Preparation, characterization, and in vivo pharmacokinetics of nanostructured lipid carriers loaded with oleanolic acid and gentiopicrin

Figures & data

Table 1 Actual values of independent variables

	Name	Units	−1	1	−a	+a	0
X1	Oleanolic acid	mg	2	4	1	5	3
X2	Gentiopicrin	mg	2	4	1	5	3
X3	Oleic acid	mL	1.19	1.53	1.02	1.7	1.36

Table 2 Formulations of nanostructured lipid carriers loaded with both oleanolic acid and gentiopicrin by central composite design

Run	X1	X2	X3	Response 1	Response 2	Response 3
F1	−a	0	0	Oleanolic acid EE (%)	Gentiopicrin EE (%)	Total EE (%)
F2	1	−1	−1
F3	0	0	0
F4	0	−a	0
F5	−1	−1	−1
F6	−1	1	1
F7	−1	−1	1
F8	0	0	a
F9	0	0	−a
F10	a	0	0
F11	0	a	0
F12	1	1	1
F13	−1	1	−1
F14	1	1	−1
F15	1	−1	1

Abbreviation: EE, entrapment efficiency.

Figure 1 Particle size and zeta potential of various surfactants.

Abbreviations: PS, particle size; ZP, zeta potential; PVP, polyvinylpyrrolidone.

Figure 2 Entrapment efficiency of samples prepared at different concentrations of Poloxamer 188.

Abbreviations: EE, entrapment efficiency; GEN, gentiopicrin; OA, oleanolic acid.

Figure 3 Effect of proportion of oleic acid on drug entrapment efficiency (A) and particle size (B).

Abbreviations: EE, entrapment efficiency; GEN, gentiopicrin; OA, oleanolic acid.

Table 3 Analysis of variance results for oleanolic acid, gentiopicrin and total entrapment efficiency, shown as R1, R2, and R3, respectively

	Source	Sum of squares	df	Mean square	F value	P value Probability > F
R1	Model	23.85	9	2.65	59.38	<0.0001
	A, oleanolic acid	1.71	1	1.71	38.25	0.0001
	C, oleic acid	0.74	1	0.74	16.48	0.0023
	AB	2.08	1	2.08	46.69	<0.0001
	A^2	5.78	1	5.78	129.42	<0.0001
	B^2	16.36	1	16.36	366.67	<0.0001
	C^2	0.24	1	0.24	5.4	0.0424
	Residual	0.45	10	0.045
	Lack of fit	0.45	5	0.089
	Pure error	0.000	5	0.000
	Cor total	24.30	19
y = 8.27 − 0.33x1 − 0.043x2 + 0.21x3 − 0.51x1x2 − 1.690e – 0.003x1x3 − 0.024x2x3 − 0.48x1^2 − 0.81x2^2 − 0.098x3^2 −1.690e − 0.003x1x3 − 0.024x2x3 − 0.48x1^2 − 0.81x2^2 − 0.098x3^2 − 1.690e − 0.003x1x3 − 0.024x2x3− 0.48x1^2 − 0.81x2^2 − 0.098x3^2
R2	Model	394.10	9	43.79	11.02	0.0004
	B, gentiopicrin	38.38	1	38.38	9.66	0.0111
	C^2	321.59	1	321.59	80.96	<0.0001
	Residual	39.72	10	3.97
	Lack of fit	39.72	5	7.94
	Pure error	0.000	5	0.000
	Total	433.82	19
y = 27.97 − 0.98x1 − 1.55x2 − 0.35x3 + 0.97x1x2 − 0.090x1x3 + 0.08x3x2x3 − 0.48x1^2 − 0.20x2^2 − 3.58x3^2
R3	Model	6.198E-003	9	6.887E-004	25.10	<0.0001
	B, gentiopicrin	5.904E-004	1	5.904E-004	21.52	0.0009
	C, oleic acid	1.490E-004	1	1.490E-004	5.43	0.0420
	AB	3.408E-004	1	3.408E-004	12.42	0.0055
	A^2	1.887E-003	1	1.887E-003	68.77	<0.0001
	B^2	4.107E-003	1	4.107E-003	149.68	<0.0001
	C^2	5.688E-004	1	5.688E-004	20.73	0.0011
	Residual	2.744E-004	10	2.744E-005
	Lack of fit	2.744E-004	5	5.487E-005
	Pure error	0.000	5	0.000
	Total	6.472E-003
y = 0.14 + 1.790e − 003x1 + 6.075e − 003x2 − 3.052e − 003x3 + 6.526e − 003x1x2 − 2.917e − 004x1x3 + 1.034e − 003x2x3 + 8.663e − 003x1^2 + 0.013x2^2 + 4.756e − 003x3^2

Figure 4 Three-dimensional map of R1 (A) and R3 (B) follows interaction between independent variables (AB).

Abbreviations: EE, entrapment efficiency; GEN, gentiopicrin; OA, oleanolic acid.

Figure 5 Particle size distribution (A) and zeta potential distribution (B) of GO-NLCs.

Abbreviation: GO-NLCs, nanostructured lipid carriers loaded with both oleanolic acid and gentiopicrin.

Figure 6 Transmission electron microscopy of optimized nanostructured lipid carriers loaded with both oleanolic acid and gentiopicrin showing spherical morphology (×87,000).

Figure 7 Differential scanning calorimetry curve of GMS, GEN, OA, and freeze-dried GO-NLCs.

Abbreviations: GMS, glycerin monostearate; GEN, gentiopicrin; OA, oleanolic acid; GO-NLCs, nanostructured lipid carriers loaded with both oleanolic acid and gentiopicrin.

Table 4 Models for drug-release fitting and correlation coefficients of nanostructured lipid carriers loaded with both oleanolic acid and gentiopicrin

Model	Equation	R^2
Gentiopicrin
Zero-order	y = 0.031x + 34.131	0.6035
First-order	y = 14.743 ln(x) − 25.938	0.9745
Higuchi	y = 3.4661x^0.4606	0.8872
Oleanolic acid
Zero-order	y = 0.0347x + 29.354	0.7057
First-order	y = 15.257 ln(x) − 31.173	0.9706
Higuchi	y = 2.8874x^0.4818	0.9305

Figure 8 In vitro release curves for GEN, OA, and GO-NLCs.

Abbreviations: GEN, gentiopicrin; OA, oleanolic acid; GO-NLCs, nanostructured lipid carriers loaded with both oleanolic acid and gentiopicrin.

Table 5 Plasma pharmacokinetic parameters for gentiopicrin and oleanolic acid after intravenous administration of gentiopicrin, oleanolic acid, and GO-NLCs

Parameter	Unit	Gentiopicrin + NS	GO-NLCs Gentiopicrin	Oleanolic acid + NS	GO-NLCs Oleanolic acid
AUC	μg · min · mL^−1	1370.400 ± 21.947**	2155.428 ± 34.349**	1052.806 ± 16.613**	1520.754 ± 25.355**
A	μg/mL	51.934 ± 0.104**	37.816 ± 0.469**	34.232 ± 0.574**	7.745 ± 0.431**
alpha	minutes	0.040 ± 0.004*	0.018 ± 0.002**	0.045 ± 0.007**	0.038 ± 0.003*
B	μg/mL	0.427 ± 0.041**	0.297 ± 0.084**	4.874 ± 0.130**	22.899 ± 0.451**
Beta	minutes	0.005 ± 0.003	0.004 ± 0.001	0.017 ± 0.001	0.017 ± 0.001
V (c)	mg · mL · μg/kg	0.191 ± 0.052**	0.262 ± 0.076**	0.255 ± 0.034**	0.326 ± 0.052**
t 1/2 α	minutes	17.157 ± 0.273**	38.115 ± 0.432**	15.529 ± 0.393**	18.164 ± 0.775**
t 1/2 β	minutes	137.814 ± 1.864**	177.516 ± 2.244**	40.658 ± 0.897**	39.890 ± 0.821**
CL	mg · min · mL · μg/kg	0.007 ± 0.001	0.005 ± 0.001*	0.010 ± 0.001*	0.007 ± 0.001

Notes:

* P<0.05;

** P<0.01.

Abbreviations: AUC, area under the concentration-time curve; NS, normal saline; GO-NLCs, nanostructured lipid carriers loaded with both oleanolic acid and gentiopicrin; CL, clearance; t 1/2 α, alpha elimination half-life; t 1/2 β, beta elimination half-life.

Figure 9 Plasma concentration-time curves for gentiopicrin and oleanolic acid after intravenous administration of gentiopicrin + normal saline, oleanolic acid + normal saline, and nanostructured lipid carriers loaded with both oleanolic acid and gentiopicrin.

Abbreviations: GEN, gentiopicrin; OA, oleanolic acid; GO-NLCs, nanostructured lipid carriers loaded with both oleanolic acid and gentiopicrin.

Figure 10 Aspartate and alanine aminotransferase levels in rats suffering acute hepatic damage in response to administration of carbon tetrachloride.

Abbreviations: GEN, gentiopicrin; OA, oleanolic acid; GO-NLCs, nanostructured lipid carriers loaded with both oleanolic acid and gentiopicrin; AST, aspartate aminotransferase; ALT, alanine aminotransferase.

Figure 11 Pathological observation of acute hepatic damage caused by carbon tetrachloride in the rat. (A) Control group, (B) carbon tetrachloride group, and (C) and group treated with nanostructured lipid carriers loaded with both oleanolic acid and gentiopicrin.