Improved solubility, dissolution rate, and oral bioavailability of main biflavonoids from Selaginella doederleinii extract by amorphous solid dispersion

Figures & data

Table 1. Formulation of TBESD solid dispersions.

Ratio	TBESD (mg)	Polymer (mg)
1:3	20	60
1:4	20	80
1:5	20	100
1:10	20	200

Table 2. The factors and levels of orthogonal test.

	A	B	C	D
1	100	100	20	55
2	95	80	15	50
3	80	65	10	45

The amount of TBESD at 20 mg.

Figure 1. Solubility of the components of (A) TBESD after forming ASD with various polymers in distilled water at room temperature; Dissolution profiles of (B) amentoflavone, (C) robustaflavone, (D) 2″,3″-dihydro-3′,3‴-biapigenin, (E) 3′,3‴-binaringenin, and (F) delicaflavone from TBESD and solid dispersion formulations, respectively (mean ± SD, n = 3).

Table 3. Solubility from TBESD and ASDs with PVP K-30 in various pH medium at room temperature (mean ± SD, n = 3).

Solubility (μg/mL)	Components
	Amentoflavone	Robustaflavone	2″,3″-Dihydro-3′,3‴-biapigenin	3′,3‴-Binaringenin	Delicaflavone
Pure extract in water	39.96 ± 2.46	15.37 ± 4.61	16.44 ± 3.47	18.71 ± 3.26	9.65 ± 1.03
ASDs in pH 1.2	434.15 ± 24.91	141.94 ± 17.15	178.65 ± 16.71	192.55 ± 21.46	112.54 ± 11.87
ASDs in pH 4.5	468.49 ± 27.16	168.39 ± 13.84	204.21 ± 19.46	243.67 ± 23.16	134.79 ± 10.13
ASDs in pH 6.8	615.69 ± 34.49	207.65 ± 21.89	246.32 ± 20.48	289.56 ± 28.13	175.16 ± 9.68
ASDs in water	678.38 ± 36.45	268.48 ± 31.73	289.16 ± 19.21	346.54 ± 14.56	187.98 ± 21.18

Table 4. Results of the orthogonal experiment (n = 3).

	A	B	C	D	EE (%)
1	1	1	1	1	0.806
2	1	2	2	2	0.840
3	1	3	3	3	0.814
4	2	1	2	3	0.803
5	2	2	3	1	0.795
6	2	3	1	2	0.799
7	3	1	3	2	0.652
8	3	2	1	3	0.810
9	3	3	2	1	0.540
K1	2.460	2.261	2.415	2.141
K2	2.397	2.445	2.183	2.291
K3	2.002	2.153	2.261	2.427
k1 (=K1/3)	0.820	0.754	0.805	0.714
k2 (=K2/3)	0.799	0.815	0.728	0.764
k3 (=K3/3)	0.667	0.718	0.754	0.809
R	0.153	0.097	0.077	0.095
Optimal parameter	A1	B2	C1	D3

K₁, K₂, and K₃ are the sum scores of level 1, level 2, and level 3 for each factor. k₁, k₂, and k₃ are the average scores of level 1, level 2, and level 3 for each factor. R is the range among the average scores for each factor, estimated by the difference between the highest and the lowest score average (R=k_max – k_min).

Figure 2. SEM images of (A) TBESD, (B) PVP K-30, and (C) TBESD-ASD; (D) DSC thermograms of TBESD, PVP K-30, PMs, and TBESD-ASD.

Figure 3. (A) PXRD patterns of TBESD, PVP K-30, PMs, and TBESD-ASD; (B) FT-IR spectra of TBESD, PVP K-30, PMs, and TBESD-ASD.

Figure 4. Chemical stability test of TBESD-ASD in difference exterior environment: (A) amentoflavone, (B) robustaflavone, (C) 2″,3″-dihydro-3′,3‴-biapigenin, (D) 3′,3‴-binaringenin, and (E) delicaflavone (mean ± SD, n = 3).

Figure 5. Mean concentration–time profiles of five active ingredients in rat plasma after a single oral administration of TBESD and solid dispersion formulations respectively (mean ± SD, n = 6).

Table 5. Pharmacokinetic parameters of five active ingredients in rats following single oral administration of TBESD and solid dispersion formulations, respectively (mean ± SD, n = 6).

PK parameters	Formulation	Amentoflavone	Robustaflavone	2″,3″-Dihydro-3′,3‴-biapigenin	3′,3‴-Binaringenin	Delicaflavone
Ke (h^–1)	TBESD	1.31 ± 0.49	1.79 ± 0.40	1.23 ± 1.56	1.44 ± 0.59	0.37 ± 0.20
	TBESD-ASDs	1.15 ± 0.77	4.37 ± 5.72	0.55 ± 0.09^#	1.04 ± 0.69	0.38 ± 0.64
t1/2 (h)	TBESD	1.00 ± 0.43	0.40 ± 0.09	1.18 ± 0.83	0.55 ± 0.20	1.06 ± 0.13
	TBESD-ASDs	1.09 ± 0.43	0.45 ± 0.41	1.30 ± 0.27*	0.74 ± 0.20	1.08 ± 0.37
Tmax	TBESD	0.23 ± 0.05	0.25 ± 0.15	0.33 ± 0.13	0.27 ± 0.12	0.22 ± 0.06
	TBESD-ASDs	0.33 ± 0.13	0.22 ± 0.06	0.29 ± 0.10	0.23 ± 0.05	0.29 ± 0.10
Cmax	TBESD	20.15 ± 13.35	18.09 ± 6.91	9.21 ± 3.93	7.48 ± 4.68	13.61 ± 5.81
	TBESD-ASDs	69.13 ± 37.63	38.87 ± 16.24*	36.16 ± 32.27^#	31.16 ± 25.82*	32.34 ± 33.36^#
AUC0–t (h·μg/mL)	TBESD	14.83 ± 6.86	10.85 ± 5.41	9.52 ± 1.75	4.16 ± 2.28	18.08 ± 2.62
	TBESD-ASDs	41.30 ± 14.10*	21.59 ± 6.61*	35.43 ± 20.94*	20.26 ± 13.63*	33.26 ± 19.80^#
AUC0–∞ (h·μg/mL)	TBESD	15.13 ± 6.68	11.66 ± 5.75	9.98 ± 1.62	5.75 ± 2.55	24.81 ± 9.30
	TBESD-ASDs	44.47 ± 13.14^#	28.75 ± 13.00*	39.43 ± 20.05*	24.53 ± 21.93*	65.35 ± 47.41^#
Cl (L/h)	TBESD	1530.83 ± 447.31	739.59 ± 257.35	909.15 ± 146.41	2170.42 ± 895.12	307.28 ± 78.42
	TBESD-ASDs	507.81 ± 171.52^#	323.06 ± 192.84*	289.20 ± 162.37^#	836.85 ± 696.73*	238.59 ± 320.60
MRT0–∞ (h)	TBESD	1.09 ± 0.29	0.61 ± 0.04	1.33 ± 0.72	0.52 ± 0.07	1.76 ± 0.44
	TBESD-ASDs	1.15 ± 0.53	0.53 ± 0.08	2.18 ± 0.80	0.89 ± 0.29^#	1.81 ± 0.95
RF		2.93	3.72	3.75	4.84	2.78

*Indicates significant difference at p < 0.05. #Indicates significant difference at p < 0.01.

Figure 6. In vivo therapeutic study and Immunohistochemistry study of different TBESD formulations in a mice model. (A) Xenograft tumor in each group of mice after treatment, (B) xenograft tumor’s growth curve of mice (mean ± SD, n = 6), (C) the average weight change of mice (mean ± SD, n = 6), and (D) representative CD34 staining (×200) of xenograft tumor in each group.

Table 6. The average weights and tumor weight-inhibitions of mice before and after treatment (mean ± SD, n = 6).

Groups	Dose (mg/kg)	Tumor weights (g)	Tumor weight-inhibitions (%)
Control	Solution	0.563 ± 0.187	–
Dox	2 mg/kg	0.244 ± 0.032**	58.44
TBESD	200 mg/kg	0.397 ± 0.049**	29.48
TBESD-ASDs	200 mg/kg	0.304 ± 0.042**	46.00

**Indicates significant difference at p<0.01.