Natural extracts into chitosan nanocarriers for rosmarinic acid drug delivery

Figures & data

Table 1. Zetasizer measurements and the association efficiency considering the different loading (%) of RA, sage, and savory nanoparticles (n = 3).

Size and surface charge					Association efficiency
Nano	Loading (%)	Size (nm)	Polydispersity	Zeta potential (mV)	Free amount concentration (µg/mL)	Total amount concentration (µg/mL)	Association efficiency (%)
RA	5	230.0 ± 48.8^a	0.155 ± 0.043	23.9 ± 5.5^b	56.6 ± 3.6	100	43.4 ± 3.5^c
	10	242.4 ± 112.3^a	0.215 ± 0.126	18.7 ± 2.3^b	94.8 ± 6.1	200	52.6 ± 3.1^c
	15	229.5 ± 40.1^a	0.142 ± 0.076	29.9 ± 9.9^b	147.9 ± 54.8	300	50.7 ± 18.3^c
	20	231.1 ± 68.3^a	0.149 ± 0.044	32.7 ± 6.4^b	186.4 ± 52.0	400	53.4 ± 2.0^c
	30	261.8 ± 50.4^a	0.171 ± 0.027	34.5 ± 4.5^b	306.0 ± 39.2	600	49.0 ± 6.5^c
	40	244.0 ± 18.0^a	0.193 ± 0.062	29.5 ± 1.6^b	390.4 ± 41.0	800	51.2 ± 3.0^c
	50	1218.0 ± 136.2^a	0.294 ± 0.132	35.0 ± 7.9^b	422.0 ± 65.0	1000	57.8 ± 1.3^c
Sage	5	330.9 ± 78.9^a	0.137 ± 0.012	20.8 ± 0.6^b	15.1 ± 0.4	100	84.9 ± 0.4^d
	10	224.3 ± 82.1^a	0.182 ± 0.061	24.3 ± 0.2^b	17.6 ± 0.6	200	91.2 ± 0.3^d
	15	200.0 ± 15.3^a	0.145 ± 0.018	21.4 ± 1.1^b	22.5 ± 1.1	300	92.5 ± 0.4^d
	20	243.0 ± 66.2^a	0.170 ± 0.123	21.7 ± 0.1^b	26.0 ± 4.0	400	93.5 ± 1.0^d
	30	271.5 ± 22.3^a	0.180 ± 0.056	27.8 ± 0.6^b	29.4 ± 3.0	600	95.1 ± 0.5^d
	40	272.9 ± 58.1^a	0.254 ± 0.099	25.1 ± 0.0^b	39.2 ± 2.1	800	95.1 ± 0.3^d
	50	278.4 ± 42.2	0.268 ± 0.092	24.9 ± 0.1^b	39.0 ± 2.6	1000	96.1 ± 0.2^d
Savory	5	233.1 ± 40.8^a	0.171 ± 0.083	23.8 ± 1.5^b	11.9 ± 0.0	100	88.1 ± 0.0^d
	10	230.7 ± 21.2^a	0.136 ± 0.094	27.8 ± 4.2^b	12.0 ± 0.1	200	94.0 ± 0.1^d
	15	182.7 ± 24.1^a	0.087 ± 0.075	28.0 ± 5.8^b	12.6 ± 0.1	300	95.8 ± 0.0^d
	20	220.8 ± 52.7^a	0.092 ± 0.024	26.7 ± 1.3^b	13.2 ± 0.2	400	96.7 ± 0.0^d
	30	188.0 ± 50.6^a	0.098 ± 0.006	28.9 ± 4.5^b	14.4 ± 0.6	600	97.6 ± 0.1^d
	40	139.4 ± 16.2^a	0.135 ± 0.088	28.4 ± 1.6^b	15.2 ± 0.4	800	98.1 ± 1.1^d
	50	229.3 ± 29.6^a	0.149 ± 0.121	26.5 ± 3.5^b	18.0 ± 0.2	1000	98.2 ± 0.1^d

^a,bThe results are given as mean of triplicate samples, each with six measurements. The same letters, in the same column indicate that no significant differences were observed between the values (p < 0.05).

^c,dThe results are given as mean of triplicate samples injections. The same letters, in the same column indicate that no significant differences were observed between the values (p < 0.05).

Figure 1. SEM micrographs of freeze-dried sage (a), savory-nanoparticles (b) both at 50% theoretical loading; and RA-nanoparticles (c), at 40% theoretical loading.

Table 2. Association efficiency, theoretical loading, and final RA content in CS nanoparticles.

Nano	Association efficiency (%)	Theoretical loading (%)	Final content in the CS nanoparticles (µg/mL)	Final RA content in CS nanoparticles (mg/mL)
RA	51.2 ± 3.0	40	800	400
Sage	96.1 ± 0.2	50	1000	100
Savory	98.2 ± 0.1	50	1000	50

Table 3. Peak temperatures in the DSC thermograms collected from CS, RA, sage and savory, physical mixtures, and nanoparticles.

	T (°C)
	Onset	Peak	EndSet
Nanos
CS	102 ± 0.55	122 ± 1.20	131 ± 0.12
RA	112 ± 0.61	130 ± 0.73	141 ± 0.24
Sage	83 ± 0.27	108 ± 0.71	118 ± 0.93
Savory	103 ± 1.11	124 ± 0.82	137 ± 0.44
Free
RA	134 ± 0.13	144 ± 0.23	149 ± 0.25
Sage	140 ± 0.21	141 ± 0.14	145 ± 1.03
Savory	141 ± 0.53	154 ± 0.51	158 ± 0.81
Physical mixture
CS-RA	90 ± 0.38	122 ± 0.83	136 ± 0.14
	147 ± 0.14	150 ± 0.70	154 ± 0.13
CS-sage	94 ± 0.91	121 ± 0.10	134 ± 0.50
	147 ± 0.67	151 ± 0.63	155 ± 0.84
CS-savory	93 ± 0.70	121 ± 0.32	132 ± 0.32
	145 ± 0.28	151 ± 0.64	154 ± 0.24

Figure 2. I. Thermogram of CS empty nanoparticles (a); thermogram of free RA (b); thermogram of RA and CS physical mixture (mixing ratio 1:1) (c); thermogram of RA encapsulated in CS nanoparticles (at a theoretical 40% loading) (d). II. Thermogram of CS empty nanoparticles (a); thermogram of free sage (b); thermogram of sage and CS physical mixture (mixing ratio 1:1) (c); thermogram of sage encapsulated in CS nanoparticles (at a theoretical 50% loading) (d). III. Thermogram of CS empty nanoparticles (a); thermogram of free savouy (b); thermogram of savory and CS physical mixture (mixing ratio 1:1) (c); thermogram of savory encapsulated in CS nanoparticles (at a theoretical 50% loading) (d).

Figure 3. I. Spectrum of CS empty nanoparticles (a); spectrum of RA in a free form (b); spectrum of physical mixture between CS-unloaded nanoparticles and RA (mixing ratio 1:1) (c); spectrum of RA encapsulation into CS nanoparticles (d). II. Spectrum of CS empty nanoparticles (a); spectrum of sage in a free form (b); spectrum of physical mixture between CS unloaded nanoparticles and sage (mixing ratio 1:1) (c); spectrum of sage encapsulation into CS nanoparticles (d). III. Spectrum of CS empty nanoparticles (a); spectrum of savory in a free form (b); spectrum of physical mixture between CS-unloaded nanoparticles and savory (mixing ratio 1:1) (c); spectrum of savory encapsulation into CS nanoparticles (d).

Table 4. Antioxidant activity measurements by ABTS and ORAC, considering the 50% loading (m/m) sage and savory nanoparticles; and 40% loading (m/m) of and RA nanoparticles for (n = 3).

	ABTS^x (eq [Asc. Ac.]g/L)/g extract		ORAC^y (µmol/eq Trolox)/g extract
	Fresh nanoparticles	Freeze-dried nanoparticles	Fresh nanoparticles	Freeze-dried nanoparticles
Nano^I
RA	0.0348 ± 0.0050^a	0.0554 ± 0.0139^a	4.8374 ± 0.1719^b	3.6520 ± 0.1770^b
Sage	0.0537 ± 0.0015^c	0.0440 ± 0.0029^c	0.6227 ± 0.0901^d	0.4251 ± 0.0069^d
Savory	0.0828 ± 0.0102^e	0.0378 ± 0.0015^f	1.5315 ± 0.2784^g	0.4526 ± 0.0087^h
Free^II
RA	0.0917 ± 0.0018		34.1218 ± 2.5733
Sage	0.1621 ± 0.0470		19.5924 ± 1.9791
Savory	0.1410 ± 0.0087		16.8117 ± 1.3605

The results are given as mean of triplicate samples, each with three measurements. The same letters, in the same line indicate that no significant differences were observed between the fresh and freeze-dried process (p < 0.05). The values are significantly different (p < 0.05) for the antioxidant methods (^{x, y}) and for the encapsulation process (^{I, II}).