Abstract: Polyphenols are known dietary antioxidants. They have recently attracted considerable interest in uses to prevent skin aging and hyperpigmentation resulting from solar UV irradiation. Prunus persica (L.) leaves are considered by-products and were reported to have remarkable antioxidant activity due to their high content of polyphenols. This study aimed at the development of a cosmeceutical anti-aging and skin whitening cream preparation using ethanol leaves extract of Prunus persica (L.) (PPEE) loaded in solid lipid nanoparticles (SLNs) to enhance skin delivery. Chemical investigation of PPEE showed significantly high total phenolic and flavonoid content with notable antioxidant activities (DPPH, ABTS, and β-carotene assays). A unique acylated kaempferol glycoside with a rare structure, kaempferol 3-O-β-4C1-(600 -O-3,4-dihydroxyphenylacetyl glucopyranoside) (KDPAG) was isolated for the first time and its structure fully elucidated. It represents the first example of acylation with 3,4-dihydroxy phenylacetic acid in flflavonoid chemistry. The in-vitro cytotoxicity studies against a human keratinocytes cell line revealed the non-toxicity of PPEE and PPEE-SLNs. Moreover, PPEE, PPEE-SLNs, and KDPAG showed good anti-elastase activity, comparable to that of N (Methoxysuccinyl)-Ala-Ala-Pro-Val-chloromethyl ketone. Besides, PPEE-SLNs and KDPAG showed significantly (p < 0.001) higher anti-collagenase and anti-tyrosinase activities in comparison to EDTA and kojic acid, respectively. Different PPEE-SLNs cream formulae (2% and 5%) were evaluated for possible anti-wrinkle activity against UV-induced photoaging in a mouse model using a wrinkle scoring method and were shown to offer a highly signifificant protective effect against UV, as evidenced by tissue biomarkers (SOD) and histopathological studies. Thus, the current study demonstrates that Prunus persica leaf by-products provide an interesting, valuable resource for natural cosmetic ingredients. This provides related data for further studying the potential safe use of PPEE-SLNs in topical anti-aging cosmetic formulations with enhanced skin permeation properties. 

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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1. Introduction

Skin aging is a complex multifactorial progressive process that catalyzes physical changes in skin and connective tissue [1]. It is classified into intrinsic and extrinsic aging. 

Extrinsic skin aging results mainly from environmental factors, such as pollutants, smoking, and life stress, or predominantly from repeated exposure to UV radiation (photoaging) [2].

Overexposure to UV radiation stimulates the overproduction of reactive oxygen species (ROS) which results in endogenous oxidative stress in skin tissues leading to the degradation of the extracellular matrix (ECM) components. ECM degradation is directly linked to skin aging and is responsible for the increase in activity of certain enzymes such as collagenase, elastase, and tyrosinase that are involved in skin aging. These dermal enzyme activations cause a decrease in the levels of elastin, and collagen which leads to loss of elasticity and strength of skin and the appearance of wrinkles [3]. Also, the induction of excessive melanin production and skin tanning results in hyperpigmentation of the skin.

Therefore, skin aging can be prevented by antioxidants with a free radical scavenging activity which may have great significance in the defense and therapeutics of age-related diseases involving ROS [4]. The other way to retard aging is using elastase, collagenase, and tyrosinase inhibitors which are very strong candidates for anti-wrinkle and skin whitening activities endorsing the preservation of skin elasticity and can be a common approach to deal with pigmentation disorders [5]. 

It has been reported that natural antioxidants isolated from plants attenuate the risk of photoaging induced by UV irradiation both in-vitro and in-vivo [6] and they are preferable as cosmeceutical ingredients than synthetic antioxidants in respect of cost and side effects. In addition, many in-vitro and in-vivo studies have shown that phenolic compounds such as phenolic acids, flavonoids, and tannins could scavenge free radicals and inhibit elastase, collagenase, and tyrosinase enzymes [7].

The use of natural antioxidants in topical preparations to protect skin against oxidative stress caused by extrinsic factors has been reported [8]. However, the majority of antioxidant molecules are innately unstable and can easily oxidize to form inactive compounds before reaching the site of action which makes them difficult to formulate in an appropriate and stable cosmetic product. These findings prove the necessity of unique delivery systems to enhance these antioxidant formulations [9]. 

Several factors affect the permeability of topically applied polyphenols; the phenolic subclass, its structure, its molecular size, and the glycoside or aglycone form, as well as the other formulation components. However, the interaction with skin components through a mixture of physical and chemical methods could improve permeability through a reversible disruption of the stratum corneum layer’s barrier structure. Encapsulation approaches are one of the permeability-enhancing methods most commonly used to easily stabilize polyphenolics during storage, increasing their antioxidant effects, dermal absorption, and penetration in cosmetic and topical therapies. Newly developed encapsulation technologies include nanoemulsions, transferases, solid lipid nanoparticles, nanocrystals, and ribosomes [10].

Solid lipid nanoparticles (SLNs) are a stable delivery system for skin products. SLNs are considered promising drug carriers for topical formulations due to their photostability, controlled release properties, occlusivity, odor masking effects, and penetration enhancement of the active constituents through the skin by increasing its hydration with no signs of skin irritation, in addition to the simplicity of production, low toxicity, and physical stability [9,11]. 

On a worldwide basis, there are about 2000 peach cultivars [12]. Prunus (Rosaceae) is a famous plant genus that includes phenolic-rich species [13–15]. Some species of this genus are cultivated in Egypt for their edible fruits or as ornamentals [16]. 

Prunus persica (L.) Batch (PP) is a small tree with glabrous twigs and a short trunk, a spreading, rounded crown [12] and commonly cultivated in West Asia, Europe, India, and North Africa [17]. Traditionally, its leaves have been used as diuretics, laxatives, vermifuges, insecticides, sedatives, for whooping cough, for the treatment of leukoderma, and as febrifuges [17]. Furthermore, pharmacological studies on leaves characterized their in-vivo anti-diabetic, spasmogenic effect, anti-inflammatory, anticoagulant, hepatoprotective, antimalarial, anti-asthmatic, and in-vitro cytotoxic, antimicrobial, and nitric oxide inhibitory with signifificant anti-oxidant activities [18–22].

Many studies have been done to investigate the phenolic profile of PP followers, fruits, and seeds, their antioxidant activities, and pharmacological and nutritional values [13,14,23,24]. However, very scarce data were available on the leaves. To date, a single report is available describing the isolation of five flavonol glycosides from an ethanol leaf extract [25]. In addition, phenolic compounds have been characterized, including phenolic acids and flflavonoids using HPLC-MS analysis with flflavonols as dominant substances among all the determined compounds [20,26].

Moreover, although PP seeds, fruits, followers, and other species have been reported to have in-vivo protective activity against UV-induced photoaging and in-vitro anti-wrinkle and skin whitening activities [15,23,27–32], this has never been reported for PP leaves extract. 

Large amounts of PP leaves are by-products derived from peach tree cultivation and the fruit canning industries [33]. The by-products of PP seeds, fruits, and other leaf varieties have been evaluated in cosmetics and as dietary food for potential valorization [32,34,35]. However, to the best of our knowledge, the leaves by-products of PP var. Florida Prince has never been investigated and could be an interesting source of phytochemicals. Compared with other industries, the cosmetic market is more accessible and expanding and can be a source for the valorization of by-products [15]. Skincare and skin aging products are some of the most important cosmetic potentials.

In a continuation of our research on polyphenolics of Egyptian edible plants [36] and since PP leaves are considered by-products and were reported to have signifificant antioxidant activity mainly due to their high flavonols content, this study aims to subject PPEE to an extensive phytochemical analysis of its phenolic profile and to incorporate PPEE into loaded SLNs as a unique skin delivery system. Furthermore, the anti-aging and skin-whitening cosmetic potential was evaluated by investigating the in-vitro antioxidant activity of PPEE, PPEE-SLNs, and the isolated constituents, along with the evaluation of their inhibitory effects against skin-related enzymes. In addition to the incorporation of PPEE-SLNs into a topical cosmeceutical anti-aging cream and to test the product safety as well as evaluation of the possible in-vivo anti-wrinkle activity of PPEE-SLNs cream formulations against UV-induced photoaging in a mouse model. This could provide data for further studying the potential use of PPEE-SLN leaves in topical formulations with enhanced skin permeation. 

2. Material and Methods 

2.1. General 

2.2. Plant Materials 

2.3. Preparation of Plant Extract 

2.4. Determination of Total Polyphenols Content (TPC) 

The Folin–Ciocalteu reagent as described by Li et al., [37] was used to estimate the amount of total phenolic content in PPEE, presented as mg equivalent gallic acid/g dry extract. 

2.5. Determination of Total Flavonoid Content (TFC) 

The aluminum chloride colorimetric method described by Bahromun et al., [38] was used to estimate the total flavonoid content. The results are presented as mg equivalent quercetin/g dry extract. 

2.6. Fractionation of PPEE and Isolation of Compound 1 

2.7. Identification of kaempferol 3-O-β-4C1-(6”-O-3,4-dihydroxyphenylacetyl glucopyranoside) (1)

2.8. In-Vitro Studies 

The human keratinocytes cell line was obtained from VACSERA CO. (The Egyptian Company for the Production of Vaccines, Sera, and Drugs, Giza, Egypt). The MTT assay was done for PPEE and PPEE-SLNs according to the method of Mostafa et al. [36]. 

The DPPH assay was performed using the method of Yardpiroon et al. [39], and Vitamin C was used as a positive control. The ABTS assay followed the method of Re et al., [40], Vitamin C was used as a standard. The β-carotene bleaching assay used the method described by Soulef et al., [14] in the presence of the positive control BHT. The value for each test sample was presented as the inhibition curve at 50% or IC50. 

The anti-aging and skin whitening activities were assessed following the methods reported by Mostafa et al. [41]. For the anti-elastase assay, 1 µg/mL of human leukocyte elastase was incubated with HEPES buffer pH 7.5 and each of the tested extracts or 1.4 mg/mL N-Methoxysuccinyl-Ala-Ala-Pro-chloromethyl ketone (standard inhibitor) in a 96-well plate for 20 min at room temperature before the addition of 100 µL of 1 mM N-Methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide as a substrate. After 40 min incubation, the absorbance was measured at 405 nm with a well devoid of extract/inhibitor serving as a blank. The tested samples were used in a concentration range of 25–300 µg/mL.

2.9. Formulation and Characterization of PPEE-SLNs

The formulation was done according to the method of Choubey et al. [11]. First, glyceryl monostearate was dissolved in a chloroform–methanol mixture (1:1) and then the ethanolic extract was dispersed in that solution. Later the organic solvents were removed using a rotovap and the solution was heated. Tween 80 was then added and the mixture was stirred at 3000 rpm for 30 min and then homogenized for 4 h before filtering and drying. Characterization of PPEE-SLNs was examined by estimating shape and surface morphology, particle size, polydispersity index (PDI), zeta potential analysis, percent entrapment efficiency (PEE), and Fourier transform infrared (FT-IR) spectroscopy studies.

2.10. Preparation of PPEE Topical Cream Formulae 

Two formulations of cream (2% & 5% of PPEE-SLNs) were prepared as shown in Table 1 according to the method of Mahawar et al. [42].

Organoleptic properties, pH of the cream, spreadability studies, viscosity test, homogeneity test, patch test (Burchard test), in-vitro skin permeation examination, and stability studies (ICH guidelines) were evaluated according to Matangi et al. [43]. A microbial limit test was carried out according to Sekar et al. [44].

2.11. In-Vivo Anti-Wrinkle Study of PPEE-SLNs Cream 

Fifty male hairless mice (HR-1) (4-week-old), 17–24 g were obtained from VACSERA. The experiment was carried out after the approval of the ethics committee at October University for Modern Sciences and Arts (MSA), Protocol number (PG1/EC1/2020PD) sample size was determined according to (G. Power) software. One week before the in-vivo anti-wrinkle study, mice were free to access food, and water and become acclimatized to the air-conditioned room (23 ± 2 ◦C). Mice (50) were divided into five groups, randomly (n = 10) as shown in Table 2. Treatment was done over 40 days, three times weekly, with 0.5 g of PPEE-SLNs cream or any other treatment and applied to the dorsal mice skin and subsequently exposed to UV (365 nm). 

Before and after topical treatments, two times/week, dorsal mice skin photos were taken. The skin wrinkle grading was evaluated using Bissett’s visual wrinkle scale [45]. The bases for the grading scale were the texture of wrinkle (fine or coarse) and keratosis, divided into five grades: worse (−2), slightly worse (−1), no change (0), slightly improved (+1), and improved (+2). 

Histological evaluation was done according to the method of Elder et al. [46]. 

2.11.4. Superoxide Dismutase (SOD) Activity

SOD activity was estimated according to the method of Ukeda et al. [47].

2.12. Statistical Analysis 

3. Results

3.1. Total Phenolic and Flavonoid Contents 

TPC was determined spectrophotometrically in PPEE as 387.5 ± 4.28 mg GAE/g extract. Also, TFC was estimated as 241.7 ± 3.25 mg QE/g extract. PPEE showed high concentrations of phenolic and flflavonoid compounds. Both results are following values previously reported for PP leaf extracts confirming that flavonoids are the main chemical constituents in PP leaves [20]. 

3.2. Isolation and Structure Elucidation of kaempferol 

3-O-β- 4C1-(600 -O-3,4-dihydroxyphenylacetyl glucopyranoside), Compound 1 

After sorting out all the known structures in that extract, the search for unique, potentially biologically active compounds became more efficient. According to the received 2-DPC analytical data the phenolics of PPEE were found to be best fractionated over an MCI gel column eluted by MeOH/H2O mixtures of decreasing polarities, a process which afforded four major column fractions. An amorphous material was separated from a hot concentrate of fraction III (desorbed from the column by 70% aqueous MeOH) on standing overnight at room temperature. Crystallization of this material twice from boiling EtOH yielded a chromatographically pure brownish-yellow amorphous powder 1. It showed chromatographic properties (dark purple spot-on PC under UV light, turning lemon yellow when fumed with ammonia vapor with moderate migration in aqueous and organic solvents) and color reactions (a lemon-yellow color with Naturstoff reagent), which suggested a kaempferol derivative bearing a free 40 hydroxyl group and an O-substituted 3-O-position. The UV spectra of 1 in MeOH (268 nm, 316 nm, 360 nm) and upon addition of the diagnostic shift reagents [48] were typical of those of 3-O-glycosylated kaempferol (positive shift with NaOAc, stable shift with NaOMe). Normal acid hydrolysis of 1 (2 N aqueous HCl, 3 h, 100 ◦C) yielded glucose (comparative paper chromatography, Co-PC), kaempferol, and 3,4-dihydroxy phenylacetic acid (UV absorption and 1H-NMR spectra, for individual samples, isolated by preparative paper chromatograms of the ethyl acetate extract of the hydrolysate). Consequently, 1 is kaempferol 3-O-(3,4-dihydroxy phenyl acetyl glucoside). The compound is recovered unchanged after being incubated with β-glucosidase enzyme for 24 h, thus proving that the glucosyl moiety is acylated. The molecular formula of 1 was concluded to be C29H26O14 from its negative HRESI mass spectrum which showed an [M-H]− ion at m/z = 597.5302 (calc. for C29H25O14, 597.5015). The ESI-MS experiment (negative-ion mode) gave a quasi-molecular ion peak [M-H]− at m/z: 597 indicating a molecular weight of 598 for 1. Further fragment ion peaks in the ESI-MS-MS spectrum were observed at m/z: [M-H]−: 447, 167, and 285 corresponding to the loss of dihydroxy phenyl acetate from the parent compound 1 while the fragment ions at m/z 285 and m/z 167 were attributed to the kaempferol and dihydroxyphenylacetic acid moieties, respectively. To determine the site of attachment of all moieties in the molecule of 1 and to allow the full assignment of all carbon and proton resonances, NMR spectroscopic analysis of 1, including 1D- 1H and 13C, and 2D- HSQC and HMBC, was then carried out. From the five signals in the sugar region between δ ppm 62.25 and 76.9 and from the anomeric carbon signal located at 99.13 ppm, it was proved that the sugar moiety must be attached to position 3 of kaempferol because this C-3 carbon signal was shifted upfield and the corresponding ortho and para-carbon signals were shifted downfield (see experimental). Similar shifts are well-known from the work of Nawwar et al. [48]. This was further confirmed by the 3 J long-range correlations recognized in the HMBC spectrum, whereby, one cross signal was found correlating the anomeric glucose proton H-100 signal at δ 5.42 to the flavonol C-3 carbon signal at δ 133.5. The β-configuration of the glucose moiety was derived from the C-l00 chemical shift at 99.13 ppm. The chemical shift values of the sugar carbons confirmed the pyranose form of this moiety [48]. The 1H-NMR spectrum of 1 was also by the proposed structure. The chemical shift of the anomeric proton signal at δ 5.42 ppm (d, J = 8 Hz) indicated that the anomeric carbon is attached to the kaempferol moiety at C-3 (δppm 133.5) and the determined coupling constant 8 Hz, prove the β-configuration of the glucose moiety. The conformation of the sugar moiety is 4C1 as followed by the β-configurations discussed above. 

Also, attachment of the 3,4-dihydroxy phenylacetic acid moiety to the C-600 methylene glucopyranose moiety followed from the downfield shift of the signal of this carbon to δ ppm 62.25 in the 13C-NMR spectrum. This was further confirmed by the cross peak in the HMBC spectrum, correlating the methylenic glucose protons signals at δ 4.04 (H-600 a) and δ 4.2 ppm (H-600 b) to the carbonyl carbon of the 3,4-dihroxyphenylacetic acid moiety at δ 176.26 ppm. These and the above-given data finally confirmed the structure of compound 1 to be the new kaempferol 3-O-β- 4C1-(600 -O-3,4-dihydroxyphenylacetyl glucopyranoside) (KDPAG), reported for the first time in nature, as it represents the first acylation with 3,4-hydroxyphenyl acetic acid in association with flflavonoid chemistry (Figure 1).

For more info: david.deng@wecistanche.com  WhatApp:86 13632399501