: MICDH, molecular inclusion complex consisting of diferuloylmethane and hydroxypropyl-β-cyclodextrin; DFM, diferuloylmethane; HCl, hydrochloric acid; PBS, phosphate-buffered saline.

Figure 4 Drug release profiles of different diferuloylmethane (DFM) formulations in 0.1 mol/L hydrochloric acid (HCl) and pH 6.8 phosphate-buffered saline (PBS) containing 2% sodium dodecyl sulfate.

Table 3 The similarities between the two release profiles of a molecular inclusion complex, consisting of diferuloylmethane (DFM) and hydroxypropyl-β-cyclodextrin (MICDH), and of free DFM in two release media, either the same or different

Release profile 1		Release profile 2		Similarity factor (f2)
Formulation	Release medium	Formulation	Release medium
MICDH	0.1 mol/L HCl	Free DFM	0.1 mol/L HCl	21.53
MICDH	pH 6.8 PBS	Free DFM	pH 6.8 PBS	20.56
MICDH	0.1 mol/L HCl	MICDH	pH 6.8 PBS	58.86
Free DFM	0.1 mol/L HCl	Free DFM	pH 6.8 PBS	96.01

Abbreviations: HCl, hydrochloric acid; PBS, phosphate-buffered saline.

Figure 5 Cell viabilities after 24 hours’ exposure to different concentrations of free diferuloylmethane (DFM) and a molecular inclusion complex consisting of DFM and hydroxypropyl-β-cyclodextrin (MICDH).

Notes: Results are presented as the mean plus or minus the standard deviation; n = 5; *P < 0.05 for MICDH compared with free DFM.

Figure 6 (A and B) Cell cycle arrest and (C and D) cell apoptosis after 24 hours’ exposure to 10 μg/mL free diferuloylmethane (DFM) and a molecular inclusion complex consisting of DFM and hydroxypropyl-β-cyclodextrin (MICDH).

Abbreviation: PI, propidium iodide.

Figure 7 (A and D) The reactive oxygen species levels, (B and E) intracellular free calcium ion levels, and (C and F) mitochondrial membrane potential after 24 hours’ exposure to 10 μg/mL free diferuloylmethane (DFM) and a molecular inclusion complex consisting of DFM and hydroxypropyl-β-cyclodextrin (MICDH).

Abbreviations: Fluo-3, Fluo-3 AM; Rho 123, rhodamine 123.

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