In vitro and in vivo skin anti-aging evaluation of gel containing niosomes loaded with a semi-purified fraction containing gallic acid from Terminalia chebula galls

Figures & data

Table 1.  Descriptions and compositions of the gel samples for rabbit skin irritation test and human skin anti-aging evaluation.

Sample formulation code	Sample descriptions	Compositions
Gel base	Gel without niosomes and the active compounds	Gel base composition (Carbopol^® 980, propylene glycol, triethanolamine, propyl paraben, methyl paraben)
BN	Gel incorporated with blank non-elastic niosomes	Gel base incorporated with 87% non-elastic niosomes
BE	Gel incorporated with blank elastic niosomes	Gel base incorporated with 87% elastic niosomes
GS	Gel incorporated with gallic acid	Gel base containing 0.5% Gallic acid in gel
GN	Gel incorporated with non-elastic niosomes loaded with gallic acid	Gel base containing 87% Non-elastic niosomes loaded with 0.5% gallic acid
GE	Gel incorporated with elastic niosomes loaded with gallic acid	Gel base containing 87% Elastic niosomes loaded with 0.5% gallic acid
SS	Gel incorporated with the semi-purified extracts containing gallic acid	Gel base containing 0.5% Semi-purified extracts containing 0.08% gallic acid in gel
SN	Gel incorporated with non-elastic niosomes loaded with the semi-purified extracts containing gallic acid	Gel base containing 87% Non-elastic niosomes loaded with 0.5% semi-purified extracts containing 0.08% gallic acid
SE	Gel incorporated with elastic niosomes loaded with the semi-purified extracts containing gallic acid	Gel base containing 87% Elastic niosomes loaded with 0.5% semi-purified extracts containing 0.08% gallic acid
Commercial product	Gel incorporated with magnesium ascorbyl phosphate	Gel base containing 6% Magnesium ascorbyl phosphate, panthenol, potassium hydroxide, propyl paraben, methyl paraben

Table 2.  DPPH radical scavenging activity of gallic acid and the semi-purified fraction of T. chebula gall extract loaded in non-elastic and elastic niosomes.

Samples	SC50 (µg/mL)
Blank non-elastic niosomes	—
Blank elastic niosomes	—
Gallic acid solution	8.84
Gallic acid loaded in non-elastic niosomes	—
Gallic acid loaded in elastic niosomes	—
Semi-purified fraction solution	6.29
Semi-purified fraction loaded in non-elastic niosomes	—
Semi-purified fraction loaded in elastic niosomes	—
Ascorbic acid	4.31
α-tocopherol	5.15
BHT	3.90

—, SC₅₀ more than 100 µg/mL.

Figure 1.  Effects of gallic acid at 50 µg/mL and the semi-purified fraction (at 50 µg/mL of gallic acid) of T. chebula gall extract loaded in non-elastic and elastic niosomes on human skin fibroblast viability at 48 h incubation. (BN, blank non-elastic niosomes; BE, blank elastic niosomes; GS, gallic acid solution; GN, non-elastic niosomes loaded with gallic acid; GE, elastic niosomes loaded with gallic acid; SS, the semi-purified fraction; SN, non-elastic niosomes loaded with the semi-purified fraction; SE, elastic niosomes loaded with the semi-purified fraction.)

Figure 2.  Effects of the gelatinolytic activity on MMP-2 expression of the semi-purified fraction, gallic acid and the standard ascorbic acid at 1–50 µg/mL: (A) zymograms; (B) % relative MMP-2 expression calculated from the following equation: relative MMP-2 expression (%) = (% MMP-2 expression of the sample / % MMP-2 expression of the control) × 100. Each value is expressed as mean ± SD. Kruskall–Wallis test was used to calculate the significant differences. *p < 0.05 and **p < 0.01 compared to each control (the untreated system).

Figure 3.  The percentages of gallic acid remaining in various gel formulations at different storage temperatures (27 ± 2, 4 ± 2 and 45 ± 2°C) for 3 months. (GS gel, gel containing the unloaded gallic acid; GN gel, gel containing non-elastic niosomes loaded with gallic acid; GE gel, gel containing elastic niosomes loaded with gallic acid; SS gel, gel containing the unloaded semi-purified fraction; SN gel, gel containing non-elastic niosomes loaded with the semi-purified fraction; SE gel, gel containing elastic niosomes loaded with the semi-purified fraction.)

Table 3.  Primary irritation index (PII) and category of irritation based on PII of various gel formulations.

Samples	Primary irritation index (PII)			Category of irritation based on PII
	24 h	48 h	72 h
Gel base	0.11	0.11	0.00	Negligible
BN gel	0.11	0.11	0.00	Negligible
BE gel	0.22	0.11	0.00	Negligible
GS gel	0.44	0.56	0.56	Slight irritation
GN gel	0.33	0.22	0.11	Negligible
GE gel	0.22	0.22	0.22	Negligible
SS gel	0.33	0.33	0.22	Negligible
SN gel	0.11	0.11	0.11	Negligible
SE gel	0.22	0.11	0.11	Negligible
5% sodium lauryl sulfate (positive control)	1.22	1.00	0.78	Slight irritation
Untreated area (negative control)	0.00	0.00	0.00	Negligible

Grading scale for skin irritation effect following OECD Test Guideline 404.

Primary irritation index (PII) = [(∑ erythema grade at 24/48/72 h + ∑ edema grade at 24/48/72 h)/3 × number of animals].

BN gel, gel containing blank non-elastic niosomes; BE gel, gel containing blank elastic niosomes; GS gel, gel containing the unloaded gallic acid; GN gel, gel containing non-elastic niosomes loaded with gallic acid; GE gel, gel containing elastic niosomes loaded with gallic; SS gel, gel containing the unloaded semi-purified fraction; SN gel, gel containing non-elastic niosomes loaded with the semi-purified fraction; SE gel, gel containing elastic niosomes loaded with the semi-purified fraction.

Table 4.  Percentage changes of skin parameter (%) after 8 weeks of applications of various gel formulations and the negative control (the untreated area).

	Parameter changes (%)
	Gel base	GN	GE	SS	SN	SE	Commercial product	Untreated area
Ravi	−19.05	−28.28	−30.95	−31.55	−39.47	−35.28	−22.43	+6.30
Rm	−24.62	−26.08	−28.66	−30.87	−29.43	−32.38	−3.38	−3.14
Ur/Uf	+22.95	+47.51	+53.92	+30.32	+28.73	+32.57	+19.89	−14.32
Uv/Ue	−14.62	−8.40	−17.69	−12.85	−21.25	−22.63	−14.93	+2.90

Ra, the average roughness; Rm, the maximum roughness; Ur/Uf, skin elastic recovery; Uv/Ue, skin elastic extension; GN gel, gel containing non-elastic niosomes loaded with gallic acid; GE gel, gel containing elastic niosomes loaded with gallic; SS gel, gel containing the unloaded semi-purified fraction; SN gel, gel containing non-elastic niosomes loaded with the semi-purified fraction; SE gel, gel containing elastic niosomes loaded with the semi-purified fraction.

−, decreased value; +, increased value.

Figure 4.  Changes of the maximum roughness or Rm (A) and the arithmetic average roughness or Ra (B) of various topical gel formulations in 31 human volunteers after topical application for 8 weeks. Student’s paired t-test was used to calculate the significant differences. *p < 0.05 compared to before application (0 wks). (GN gel, gel containing non-elastic niosomes loaded with gallic acid; GE gel, gel containing elastic niosomes loaded with gallic acid; SS gel, gel containing the unloaded semi-purified fraction; SN gel, gel containing non-elastic niosomes loaded with the semi-purified fraction; SE gel, gel containing elastic niosomes loaded with the semi-purified fraction.)

Figure 5.  Comparison of the skin roughness before (left) and after application for 8 weeks (right) and % changes of the arithmetic average roughness (Ra) values of various topical formulations. (GN gel, gel containing non-elastic niosomes loaded with gallic acid; GE gel, gel containing elastic niosomes loaded with gallic acid; SS gel, gel containing the unloaded semi-purified fraction; SN gel, gel containing non-elastic niosomes loaded with the semi-purified fraction; SE gel, gel containing elastic niosomes loaded with the semi-purified fraction.)

Figure 6.  Changes of the skin elastic recovery or Ur/Uf (A) and changes of the skin elastic extension or Uv/Ue (B) of various topical gel formulations in 31 human volunteers after application for 8 weeks. Student’s paired t-test was used to calculate the significant differences. *p < 0.05 compared to before application (0 wks). (GN gel, gel containing non-elastic niosomes loaded with gallic acid; GE gel, gel containing elastic niosomes loaded with gallic acid; SS gel, gel containing the unloaded semi-purified fraction; SN gel, gel containing non-elastic niosomes loaded with the semi-purified fraction; SE gel, gel containing elastic niosomes loaded with the semi-purified fraction.)

Figure 7.  Comparison of moisture (A), melanin content (B) and erythema index (C) on skin surface of various topical formulations at initial and after 8 weeks of application. Student’s paired t-test was used to calculate significant differences. No significant difference was observed between before and after 8 weeks of application (p > 0.05).