3.3. In-Vitro Studies

3.3.1. Evaluation of Cytotoxicity in Human Keratinocytes

According to relevant studies,cistanche is a common herb that is known as "the miracle herb that prolongs life". Its main component is cistanoside, which has various effects such as antioxidant, anti-inflammatory, and immune function promotion. The mechanism between cistanche and skin whitening lies in the antioxidant effect of cistanche glycosides. Melanin in human skin is produced by the oxidation of tyrosine catalyzed by tyrosinase, and the oxidation reaction requires the participation of oxygen, so the oxygen-free radicals in the body become an important factor affecting melanin production. Cistanche contains cistanoside, which is an antioxidant and can reduce the generation of free radicals in the body, thus inhibiting melanin production.

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The MTT assay was used to test whether PPEE, PPEE-SLNs, and KDPAG could affect cell viability using human keratinocytes. Different concentrations of PPEE, PPEE-SLNs, extract-free-SLNs, and KDPAG at 0.0625– 1mg/mL were incubated over 24 h with a human keratinocytes cell line (Figure 2). Over 24 h, there was no observed signifificant reduction in human keratinocytes cell viability in the concentration range of 0.0625–0.5 mg/mL (p < 0.05). However, after treatment with 1 mg/mL of PPEE, PPEE-SLNS, Extract-free-SLNs, and KDPAG over 24 h, a signifificant reduction in cell viability of human keratinocytes was observed, 93%, 90%, 95%, and 89% of the cells survived, respectively. Thus, concentrations of PPEE, PPEE-SLNs, and KDPAG equal to or lower than 0.5 mg/mL were used for subsequent studies.

The antioxidant activity was evaluated using DPPH, ABTS systems, and the β-carotene bleaching test. In all tested samples, a concentration-dependent manner was found. In the DPPH assay, KDPAG showed the highest scavenging activity followed by PPEE-SLNs and then PPEE. The IC50 values were 6.35 ± 3.40 µg/mL, 8.79 ± 2.70 µg/mL, and 10.5 ± 1.81 µg/mL respectively compared to vitamin C used as a standard with an IC50 of 2 ± 0.01 µg/mL. This trend was observed also against ABTS radicals with a value of 3.91 ± 1.43 µg/mL, 4.29 ± 1.12 µg/mL, and 6.10 ± 0.62 µg/mL, respectively, compared with the standard Vit. C with an IC50 of 0.96 ± 0.02 µg/mL confirming the results of the DPPH assay. Such scavenging activity for PP leaf extracts and their fractions has already been pointed out for both assays but with higher values than the obtained ones [14,49]. These findings suggest that the activity of antioxidants is affected by environmental conditions, plant parts, stage of maturity, method of harvesting, and solvents used for extraction. These results are consistent with the literature as PP leaf extract was found to have a better antioxidant capacity than seed, peel, pulp, and fruit extracts in both assays [14,20,50]. Also, the reported high flavonoid content of PP leaves, in particular, flavonols was thought to be responsible for the antioxidant activity [20].

PPEE, PPEE-SLNs, and KDPAG showed anti-elastase, anti-collagenase, and anti-tyrosinase activity with a high% inhibition at 300 µg/mL and relatively low IC50 values (Table 3) compared to their respective positive controls.

3.4. Evaluation of PPEE-SLNs and PPEE-SLNs Cream 

The prepared PPEE-SLNs formulations showed particle sizes of 170 nm to 176 nm; this indicates that the addition of surfactant to solid lipid nanoparticle systems causes the interfacial film to condense and stabilize. Formulations show low values of polydispersity index (0.230–0.450) indicating homogeneity of particle size distribution with ZP between −21.8 and −22.0 ensuring high stability products (Figure 3a,b). The recorded 70–77% of PPEE-SLNs encapsulation efficacy is due to the type of lipid (glycerol monostearate) which is capable of closing the surface pores in the beads. TEM & SEM micrographs revealed the formation of nanoparticles of narrow particle size distribution, smooth, spherical, and homogenous nanovesicles (Figure 3c,d). In FTIR, PPEE exhibits characteristic peaks at 3352 cm−1 corresponding to the aromatic secondary amine N-H stretching, 2974.23 cm−1 corresponding to aromatic C-H stretching, 1735.93 cm−1 corresponding to C=O stretching, and 1257.59 cm−1 corresponding to C-N aliphatic amine stretching as appeared in (Figure 4). The FTIR spectra of PPEE and lipid physical mixture exhibit the same characteristic peaks due to the aromatic secondary amine N-H stretching at 3348.42 cm−1, C=O stretching at 1735.93 cm−1, and C-N aliphatic amine stretching at 1257.59 cm−1. Thus, it is evident that all the characteristic peaks that were present in the spectra of PPEE replicated almost in the same region in the spectra of the PPEE-SLNs physical mixture indicating that there is no signifificant interaction between the drugs and the lipids. PPEE-SLNs creams (2% and 5%) were yellowish with a smooth appearance, smooth surface, and a suitable pH ranging from 5–5.8 ± 0.15 which confirms the compatibility of the formulations with skin secretions, not irritant to human skin, no presence of redness or edema, easily washable with water and good spreadability value ranged from 10–13 g cm/s, with viscosity in the range of 500 ± 6.24 to 600 ± 7.52 CPS at 10 rpm, no evidence of phase separation and good consistency during the study period. The total amount of bacteria and molds was less than 100 CFU/mL and in the acceptance range for skincare products. The 2% cream showed an initial burst release of PPEE-SLNs of about 15.21 ± 1.44% during the first 1 h; following that, the PPEE-SLNs entrapped into the cream were released gradually; 60.32 ± 2.54% were released after 12 and 62 ± 1.44% almost after 24 h respectively. On the other hand, the 5% cream showed an initial burst release of PPEE-SLNs of about 20.21 ± 2.70% during the first 1 h; following that, the PPEE-SLNs entrapped into the cream were released gradually, 77.12 ± 2.88% were released after 12 and 80 ± 2.91% almost after 24 h respectively. Both cream formulae showed extended release of PPEE over 24 h (Figure 5). Moreover, our research study presented that formulations of cream (2% and 5%) are stable for two months.

3.5. In-Vivo Studies 

After twenty days of treatment with the cream, the anti-wrinkle scores were assessed. The anti-wrinkle effect of PPEE-SLNs cream was dose-dependent, and the effect observed in the 5% PPEE-SLNs cream group (G5) was comparable to the result in the positive control group (G3), which was treated with a market product (Figure 6) and showed significantly better anti-wrinkle scores compared with standard anti-wrinkle cream. The photographs are presented in Figure 7. Groups (G3–G5) showed very smooth and improved skin surfaces. There were no skin changes in G1 animals. In contrast, G2 showed thick and deep wrinkles.

The elastic fibers were significantly decreased by UV irradiation compared with those of the normal group. Skin images for untreated and treated mice were observed by microscope (Figure 8). G1 had normal skin thickness of both dermis and epidermis and contained fibroblasts, in addition to, normal elastic fibers thickness with no fragmentation. Compared with G1, skin from the UV-irradiated mice (G2) showed a signifificant increase in the thickening of both the epidermis and dermis as well as a decrease in the formation of fibroblast and the elastic fibers by UV irradiation. The topical application of 5% (G5) and 2% (G4) PPEESLNs cream was comparable to that of G3 that received the commercial product. All G4 and G5 significantly decreased the thickness of the dermis and epidermis, and increase the content of fibroblasts as their number determines the content of collagen fibers, thus helping to repair damaged skin and reducing the aging effect on the skin (Figure 9a,b). Also, both groups showed preventive effects against the degradation of elastic fibers by UV irradiation, so we can conclude that treatment with either dose of PPEE-SLNs cream showed a protective effect against UV irradiation.

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