Silymarin nanoemulsion against human hepatocellular carcinoma: development and optimization

Figures & data

Table 1. Treatment schedule of silymarin formulations in male Wistar rats.

Groups	Drugs	Treatment schedule
I	Standard suspension	Silymarin 42 mg/kg p.o. single dose given to male Wistar rats
II	Conventional suspension	Conventional formulation of silymarin 35 mg/kg p.o. single dose given to male Wistar rats
III	Silymarin nanoformulation	Optimized nanoemulsion of silymarin 20 mg/kg p.o. single dose given to male Wistar rats

Figure 1. Silymarin showing maximum solubility in sefsol 218.

Figure 2. Pseudoternary phase diagrams of silymarin nanoemulsions, F1 (S_mix 1:0), F2 (S_mix 1:1), F3 (S_mix 2:1), and F4 (S_mix 1:2).

Table 2. Thermodynamic stability tests of prepared nanoformulation.

					Thermodynamic stability tests			Dispersibility
Formulation	Smix ratio	%Oil	%Smix	%Water	Heating/cooling	Centrifugation	Freeze–Thaw	0.1 N HCl	H2O	Inference
NE 1	1:0	1.87	13.12	85	X	–	–	–	–	FAILED
NE 2		5	45	50	✓	✓	✓	C	C	FAILED
NE 3		10	30	60	✓	✓	✓	A	A	PASSED
NE 4	1:1	3	23	74	✓	✓	✓	A	A	PASSED
NE 5		7.02	28.07	64.91	✓	✓	✓	C	C	FAILED
NE 6		11.24	33.71	55.06	✓	✓	✓	B	B	FAILED
NE 7		14.93	44.78	40.30	✓	✓	X	–	–	FAILED
NE 8		18.18	36.36	45.45	✓	✓	X	–	–	FAILED
NE 9	2:1	5.80	28.99	65.22	✓	✓	✓	A	A	PASSED
NE 10		9.09	45.45	45.45	✓	✓	✓	B	B	FAILED
NE 11		13.33	66.67	20	✓	✓	✓	C	C	FAILED
NE 12		16.67	66.67	16.67	X	–	–	–	–	FAILED
NE 13		20.69	48.28	31.03	✓	✓	X	–	–	FAILED
NE 14	1:2	2.50	17.50	80	✓	✓	✓	A	A	PASSED
NE 15		6	14	80	✓	✓	✓	C	C	FAILED
NE 16		10.53	24.56	64.91	✓	✓	✓	B	B	FAILED
NE 17		15	35	50	X	–	–	–	–	FAILED

Figure 3. Silymarin-loaded nanoemulsion: (A) droplet size, (B) transmission electron microscopy, and (C) zeta potential.

Figure 4. Drug release study of silymarin nanoemulsion and its comparison with marketed suspension.

Table 3. Droplet size, poydispersity index, zeta potential, viscosity, refractive index, conductivity, and percent transmittance of selected silymarin nanoemusion formulations.

Formulation	Droplet size ± SD (nm)	Polydispersity index	Zeta potential (mV)	Viscosity ± SD (cps)	Refractive index ± SD	Conductivity ± SD (μS/cm)	Percent transmittance
NE 3	29.34 ± 0.634	0.191 ± 0.008	−29.3 ± 0.345	12.34 ± 0.927	1.369 ± 0.0259	430.340 ± 1.873	98.15 ± 0.91
NE 4	79.02 ± 1.651	0.449 ± 0.022	−12.1 ± 0.131	11.83 ± 0.924	1.320 ± 0.0729	461.579 ± 1.214	98.47 ± 1.07
NE 9	21.24 ± 0.291	0.104 ± 0.016	−17.9 ± 0.527	9.59 ± 0.764	1.316 ± 0.0151	480.432 ± 1.837	99.52 ± 0.54
NE 14	31.31 ± 0.298	0.168 ± 0.022	−22.6 ± 0.751	10.9 ± 3.165	1.360 ± 0.0062	455.019 ± 2.045	99.02 ± 0.06

All data expressed as mean ± SD (n = 3).

Table 4. Pharmacokinetic parameters obtained after oral administration of silymarin nanoemulsion and its comparison with standard and conventional suspensions in male Wistar rats (n = 6).

Formulation	Cmax^a (μg/ml)	Tmax^b (h)	AUC 0 → all (μg h/ml)^c
Standard suspension	5.24 ± 4.31	3.0 ± 0.31	17.82 ± 7.32
Conventional suspension	10.36 ± 1.06^#	2.0 ± 0.62^#	37.43 ± 2.89^#
Silymarin nanoemulsion (NE9)	38.62 ± 2.43^##^,*	0.5 ± 0.45^##^,*	308.51 ± 4.23^##^,**

^a Peak of maximum concentration.

^b Time of peak concentration.

^c Area-under-the-concentration–time profile curve.

Mean ± SD (n = 3).

## p < .01 and #p < .05 considered significant, when compare with standard suspension and conventional suspension.

*p < .05 and **p < .01 considered significant, when compared with conventional suspension.

Figure 5. Plasma concentration of various silymarin formulations in male Wistar rats after oral administration.

Figure 6. (A and B) In vitro cytotoxicity test. (C and D) Intracellular reactive oxygen species generation. (E and F) Chromatin condensation of optimized silymarin nanoemulsion.

Figure 7. Effects of silymarin nanoemulsion on Chang liver cells (non-tumor cells).