Proliposomes for oral delivery of total biflavonoids extract from Selaginella doederleinii: formulation development, optimization, and in vitro–in vivo characterization

Figures & data

Table 1 The factors and levels of orthogonal test

	A	B	C	D
1	10	15:1	15:1	15:1
2	20	10:1	10:1	10:1
3	30	5:1	5:1	5:1

Note: The amount of TBESD at 10 mg.

Abbreviation: TBESD, total biflavonoids extract from Selaginella doederleinii.

Table 2 Effects of different cryoprotectants on the characteristics and EE of P-TBESD (mean ± SD, n=3)

Compound	Appearance	Particle size (nm)	PDI	EE (%)
None	Collapse	237.17±3.20	0.193	89.33±3.20
Lactose	Collapse	330.13±0.52	0.227	85.14±1.11
Xylitol	A little collapse	339.30±2.30	0.233	81.28±0.49
Mannitol	Smooth, unshrinking	472.97±9.35	0.258	88.51±5.12
Isomalto-oligosaccharides	Smooth, unshrinking	251.93±2.54	0.189	91.24±1.73

Abbreviations: PDI, polydispersity index; TBESD, total biflavonoids extract from Selaginella doederleinii; EE, entrapment efficiency; P-TBESD, TBESD proliposomes.

Figure 1 HPLC chromatograms of (A) blank liposome, (B) reference sample and (C) TBESD liposomes (1. amentoflavone；2. robustflavone；3. 2’’,3’’-dihydro-3’,3’’’-biapigenin；4. 3’,3’’’-binaringenin；5. Delicaflavone).

Abbreviations: TBESD, total biflavonoids extract from Selaginella doederleinii; HPLC, high-performance liquid chromatography.

Figure 2 Characterization of P-TBESD. (A) Transmission electron micrograph of P-TBESD. (B) Size distribution of P-TBESD. (C) Zeta potential of P-TBESD.

Abbreviations: P-TBESD, TBESD proliposomes; TBESD, total biflavonoids extract from Selaginella doederleinii.

Figure 3 Mean concentration-time profiles of five active ingredients in rat plasma after a single oral administration of TBESD and proliposome formulations, respectively (mean ± SD, n=6).

Abbreviation: TBESD, total biflavonoids extract from Selaginella doederleinii.

Figure 4 In vivo therapeutic study and histopathology 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). (D) Representative histological images (×200) of various organ tissues of tumor-bearing male BALB/c mice treated with Fluorouracil, TBESD or P-TBESD.

Abbreviation: TBESD, total biflavonoids extract from Selaginella doederleinii; P,TBESD, TBESD proliposomes.

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

	A	B	C	D	EE (%)
1	1	1	1	1	65.17
2	1	2	2	2	86.56
3	1	3	3	3	68.93
4	2	1	2	3	69.55
5	2	2	3	1	62.75
6	2	3	1	2	73.01
7	3	1	3	2	80.81
8	3	2	1	3	87.67
9	3	3	2	1	78.84
K1	220.65	215.53	225.85	206.76
K2	205.31	236.98	234.94	240.38
K3	247.32	220.77	212.49	226.15
k1 (=K1/3)	73.55	71.84	75.28	68.92
k2 (=K2/3)	68.44	78.99	78.31	80.13
k3 (=K3/3)	82.44	73.59	70.83	75.38
R	14.00	7.15	7.48	11.21
Optimal parameter	A3	B2	C2	D2

Notes: 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 = kmax-kmin).

Abbreviation: EE, entrapment efficiency.

Table 4 Pharmacokinetic parameters of five active ingredients in rats following single oral administration of TBESD and proliposome 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	0.78±0.26	1.63±0.38	1.17±1.15	1.26±0.49	0.66±0.08
	Liposomes	0.54±0.18	0.65±0.31**	0.57±0.13**	1.26±0.67	0.85±0.41
T1/2 (h)	TBESD	1.00±0.43	0.44±0.13	1.01±0.65	0.64±0.30	0.93±0.34
	Liposomes	1.39±0.41*	1.25±0.49**	1.27±0.33	0.65±0.25	1.06±0.13
Tmax	TBESD	0.23±0.05	0.25±0.15	0.29±0.17	0.27±0.12	0.22±0.06
	Liposomes	0.27±0.12	0.20±0.07	0.27±0.12	0.33±0.13	0.23±0.05
Cmax	TBESD	21.81±17.41	18.09±6.91	8.88±4.39	7.15±4.96	13.61±5.81
	Liposomes	117.76±33.76**	35.54±20.01*	49.35±36.65**	51.35±26.32**	35.21±13.03**
AUC0-∞ (μg/mL·h)	TBESD	14.33±8.36	11.18±5.23	9.37±1.84	4.10±2.34	17.47±3.20
	Liposomes	95.89±42.44**	58.52±58.06**	71.35±50.48**	39.16±17.70**	33.34±15.40**
Vd (L/kg)	TBESD	2277.27±1312.85	445.37±208.37	1292.64±133.83	2096.29±1067.65	571.40±126.35
	Liposomes	446.06±101.75**	328.62±244.52	452.77±282.70**	268.63±121.95**	291.15±75.17**
CL (L/h)	TBESD	1543.03±483.41	677.54±189.18	923.64±150.91	2334.92±1132.08	372.72±609.21
	Liposomes	244.68±100.90**	182.35±95.54**	219.41±186.73**	305.80±124.77**	248.80±127.94*
MRT0-∞ (h)	TBESD	0.96±0.34	0.79±0.15	1.65±1.12	0.53±0.08	1.81±1.22
	Liposomes	1.77±1.02**	2.94±2.59**	3.68±1.94*	0.83±0.15**	2.13±0.69
RF		6.69	5.23	7.61	9.55	1.91

Notes: *Indicates significant difference at p<0.05. **Indicates significant difference at p<0.01.

Abbreviation: TBESD, total biflavonoids extract from Selaginella doederleinii.

Table 5 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	0.376±0.049	–
Fluorouracil	25 mg/kg	0.163±0.059**	56.75
TBESD	300 mg/kg	0.278±0.035**	25.96
P-TBESD	300 mg/kg	0.192±0.047**	48.85

Notes: Compared with control: *P<0.05, **P<0.01.

Abbreviation: P-TBESD, TBESD proliposomes; TBESD, total biflavonoids extract from Selaginella doederleinii.

Table S1 The standard curve results of biflavonoids

Analyte	Linear range	Regression equation(w=1/x^2)	R
(μg/mL)
Amentoflavone	1-200	y=44715.0x+235.87	0.99955
Robustaflavone	0.5-100	y=22648.9x+257.41	0.99959
2'',3''-Dihydro-3',3'''-biapigenin	0.5-100	y=9947.6x+42.18	0.99965
3',3'''-Binaringenin	0.5-100	y=27526.5x+100.18	0.99954
Delicaflavone	0.5-100	y=29624.5x+117.95	0.99994

Table S2 Precision and repeatabilities of biflavonoids from P-TBESD (n=6)

Analyte	Con. (μg/mL)	Precision		Repeatabilities
		Mean ± SD	RSD (%)	RSD (%)
Amentoflavone	3	3.00 ± 0.08	2.51	2.91
	80	80.42 ± 1.51	1.88	2.01
	160	160.50 ± 1.84	1.15	1.26
Robustaflavone	1.5	1.48 ± 0.03	2.13	2.72
	40	40.36 ± 0.68	1.69	2.03
	80	80.40 ± 0.93	1.16	1.84
2'',3''-Dihydro-3',3'''-biapigenin	1.5	1.51 ± 0.03	2.05	2.68
	40	40.07 ± 0.77	1.91	1.78
	80	80.15 ± 0.78	0.98	1.41
3',3'''-Binaringenin	1.5	1.50 ± 0.02	1.25	2.47
	40	39.97 ± 0.42	1.06	2.04
	80	79.81 ± 0.88	1.11	1.83
Delicaflavone	1.5	1.51 ± 0.03	2.09	2.79
	40	40.08 ± 0.29	0.72	2.11
	80	79.83 ± 0.95	1.19	1.72

Abbreviations: P-TBESD, TBESD proliposomes; TBESD, total biflavonoids extract from Selaginella doederleinii.

Figure S1 Cytotoxicity of HT-29 cells after incubated with free TBESD and P-TBESD for 48 h. (n=5).