Enhanced oral bioavailability of silymarin using liposomes containing a bile salt: preparation by supercritical fluid technology and evaluation in vitro and in vivo

Figures & data

Figure 1 Schematic diagram of the apparatus used for solution-enhanced dispersion by supercritical fluids.

Notes: (A) 1, CO₂ cylinder; 2, refrigerator; 3, high-pressure pump; 4, stabilization tank; 5, pressure sensor; 6, temperature sensor; 7, nozzle; 8, filter; 9, view vessel; 10, air bath; 11, high-pressure constant flow pump; 12, graduated flask; 13, separator; 14, wet gas meter. (B) The structure of the nozzle.

Abbreviations: P, pressure; T, temperature.

Table 1 Coded levels and measured responses for the 20 experiment formulation runs

No	A	B	C	Size (nm) (Y1)	EE% (Y2)	DL/% (Y3)
1	1:3	0.8	12	201.8	78.78	2.75
2	2:3	0.8	12	369.6	41.87	3.25
3	1:3	1.2	12	379.0	80.95	2.17
4	2:3	1.2	12	215.2	42.30	2.68
5	1:3	0.8	18	233.2	71.72	2.36
6	2:3	0.8	18	386.8	60.54	4.49
7	1:3	1.2	18	178.2	68.12	1.78
8	2:3	1.2	18	224.3	59.99	3.66
9	1:5	1.0	15	318.9	77.18	0.83
10	3:4	1.0	15	313.6	43.08	3.35
11	1:2	0.7	15	311.6	69.02	4.36
12	1:2	1.4	15	184.7	71.50	3.67
13	1:2	1.0	10	190.1	84.81	4.45
14	1:2	1.0	20	286.6	76.91	3.81
15	1:2	1.0	15	165.6	92.97	4.77
16	1:2	1.0	15	174.8	91.36	4.69
17	1:2	1.0	15	169.4	90.70	4.66
18	1:2	1.0	15	171.1	90.96	4.67
19	1:2	1.0	15	172.4	91.78	4.72
20	1:2	1.0	15	170.7	91.27	4.68

Notes: A, the ratio of silymarin to phospholipids; B, flow rate of solution; C, pressure.

Abbreviations: EE, entrapment efficiency; DL, drug loading.

Table 2 Comparison of the experimental and predicted values of SM-Lip-SEDS under predicted optimum conditions

Measured responses	Predicted values	Experimental values	Bias (%)
Y1 (size)	168.21 nm	160.50 nm	4.80
Y2 (EE)	92.29%	91.38%	0.99
Y3 (DL)	4.65%	4.73%	−1.72

Note: Bias was calculated as (predicted value – observed value)/predicted value ×100%.

Abbreviations: SM, silymarin; SEDS, solution-enhanced dispersion by supercritical fluids; EE, encapsulation efficiency; DL, drug loading.

Figure 2 Response surface models showing the influence of the factors on the responses.

Notes: Three-dimensional response surface plots showing the effects of (I) the ratio of silymarin to phospholipids, (II) flow rate of solution, and (III) pressure on the responses of Y₁ (A), Y₂ (B), and Y₃ (C). Y₁ is the response of the mean particle size. Y₂ is the response of entrapment efficiency. Y₃ is the response of drug loading.

Figure 3 Size distribution, zeta potential, and transmission electron microscopic images of liposomes (Lip) prepared by the solution-enhanced dispersion supercritical fluids (SEDS), thin-film dispersion (TFD), and reversed-phase evaporation (RPE) methods.

Abbreviation: SM, silymarin.

Figure 4 A comparison of entrapment efficiency and drug loading of liposomes prepared by different methods.

Notes: Entrapment efficiency (A) and drug loading (B) of liposomes prepared by the (a) thin-film dispersion method, (b) reversed-phase evaporation method, and (c) solution-enhanced dispersion by supercritical fluids (SEDS) method. *P<0.05 compared to SEDS (mean ± SD, n=3).

Abbreviation: SD, standard deviation.

Figure 5 X-ray diffraction analyses of (A) silymarin, (B) sodium glycocholate, (C) hydrogenated soybean phosphatidylcholine, (D) soybean phosphatidylcholine, (E) physical mixture of SM, SGC, and phosphatidylcholines, and (F) SM liposomes.

Figure 6 Differential scanning calorimetry analyses of (A) silymarin, (B) sodium glycocholate, (C) hydrogenated soybean phosphatidylcholine, (D) soybean phosphatidylcholine, (E) physical mixture of SM, SGC, and phosphatidylcholines, and (F) SM liposomes.

Abbreviations: SM, silymarin; SGC, sodium glycocholate.

Figure 7 Sonication-induced leakage of silymarin (SM) from liposomes (lip) prepared by the solution-enhanced dispersion supercritical fluids (SEDS), thin-film dispersion (TFD), and reversed-phase evaporation (RPE) methods (n=3).

Abbreviation: s, seconds.

Figure 8 In vitro release of silymarin (SM) in sodium phosphate buffer solution (pH =6.8).

Note: The cumulative release of SM is shown from SM powder, SM liposomes prepared by the thin-film dispersion (TFD) method, SM liposomes prepared by the reversed-phase evaporation method (RPE), and SM liposomes prepared by the solution-enhanced dispersion by supercritical fluids (SEDS) method (n=3).

Abbreviation: h, hour.

Table 3 The main pharmacokinetic parameters of the indicated preparations in male Wistar rats (n=6)

Formulation of SM	Tmax (h)	Cmax (μg/mL)	AUC0−t (μg/mL h)
SM powder	1.417±0.204	0.640±0.132	3.824±0.355
Commercial product	0.916±0.129	0.703±0.072	7.753±0.576*
SM-Lip-SEDS	7.667±0.816*	1.296±0.137*	18.406±1.481*
SM-Lip-RPE	5.667±0.816*	1.162±0.103*	15.331±2.181*
SM-Lip-TFD	5.500±1.225*	0.931±0.115*	14.054±1.417*

Note:

* P<0.05, compared to the SM powder (conventional powder formulation).

Abbreviations: SM, silymarin; h, hour; Lip, liposomes; SEDS, solution-enhanced dispersion by supercritical fluids; RPE, reversed-phase evaporation; T_max, time peak plasma concentration; C_max, peak plasma concentration; AUC_0−t, area under the plasma concentration-time curve; TFD, thin-film dispersion.

Figure 9 In vivo plasma concentration–time profiles.

Note: Levels of silymarin (SM) following oral administration of an aqueous SM suspension, commercial SM product, and SM liposomes (lip) prepared by different methods (n=6).

Abbreviations: h, hour; SEDS, solution-enhanced dispersion by supercritical fluids; TFD, thin-film dispersion; RPE, reversed-phase evaporation.