Nanoemulsion as a strategy for improving the oral bioavailability and anti-inflammatory activity of andrographolide

Figures & data

Figure 1 Chemical structure of andrographolide.

Figure 2 Solubility tests of andrographolide in different vehicles. Results were expressed as mean ± standard deviation (n=3). *Significantly different compared with the water group at p<0.05.

Table 1 Composition and characterization of various AG-NE formulations

Excipients (g)	AG-NE composition
	F1	F2	F3	F4	F5	F6
Andrographolide	0.03	0.03	0.03	0.03	0.03	0.03
α-tocopherol	2	2	–	–	–	–
Triacetin	–	–	2	2	–	–
Limonene	–	–	–	–	2	2
Ethanol	2	2	2	2	2	2
Tween 20	2	–	2	–	2	–
Cremophor EL^®	–	2	–	2	–	2
Water	6	6	6	6	6	6
Droplet size (nm)	2,298±183	122±11	1,220±325	6,600±523	533±51	140±53
Viscosity (cps)	472	28	43	42	25.2	20

Notes: Droplet size expressed as mean ± standard deviation (n=3 for each formulation). The – indicates absence of the excipient.

Abbreviations: AG-NE, andrographolide-loaded nanoemulsion; cps, centipoise.

Figure 3 (A) Size distribution of the optimized andrographolide-loaded nanoemulsion (AG-NE) droplets determined by photon correlation spectroscopy. (B) Transmission electron microscopy image of the AG-NE.

Table 2 AG content, AG remained, and droplet size of the optimized AG-NE (F2) stored at 4°C and 25°C over a period of 90 days

Parameters	Test period (d)
	0	7	14	30	60	90
4°C
AG content (mg/mL)	3.95±0.05	3.93±0.12	3.92±0.15	3.89±0.11	3.88±0.16	3.87±0.21
AG remained (%)	100.0	99.5	99.2	98.5	98.2	98.0
Droplet size (nm)	122±13	122±7	123±11	121±15	123±12	124±9
25°C
AG content (mg/mL)	3.95±0.05	3.94±0.08	3.94±0.12	3.87±0.09	3.83±0.16	3.80±0.18
AG remained (%)	100.0	99.8	99.8	97.2	97.0	96.3
Droplet size (nm)	122±13	127±16	129±17	132±14	132±16	132±16

Note: AG content and droplet size expressed as mean ± standard deviation (n=3 for each test time).

Abbreviations: AG, andrographolide; AG-NE, andrographolide-loaded nanoemulsion; d, days.

Figure 4 High-performance liquid chromatograms of (A) blank rat plasma, (B) standard andrographolide solution (0.5 μg/mL) and internal standard (ISTD) solution spiked with blank rat plasma, and (C) rat plasma sample at 5 min after oral administration of 100 mg/kg andrographolide-loaded nanoemulsion. Peak identification: (1): 10 μg/mL ISTD and (2): andrographolide.

Figure 5 Concentration of andrographolide (AG) in AG suspension, AG ethanol solution, and the optimized andrographolide-loaded nanoemulsion (AG-NE) groups of different small intestine regions after incubation in AG solution (50 μg/mL) at 37°C for 1 h. Results were expressed as mean ± standard deviation (n=3). Different letters (a–d) indicate a significant difference at p<0.05.

Table 3 Pharmacokinetic parameters of AG after intravenous administration of AG solution and after oral administration of AG suspension and the optimized AG-NE in male rats

Parameters	AG solution (5 mg/kg, IV)	AG suspension (300 mg/kg, PO)	AG-NE (100 mg/kg, PO)
Tmax (h)	–	0.75±0.18	0.08
C0 or Cmax (μg/mL)	62.68±1.09	0.73±0.08	3.78±0.88*
t1/2 (h)	1.53±0.77	1.22±0.18	2.28±0.58*
AUC0–t (h μg/mL)	9.32±1.73	1.66±0.21	3.21±0.26*
AUC0–∞ (h μg/mL)	10.00±1.68	1.87±0.17	3.70±0.24*
Relative bioavailability (%)	–	–	594.28±38.59

Notes: Data presented as mean ± standard deviation (n=6 for each group).

* Significantly different compared between AG-NE and AG suspension groups (p<0.05). T_max, time of occurrence for maximum AG concentration; C₀/C_max, maximum concentration of AG; t_1/2, half-life; AUC_0–t, area under the plasma concentration-time curve from zero hours to time; AUC_0–∞, area under the plasma concentration-time curve from zero hours to infinity.

Abbreviations: AG, andrographolide; AG-NE, andrographolide-loaded nanoemulsion; IV, intravenous injection; PO, oral administration.

Figure 6 The plasma concentrations–time profiles of andrographolide (AG) after drug administration of AG solution (5 mg/kg, intravenous [IV] injection), AG suspension (300 mg/kg, PO), and the optimized andrographolide-loaded nanoemulsion (AG-NE) (100 mg/kg, PO) in rats. Results expressed as mean ± standard deviation (n=6).

Abbreviation: PO, oral administration.

Figure 7 Effects of pre-treatment with blank suspension, andrographolide (AG) suspension, blank nanoemulsion (NE), and the optimized andrographolide-loaded nanoemulsion (AG-NE) on (A) ulcer index in the intestinal mucosa and (B) histological damage score of mice on its indomethacin-induced intestinal lesions. Group I: sham group; Others were pretreated with formulation by oral administration 1 h before treatment with indomethacin as follows: Group II was pretreated with blank suspension; group III was pretreated with AG suspension (300 mg/kg); group IV was pretreated with blank NE; and group V was pretreated with AG-NE (100 mg/kg). Results were expressed as mean ± standard deviation (n=4). Different letters (a–d) indicate a significant difference at p<0.05.

Figure 8 Representative images of small intestines. (A) The sham group and effect of pretreatment with (B) blank suspension, (C) andrographolide suspension, (D) blank nanoemulsion, and (E) the optimized andrographolide-loaded nanoemulsion by oral administration 1 h before treatment with indomethacin on histological appearance of indomethacin-induced small intestine lesions in mice (original magnification ×200).