Effects of homogenization process parameters on physicochemical properties of astaxanthin nanodispersions prepared using a solvent-diffusion technique

Figures & data

Table 1 Central composite design and response variables for processing of astaxanthin nanodispersions

Sample number	Time (minutes)	Speed ×1,000 (rpm)	Particle size* (nm)	PDI*	Astaxanthin loss* (% w/w)
1	10.0	5.0	92.0±2.10g	0.275±0.0018e	35.3±0.77d
2	10.0	1.0	133.4±0.18c	0.217±0.0080g	15.3±2.60g
3	10.0	9.0	112.3±1.68e	0.507±0.0026b	58.9±3.30c
4	10.0	5.0	91.2±0.92g	0.276±0.0065e	35.0±0.02d
5	20.0	5.0	135.1±0.98b	0.467±0.0019c	65.8±1.90b
6	0.5	5.0	106.2±1.87f	0.434±0.0021d	8.9±0.00h
7	10.0	5.0	91.3±1.55g	0.276±0.0557e	35.7±0.45d
8	17.1	7.8	82.7±2.00i	0.602±0.0077a	70.2±0.01a
9	10.0	5.0	91.4±0.86g	0.275±0.0025e	35.5±0.44d
10	2.9	7.8	96.2±1.02h	0.434±0.0010d	28.4±0.02f
11	17.1	2.2	146.5±0.54a	0.240±0.0094f	29.8±1.00e
12	10.0	5.0	91.4±0.42g	0.276±0.0101e	35.8±0.80d
13	10.0	5.0	91.2±1.05g	0.276±0.0051e	35.1±0.12d
14	2.9	2.2	116.9±1.51d	0.239±0.0062f	7.3±1.08i

Notes:

* Experimental values of responses. Values are shown as the mean ± standard deviation (n=3).

a–i Different letters show statistically significant differences (P<0.05) between response values in which the comparison tests were performed between similar responses.

Abbreviation: PDI, polydispersity index.

Figure 1 Representative high-performance liquid chromatograms of dissolved astaxanthin in different concentrations (stock solutions) and extracted astaxanthin from suggested optimum astaxanthin nanodispersions (prepared using homogenizer for 7 minutes and 6,000 rpm).

Abbreviation: mAU, milli-absorbance units.

Table 2 Homogenization conditions and physicochemical characteristics of astaxanthin nanodispersions used for bioavailability studies

Sample name	Homogenization conditions			Particle size (nm)	PDI	Astaxanthin loss (% w/w)
	Time (minutes)	Speed ×1,000 (rpm)
A	10	6.6	Predicted	87.5	0.350	44.2
(Optimum based on minimum particle size, individual optimization)			Experimental	85.1±4.80c	0.382±0.0364a	47.5±5.20a
B	11.5	1	Predicted	142.7	0.172	16.2
(Optimum based on minimum PDI, individual optimization)			Experimental	145.8±5.98a	0.185±0.0259c	18.1±2.64c
C	0.5	1	Predicted	128.2	0.325	0.5d
(Optimum based on minimum astaxanthin loss, individual optimization)			Experimental	132.2±4.43b	0.341±0.0203ab	0.2±2.90d
D	7	6	Predicted	88.9	0.322	32.4
(Optimum based on minimum particle size, PDI and astaxanthin, multiple optimization)			Experimental	90.1±2.85c	0.315±0.0105b	30.4±5.95b

Notes: Experimental values are shown as the mean ± standard deviation (n=3).

a–d Different letters show statistically significant differences (P<0.05) between experimental response values in which the comparisons tests were performed between similar responses.

Abbreviation: PDI, polydispersity index.

Table 3 Regression coefficients, R², adjusted R², and probability values for the final reduced models

Regression coefficienta	Mean particle size	PDI	Astaxanthin concentration
b0	144.796	0.406913	−5.6278
b1	−1.236	−0.039935	1.3685
b2	−15.736	−0.022332	3.0433
b1^2	0.0245	0.001646	–
b2^2	1.609	0.004425	–
b12	−0.542	0.002100	0.2427
R^2	0.896	0.969	0.989
P-value (regression)	0.003	0.000	0.000

Notes:

a b₀ is a constant; b_i, b_ii and b_ij are the linear, quadratic, and interaction coefficients of the quadratic polynomial equation, respectively. 1, homogenization time; 2, homogenization speed.

Abbreviation: PDI, polydispersity index.

Table 4 Significance probability (P-value, F-ratio) of regression coefficients in final reduced second-order polynomial models

Main effects		Main effects		Quadratic effects		Interacted effects
		x1	x2	x11	x22	x1x2
Mean particle size (Y1, nm)	P-value	0.051a	0.014	0.009	0.006	0.046
	F-ratio	10.952	10.719	13.075	15.359	5.876
PDI (Y2)	P-value	0.000	0.209a	0.000	0.015	0.027
	F-ratio	42.224	1.915	52.345	10.304	7.823
Astaxanthin loss (Y3, g/100 g)	P-value	0.002	0.001	NS	NS	0.003
	F-ratio	1.677	18.063	0.000	0.000	16.492

Note:

a NS, not statistically significant at P>0.05; X₁, homogenization time; X₂, homogenization speed.

Abbreviation: PDI, polydispersity index.

Figure 2 Response surface plots for (A) mean particle size, (B) PDI, and (C) astaxanthin loss from astaxanthin nanodispersions as a function of significant (P<0.05) interaction effects between time and speed of homogenizer.

Abbreviation: PDI, polydispersity index.

Figure 3 Fitted line plots between the experimental and predicted values for (A) mean particle size, (B) PDI and (C) astaxanthin loss; dashed lines (- -) and dotted lines (….) show 95% CI and 95% PI, respectively.

Abbreviations: CI, confidence intervals; PDI, polydispersity index; PI, predicted intervals.

Figure 4 Transmission electron micrographs of astaxanthin nanodispersion prepared in optimum homogenization conditions (6,000 rpm and 7 minutes).

Figure 5 Cellular uptake of astaxanthin from individual and multiple suggested astaxanthin nanodispersions. (A) Optimum astaxanthin nanodispersions based on minimum particle size, individual optimized. (B) Optimum astaxanthin nanodispersion based on minimum polydispersity index, individual optimized. (C) Optimum astaxanthin nanodispersion based on minimum astaxanthin loss, individual optimized. (D) Optimum astaxanthin nanodispersions based on minimum particle size, polydispersity index, and astaxanthin loss, multiple optimized. Astaxanthin (bulk form) is astaxanthin powder dissolved in 0.5% tetrahydrofuran.

Notes: ^a–dDifferent letters show statistically significant differences (P<0.05) between experimental response values in which the comparisons tests were performed between similar responses.