Nanosuspensions as carriers of Annona muricata acetogenins: antibacterial activity against Enterococcus faecalis and listeria monocytogenes

Figures & data

Table 1. The Box-Behnken experimental design to formulate nanosuspensions (NSps) as carriers of acetogenins (βCDSL-ACGs-NSps) with the testing and predicted response variables.

Treatment	NSps preparation conditions			Particle size (nm)		Polydispersity index
	XSL (mg/mL)	XCD (g/100 g)	XPM (s)	^1Experimental	^2Predicted	^1Experimental	^2Predicted
1	15	0.05	MA	234.80 ± 1.50^j	234.74	0.22 ± 0.002^h	0.21 ± 0.0014^i
2	5	0.1	LA	188.30 ± 0.30^g	188.32	0.24 ± 0.006^f	0.24 ± 0.0006^f
3	10	0.05	LA	272.36 ± 0.66^k	272.48	0.27 ± 0.005^e	0.27 ± 0.0010^d
4	10	0.05	US	145.92 ± 0.55^a	145.98	0.24 ± 0.005^f	0.24 ± 0.0010^f
5	10	0.2	LA	153.91 ± 0.75^c	153.61	0.36 ± 0.005^c	0.36 ± 0.0006^a
6	15	0.1	US	172.85 ± 0.55^f	172.71	0.24 ± 0.009^f	0.24 ± 0.0010^f
7	10	0.1	MA	156.50 ± 0.40^d	156.49	0.40 ± 0.006^b	0.30 ± 0.0006^bc
8	5	0.05	MA	201.04 ± 0.77^h	201.21	0.24 ± 0.003^f	0.24 ± 0.0010^f
9	15	0.2	MA	201.20 ± 0.50^h	201.21	0.15 ± 0.007^j	0.15 ± 0.0006^k
10	15	0.1	LA	148.43 ± 1.47^b	148.37	0.30 ± 0.007^d	0.30 ± 0.0006^bc
11	10	0.2	US	144.67 ± 2.10^a	144.72	0.24 ± 0.007^fg	0.24 ± 0.0006^g
12	5	0.2	MA	210.35 ± 0.35^i	210.53	0.20 ± 0.004^i	0.20 ± 0.0010^j
13	10	0.1	MA	203.81 ± 2.66^h	203.66	0.43 ± 0.002^a	0.30 ± 0.0006^bc
14	10	0.1	MA	203.50 ± 2.60^h	203.66	0.20 ± 0.004^i	0.30 ± 0.0006^c
15	5	0.1	US	161.79 ± 1.80^e	161.81	0.23 ± 0.005^g	0.23 ± 0.0006^h

X_SL, soy lecithin; X_CD, hydroxypropyl-beta-cyclodextrin; X_PM, dispersion method. Data are expressed as mean ± SD (n = 3). Different lowercase letters indicate significant statistical differences between treatments (α = 0.05). LA, low agitation (500 rpm); MA, middle agitation (10,000 rpm); US, ultrasound (50 W, 40 kHz). ¹Particle size was obtained from the mean of 20 measurements in triplicate and expressed in nm. ²Values were predicted by a second-degree polynomial equation, R² = 0.90.

Table 2. Analysis of variance and regression coefficients of polynomial quadratic models predicted with the formulation conditions of acetogenin-loaded nanosuspensions on particle size.

Source	Analysis of variance				Regression coefficients
	^1SS	^2DF	^3MS	F Value	Acetogenins content β-coefficient
Mean/	-	-	-	-	1.50189^+
XSL	0.013972	1	0.013972	0.12497^+	0.20967^+
XSL^2	0.042655	1	0.042655	22.46787^++	−0.00990^+
XCD	0.024711	1	0.024711	34.8151^+	2.37065^++
XCD^2	0.030751	1	0.030751	43.32587^+	−6.075351^++
XPM	0.003831	1	0.003831	5.3976^++	0.37706^+
XPM^2	0.000910	1	0.000910	1.28258^+	−0.05991^+
XSL* XCD	0.019419	4	0.004855	6.8400^+	−0.99307^+
XSL* XCD^2	0.077372	1	0.046466	8.7732^+	1.54104^+
XSL^2* XCD	0.139790	1	0.139790	1.1972^+	0.02749^+
XSL* XPM	0.120555	3	0.040185	0.97938^+	−0.06949^+
XSL^2* XPM	0.039816	1	0.039816	2.3594^+	0.00381^+
XCD*XPM	0.077493	2	0.038747	54.5904^+	0.81586^+
Lack of Fit	0.003831	1	0.003831	5.3976^++
Pure Error	0.044006	62	0.000710
R-square	0.90
R-adjust	0.89
Total SS	0.62514

X_SL, soybean lecithin; X_CD, hydroxypropyl-β-cyclodextrin; X_PM, dispersion method; R², regression coefficient; ¹Sum of the square. ² Degree of freedom. ³Means square. ⁺Significant (α < 0.01). ⁺⁺Non significant (α > 0.01).

Figure 1. Response surface plots of the particle size from nanosuspension loaded with acetogenins obtained with low agitation (500 rpm) (a), High agitation (1000 rpm) (b), ultrasound, (c) and pareto plot (d). X_CD, hydroxypropyl-β-cyclodextrin (%); X_SL, soy lecithin (mg/mL); X_PM, dispersion method.

Table 3. Optimal experimental conditions to formulate nanosuspensions as carriers of acetogenins (βCDSL-ACGs-NSps) and experimental validation.

Independent variables	Optimal conditions
Soy lecithin (mg/g)	10
Hydroxypropyl-β-cyclodextrin (g/100 g)	.1625
Preparation method	Ultrasound
Optimal response (nm)	135.67
−95% Confidence limit	15.22
+95% Confidence limit	123.67
Experimental validation	Response variables
Particle size (nm)	133.37 ± 4.62
Polydispersity index	.20 ± .02
Z Potential (mV)	−37 ± .02
Entrapment efficiency (%)	85.33 ± 4.03
Encapsulation efficiency (%)	56.09 ± .47

−95% confidence limits, lower limit; +95% confidence limit, upper limit; the confidence interval is the difference between the upper and lower limits. All values are the mean ± SD of three determinations *p < .05.

Figure 2. Fourier-transform infrared spectroscopy (a), X-ray diffraction (b), and ultraviolet-visible diffuse reflectance spectroscopy (c) of isolated acetogenins (ACGs) and the optimized nanosuspension loaded with acetogenins (OβCDSL-ACGs-NSps) optimized nanosuspension loaded with acetogenins. βCD-SL complex, hydroxypropyl-β-cyclodextrin complexed with soy lecithin.

Figure 3. Particle size distribution of the optimized nanosuspension loaded with acetogenins (OβCD-ACGs-NSps)(a), particle morphology by atomic force microscopy of OβCD-ACGs-NSps (b), and 3D micrograph of OβCD-ACGs-NSps (c).

Figure 4. Representative HPLC-chromatogram of acetogenins extracted from optimized nanosuspension (OβCD-ACGs-NSps).

Figure 5. Cumulative release of ACGs from the optimized nanosuspension (OβCD-ACGs-NSps) and prediction of the overall release behavior according to the Korsmeyer-Peppas model (a), bacterial inhibition of E. faecalis (b) and L. monocytogenes (c) of OβCD-ACGs-NSps. k, release constant; n, release exponent; R², coefficient of determination; DMSO, Dimethylsulfoxide; βCD-SL, hydroxypropyl-β-cyclodextrin complexed with soy lecithin; antibiotic, trimethoprim with sulfamethoxazole (3200 μg/mL).

Table 4. Acgs release kinetics for zero-order, Higuchi and Korsmeyer-Peppas model and n-value.

Model	Zero-order	Higuchi equation	Korsmeyer-Peppas model
Equation	Mt = kt	Mt = k × t^0.5	Mt = kt^n
R^2	−0.14	0.59	.90
Adjusted R^2	−0.14	0.59	.89
n value	–	–	.20

M_t in all equations is the concentration of ACGs released at time t. All values are the mean ± SD of three determinations.