High-Payload Nanosuspension of Centella asiatica Extract for Improved Skin Delivery with No Irritation

Figures & data

Figure 1 Chemical structure of (A) AS, (B) AA, and (C) MA.

Abbreviations: AS, asiaticoside; AA, asiatic acid; MA, madecassic acid.

Table 1 Effect of Suspending Agent on the Particle Size, and Size Homogeneity of CA-Loaded NSs

Stabilizers^a	Immediately After Preparation		After Centrifugation at 13,000 rpm for 10 min
	Particle Size (nm)^b	Homogeneity (PDI)^b,c	Particle Size (nm)^b	Homogeneity (PDI)^b,c
Polymers
PVP K30	201.4 ± 5.0	0.24 ± 0.01	219.7 ± 12.6	0.29 ± 0.03
Carbopol 934NF	177.7 ± 0.5	0.21 ± 0.02	234.0 ± 1.1	0.25 ± 0.01
Na. CMC	347.3 ± 4.9	0.20 ± 0.01	361.8 ± 3.9	0.16 ± 0.02
Methylcellulose	271.3 ± 3.6	0.29 ± 0.00	378.2 ± 4.0	0.32 ± 0.01
HPMC E50LV	144.6 ± 5.8	0.28 ± 0.01	318.3 ± 1.3	0.26 ± 0.01
Kollidon VA64	224.2 ± 2.0	0.20 ± 0.00	255.3 ± 2.0	0.24 ± 0.01
Surfactants
Tyloxapol	326.1 ± 5.9	0.35 ± 0.05	318.2 ± 3.7	0.36 ± 0.02
Kolliphor HS15	217.7 ± 4.0	0.24 ± 0.02	215.7 ± 7.5	0.28 ± 0.01
Poloxamer 188	321.4 ± 11.7	0.35 ± 0.04	548.5 ± 19.2	0.51 ± 0.02
Kolliphor EL	275.8 ± 2.8	0.26 ± 0.04	268.0 ± 3.6	0.29 ± 0.03
Lecithin	260.5 ± 4.0	0.33 ± 0.04	346.2 ± 2.3	0.31 ± 0.00
PEG 40 stearate	273.7 ± 1.6	0.26 ± 0.01	399.3 ± 2.4	0.43 ± 0.01
Polysorbate 20	281.3 ± 1.8	0.29 ± 0.05	415.3 ± 5.8	0.41 ± 0.04
Polysorbate 80	278.5 ± 2.0	0.29 ± 0.03	455.3 ± 28.6	0.49 ± 0.09
Kolliphor RH 40	308.2 ± 3.2	0.30 ± 0.05	430.6 ± 12.6	0.38 ± 0.04

Notes: ^aThe concentration of CA and stabilizer in the NS formulas were set to 100 mg/mL and 5 mg/mL, respectively, with milling speed of 1500 rpm for 4 h. All samples were uniform after preparation, and even after centrifugation at 13,000 rpm for 10 min; ^bData are expressed as mean ±SD (n=3); ^cCalculated by dividing weight average molecular weight by number average molecular weight.

Abbreviations: CA, Centella asiatica; NS, nanocrystal suspensions.

Figure 2 Effect of formulation variables on the median diameter and homogeneity of nanocrystals fabricated using bead-milling technology. (A) Effect of PVP K30 concentration and milling speed on the crystal size and homogeneity; (B) Effect of CA concentration and aqueous vehicle (DW and BG 50 v/v% solution) on the crystal size and homogeneity.

Notes: (A) CA concentration and milling time were fixed to 100 mg/mL and 4 h, respectively. (B) PVP K30 concentration, milling speed, and milling time were fixed to 5 mg/mL, 1800 rpm, and 4 h, respectively. Three batches of each sample were prepared, and the data are presented as mean ± SD (n=3).

Abbreviations: CA, Centella asiatica; DW, distilled water; BG, butylene glycol.

Table 2 Inter-Day Validation of HPLC Methods for AS, AA, and MA

	AS	AA	MA
Accuracy (%)^a
5 µg/mL	101.4	N.D.^b	103.5
10 µg/mL	97.5	98.4	103.3
25 µg/mL	101.6	100.5	102.8
100 µg/mL	99.6	99.7	100.3
Precision (%)^c
5 µg/mL	2.25	N.D.	1.62
10 µg/mL	3.90	3.70	2.83
25 µg/mL	2.00	1.58	2.00
100 µg/mL	2.37	2.46	2.74
LOD (µg/mL)	1.32	1.90	1.90
LOQ (µg/mL)	4.01	5.75	5.75

Notes: ^aCalculated using the following formula: concentration/nominal concentration × 100; ^bnot determined; ^cstandard deviation of calculated concentration/average of calculated concentration) × 100.

Abbreviations: AS, asiaticoside; AA, asiatic acid; MA, madecassic acid; LOD, limit of detection; LOQ, limit of quantification.

Table 3 Physicochemical Characteristics of the CA-Loaded NS Formulations and Marketed Product (Madeca Cream)

	1%NS	5%NS	10%NS-1	10%NS-2	Madeca Cream^b
Compositions
CA (mg)	10.0	50.0	100.0	100.0
PVP K30 (mg)	5.0	5.0	5.0	5.0
BG (mL)	-	-	-	0.5
DW (mL)	1.0	1.0	1.0	0.5
Physicochemical characteristics
AS content (mg/mL)^a	3.49 ± 0.03	17.19 ± 0.48	33.75 ± 0.12	29.90 ± 0.66	2.77 ± 0.04
MA content (mg/mL)^a	3.25 ± 0.02	15.44 ± 0.67	30.37 ± 1.16	26.90 ± 0.04	3.03 ± 0.20
AA content (mg/mL)^a	2.49 ± 0.01	10.23 ± 0.32	18.79 ± 0.74	15.43 ± 0.08	1.72 ± 0.21
Particle size (nm)^a	493.5 ± 0.8	268.4 ± 2.1	201.4 ± 5.0	379.4 ± 6.3	–^c
Homogeneity (PDI)^a	0.17 ± 0.01	0.24 ± 0.00	0.24 ± 0.01	0.20 ± 0.30	–^c
pH^a	5.72 ± 0.07	5.40 ± 0.01	5.32 ± 0.05	5.65 ± 0.01	–^c

Notes: ^aData are expressed as mean ± SD (n=3); ^bThe exact composition of Madeca cream is unknown, and only the content of AS, MA, and AA contained in cream formula was evaluated; ^cNot determined.

Abbreviations: CA, Centella asiatica; BG, butylene glycol; NS, nanocrystal suspensions; AS, asiaticoside; AA, asiatic acid; MA, madecassic acid.

Figure 3 Morphological and physical characteristics of CA-loaded NS. Representative microscopic images of (A) CA raw material, and (B) high-payload NS (10%NS-1). (C) DSC curves of (a) raw material, (b) CA-free vehicle, (c) 10%NS-1, (d) 10%NS-2, (e) MA, (f) AS, and (g) AA. (D) XRD patterns of (a) raw material, (b) CA-free vehicle, (c) 10%NS-1, and (d) 10%NS-2.

Abbreviations: CA, Centella asiatica; NS, nanocrystal suspensions; DSC, differential scanning calorimeter; MA, madecassic acid; AS, asiaticoside; AA, asiatic acid; XRD, X-ray diffractometer.

Figure 4 In vitro dissolution profile of (A) AS, (B) AA, and (C) MA from CA-loaded NSs, raw material, and the marketed cream formula (Madeca cream) under sink condition.

Notes: Sink condition was provided by adding 1% (w/v) of SLS into 10 mM phosphate-buffered saline (pH 6.8). Data are expressed as mean ± SD (n=3).

Abbreviations: AS, asiaticoside; AA, asiatic acid; MA, madecassic acid; CA, Centella asiatica; NS, nanocrystal suspensions; SLS, sodium lauryl sulfate.

Figure 5 Accumulation of (A) AS, (B) AA, and (C) MA in porcine dorsal skin after 24 h post-application of CA-loaded NSs (1%NS, 5%NS, 10%NS-1, and 10%NS-2) or the marketed cream formula in a Franz diffusion cell model.

Notes: Data are expressed as mean ± SD (n = 4). Statistical analysis was conducted using one-way ANOVA test. *p < 0.05, compared to Madeca cream; **p < 0.05, compared to 1%NS; ***p < 0.05, compared to 5%NS.

Abbreviations: AS, asiaticoside; AA, asiatic acid; MA, madecassic acid; CA, Centella asiatica; NS, nanocrystal suspensions.

Table 4 Scoring of Severity of Erythema or Oedema After Topical Daily Application of Vehicle, High-Payload NSs (10%NS-1 and 10%NS-2), or the Marketed Cream Onto the Dorsal Legion of Normal Rats (n = 5 per Each Group)

	Vehicle	10%NS-1	10%NS-2	Madeca Cream
Erythema
Day 1	0	0	0	0
Day 2	0	0	0	0
Day 3	0	0	0	0
Day 4	0	0	0	0
Day 5	0	0	0	0
Oedema
Day 1	0	0	0	0
Day 2	0	0	0	0
Day 3	0	0	0	0
Day 4	0	0	0	0
Day 5	0	0	0	0

Note: Data are expressed as the sum of individual scores.

Abbreviation: NS, nanocrystal suspensions.

Figure 6 Representative images of dorsal skin of normal rats after daily application of CA-free vehicle, high-payload NSs (10%NS-1 or 10%NS-2), or marketed cream for 5 days.

Abbreviations: CA, Centella asiatica; NS, nanocrystal suspensions.