Preparation and Characterization of Callus Extract from Pyrus Pyrifolia and Investigation of Its Effects on Skin Regeneration

cosmetics

Article Preparation and Characterization of Callus Extract from Pyrus pyrifolia and Investigation of Its Effects on Skin Regeneration

Dae Eung Park †, Deepak Adhikari †, Rudra Pangeni , Vijay Kumar Panthi, Hyun Jung Kim * and Jin Woo Park *

College of Pharmacy and Natural Medicine Research Institute, Mokpo National University, Muan-gun, Jeonnam 58554, Korea; [email protected] (D.E.P.); [email protected] (D.A.); [email protected] (R.P.); [email protected] (V.K.P.) * Correspondence: [email protected] (H.J.K.); [email protected] (J.W.P.); Tel.: +82-61-450-2686 (H.J.K.); +82-61-450-2704 (J.W.P.) † These authors contributed equally to this work.

Received: 1 November 2018; Accepted: 12 December 2018; Published: 15 December 2018

Abstract: In the present study, an aqueous extract was prepared using calli from the in vitro-derived leaves of Pyrus pyrifolia cultured in Murashige and Skoog medium containing picloram for a plant growth regulator. The major biological components in the callus extract were identified as uridine (1), adenosine (2), and guanosine (3). In terms of the antioxidant activity, at 300 µg/mL, the extract exhibited free radical scavenging activity of 76.9% ± 2.88% in the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, comparable to that of 44 µg/mL ascorbic acid (82.5% ± 3.63%). In addition, the IC50 values for inhibition of advanced glycation end product formation from collagen and elastin were 602 ± 2.72 and 3037 ± 102.5 µg/mL, respectively. The extract significantly promoted keratinocyte and fibroblast cell proliferation in a dose-dependent manner. Moreover, fibroblasts treated with 1.36 µg/mL extract exhibited a 1.60-fold increase in procollagen type I C-peptide level compared to controls. The in vitro wound recovery rates of keratinocytes and fibroblasts were also 75% and 38% greater, respectively, than those of serum-free controls at 9 and 36 h after extract treatment (1.36 µg/mL). Additionally, the extract flux across the human epidermis increased by 1598% after its incorporation into elastic nanoliposomes (NLs). Therefore, elastic NLs loaded with Pyrus pyrifolia callus extract have potential use as skin rejuvenators and antiaging ingredients in cosmetic formulations.

Keywords: Pyrus pyrifolia; callus extract; skin regeneration; topical delivery; antiaging cosmetics

1. Introduction Cutaneous aging is a complicated biological process caused by various intrinsic and extrinsic factors, which bring about gradual destruction of structural integrity of the skin as well as its physiological functions [1,2]. Extrinsic aging involves a number of factors, such as continuous exposure to environmental insults, e.g., ultraviolet (UV) radiation. Such insults result in oxidative damage to the lipids, proteins, and DNA of the skin via the generation of free radicals that provoke cross-liking of collagen, thus leading to a loss of skin elasticity. Free radicals also damage the dermal connective tissue, resulting in premature skin aging (photoaging) [3–5]. Furthermore, persistent exposure to reactive oxygen species (ROS) has been shown to initiate skin aging, including wrinkle formation and melanogenesis [6]. The origin of intrinsic aging is commonly called the “biological clock.” One theory is based on the observation that diploid cells, such as ﬁbroblasts, have a ﬁnite lifespan in culture [7,8].

Cosmetics 2018, 5, 71; doi:10.3390/cosmetics5040071 www.mdpi.com/journal/cosmetics Cosmetics 2018, 5, 71 2 of 25

This results in cellular senescence, leading to altered gene expression and then to degenerative changes in tissues. Another intrinsic mechanism that contributes to skin aging is damage due to free radicals accumulated during the lifespan of an individual [9]. The Maillard glycation theory is a further general mechanism of intrinsic aging. Accumulation of the reaction products of protein glycation, produced by nonenzymatic reaction of proteins with glucose or other reducing sugars in living organisms, can induce structural and functional modifications of tissue proteins [10]. Many studies have demonstrated a significant role of glycation in the normal aging process as well as in the pathogenesis of aging-related diseases, such as diabetes, atherosclerosis, end-stage renal disease, rheumatoid arthritis, and neurodegenerative diseases [11–13]. Moreover, glycation has several adverse effects on the characteristics of collagen. The accumulation of Maillard reaction products has been shown to lead to stiffer and more brittle collagen, with consequent influences on the properties of the extracellular matrix (ECM) and ECM–cell interactions [14,15]. Thus, targeting glycation should have broad and beneficial effects on aging and aging-related diseases. The pear, Pyrus pyrifolia, is one of the most widely consumed fruits in the world and has also been used as folk medicine in Korea and China because of its biological activities, including antitussive, anticonstipative, antiulcer, and antioxidative effects [16–20]. In addition, many phenolic compounds, such as arbutin, chlorogenic acid, quercetin, catechins, kaempferol, and procyanidins, have been characterized in pear fruits, and various hydroxycinnamoyl malic acids and their ethyl esters, hydroxycinnamoyl malates, procyanidins, and triterpenes have also been found in pear peel [21–26]. In particular, steroids (β-sitosterol, daucosterol, and α-amyrin), quercitrin, and triterpenoids (oleanolic acid, ursolic acid, and 2β,19α-hydroxyurosolic acid) have been isolated and identified as anti-inflammatory and/or antimicrobial compounds in Pyrus bretschneideri R. (Chinese pear) [16,17]. The aqueous ethanoic extract of the bark from P. pyrifolia was also shown to have positive glycation inhibitory and antioxidative efficacies [27]. Plant resources have long been studied as natural antioxidants and enzyme inhibitors related to inflammation, melanin hyperpigmentation, and a variety of skin disorders. Therefore, these resources are valuable in cosmetic formulations which aim to inhibit or impede oxidative damage to collagen induced by free radicals [28]. Antioxidants, including various bioactive compounds derived from natural products, have been applied to inhibit aging. However, the provision of fresh materials, regardless of the season and the plant reproductive cycle, have always been barriers to their application in the pharmaceutical and cosmetics industries. These factors make the extraction of bioactive compounds from plant sources highly inefficient and emphasize the need for novel approaches to produce their secondary metabolites. To overcome these limitations, in vitro plant tissue and cell culture have been suggested as valid alternatives [28–31]. The in vitro propagation and cell suspension cultures not only offer viable methods for the mass multiplication of scarce and endangered species, but are also important in the quality-controlled production of desired plant-derived bioactive components, including secondary metabolites with high levels of uniformity from batch to batch without risk of pathogenic or environmental contamination and for screening of natural inhibitors against various biological disorders [32,33]. Thus, plant cell culture represents an efficient way to produce several valuable natural products, such as commercially important products, including pigments (e.g., anthocyanins and betacyanins) and anti-inflammatory agents (e.g., berberine and rosmarinic acid) [33,34]. Extracts from plant cell cultures also include a board range of active ingredients, and in many cases they are present in high concentrations compared with those produced in vivo. However, for use in topically applied cosmetic products, bioactive compounds for preventing skin aging should be well tolerated and nonirritating, with no toxicity or side effects. In addition, the active compounds should be able to penetrate into the desired site of action across the stratum corneum (SC), and ultimately reach the living parts of the epidermis and/or dermis where glycation exhibits its harmful effects. Therefore, many studies have attempted to enhance the efficacy, stability, and solubility of bioactive compounds present in the plant extracts by incorporating them into colloidal systems, such as nanostructured lipid Cosmetics 2018, 5, 71 3 of 25 carriers including solid lipid nanoparticles, polymeric nano-sized micelles, and microemulsions and nanoemulsions, to facilitate their skin penetration and/or protect them from degradation [35]. To produce various bioactive compounds, this study aimed to prepare and characterize an aqueous extract of P. pyrifolia callus as an antiaging cosmetic ingredient. In addition, topical nanocarriers based on nanoliposomes (NLs) were designed to enhance skin permeation of the callus extract. Calli were established from leaf explants of P. pyrifolia under controlled conditions, and molecular markers in the aqueous extract from in vitro-derived calli were identified by liquid chromatography mass spectrometry (LC-MS). Thereafter, the antioxidant activity of the callus extract was characterized by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,20-azinobis-(3-ethylbenzoline-6-sulfonate) (ABTS) free radical scavenging assays, and the reduction of transition metal ions by ferric-reducing antioxidant power (FRAP) test. In addition, the inhibitory effect of the callus extract on ECM protein glycation was assessed by collagen and elastin glycation assays. To evaluate the skin regeneration efficacy of the P. pyrifolia callus extract, its abilities to stimulate proliferation and migration of keratinocytes and fibroblasts, followed by promotion of collagen synthesis by human fibroblasts, were measured. Finally, improvement of the skin permeability of the callus extract was assessed after its incorporation into elastic NLs.

2. Materials and Methods

2.1. Materials Murashige and Skoog (M&S) medium, picloram, 2,4-dichlorophenoxyacetic acid, and 6-benzylaminopurine were purchased from Duchefa Bohemia B.V. (Haarlem, The Netherlands). Uridine, adenosine, and guanosine were obtained from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan). DPPH, ABTS, potassium persulfate, 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), ascorbic acid, FeCl3, FeSO4, cholesterol, and polysorbate 80 (Tween 80) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Hydrogenated lecithin (Lipoid P75-3) and hydrogenated phosphatidylcholine (Lipoid P100-3) were provided by Lipoid (Ludwigshafen, Germany). Other chemicals were obtained from Merck KGaA (Darmstadt, Germany) and Thermo Fisher Scientiﬁc (Waltham, MA, USA).

2.2. Preparation and Characterization of P. pyrifolia Callus Extract

2.2.1. Preparation of Plant Material P. pyrifolia plant seeds were soaked in pure water overnight and then immersed in 70% (v/v) ethanol for 90 s. After sterilizing their surfaces with a 4% (w/v) sodium hypochlorite solution for 15 min, the seeds were grown aseptically in M&S basal medium supplemented with 3% (w/v) sucrose and 0.4% (w/v) gelite. The pH of the medium was adjusted to 5.8 with 1 N NaOH before autoclaving at 121 ◦C for 15 min. Next, the seeds were maintained up to sprouting for 16 h in the light (2000 lx; 25 ◦C ± 1 ◦C) and 8 h in the dark (16 ◦C ± 1 ◦C) for 3–7 days. To induce callus formation, a 5 × 5-mm wound was created on the young leaves from the germination using a scalpel and placed onto M&S medium containing 3% (w/v) sucrose and 0.26% (w/v) gelite in combination with growth regulator (2 mg/L of picloram), followed by incubation in the dark at 26 ◦C ± 2 ◦C for 8 weeks. After collecting the calli, they were transplanted in the fresh M&S medium with 2 mg/L of picloram, and further incubated in the dark in a growth chamber at 26 ◦C ± 2 ◦C for 4 weeks. To raise the biomass of callus, cell suspension cultures were initiated by cultivating the calli in 500-mL flasks with 200 mL of M&S medium containing 3% (w/v) sucrose and 2 mg/L of picloram on a rotary shaker (120 rpm) in the dark at 25 ◦C ± 1 ◦C and then subcultured once every 3 weeks. After gathering and air-drying the calli at 50 ◦C, they were pulverized into a fine powder. Next, an aqueous callus extract was prepared by boiling 200 g of the callus powder in 1 L of water for 4 h, followed by filtration through a filter paper (pore size: 0.4 µm) and freeze-drying. Cosmetics 2018, 5, 71 4 of 25

2.2.2. Isolation and Identification Procedure The dried extract (9.98 g) was separated by flash chromatography using RP cartridges (120 g, KP-C18-HS; Biotage, Uppsala, Sweden) and eluted with methanol-water mixture (0:100 to 40:60) for 60 min at 40 mL/min to afford 13 fractions. Fractions 3–6 were further purified with a semi-preparative high-performance liquid chromatography (HPLC) system composed of a Waters 600 HPLC controller and pump with a 2998 photodiode array detector using SunFire Prep C18 OBD (150 × 19 mm, 5 µm; Waters, Milford, MA, USA). The mobile phase consisted of water containing 0.1% (v/v) formic acid (A) and methanol (B), and was applied in linear gradient mode from 100% to 93% A at a flow rate of 7.0 mL/min for 40 min to yield uridine (1, 19.9 mg), adenosine (2, 28.1 mg), and guanosine (3, 12.0 mg). Nuclear magnetic resonance (NMR) data were obtained using a Varian VNMRS600 spectrophotometer (Varian, Palo Alto, CA, USA). LC-MS was performed using an Agilent 1260 Infinity system combined with an Agilent 6120 quadruple LC-MS system in positive ESI mode (Agilent Technologies, Santa Clara, CA, USA) with the following parameters: capillary voltage, 3.0 kV; nebulizer (N2) pressure, 35 psi; drying gas temperature, 250 ◦C; drying gas flow, 12.0 L/min.

1 0 Uridine (1): H NMR (600 MHz, D2O) δ 7.87 (1H, d, J = 8.4 Hz, H-6), 5.91 (1H, d, J = 4.8 Hz, H-1 ), 5.89 (1H, d, J = 8.4 Hz, H-5), 4.34 (1H, t, J = 5.1 Hz, H-20), 4.22 (1H, t, J = 5.4 Hz, H-30), 4.13 (1H, m, H-40), 0 0 13 3.91 (1H, dd, J = 12.9, 2.7 Hz, H-5 ), 3.80 (1H, dd, J = 12.9, 4.5 Hz, H-5 ); C NMR(150 MHz, D2O): 163.9 (C-4), 149.4 (C-2), 139.5 (C-6), 100.0 (C-5), 87.0 (C-10), 81.9 (C-40), 71.3 (C-20), 67.1 (C-30), 58.4 (C-50); ESI-MS m/z 245.3 [M + H]+.

1 Adenosine (2): H NMR (600 MHz, D2O) δ 8.17 (1H, s, H-8), 8.01 (1H, s, H-2), 5.86 (1H, d, J = 6.0 Hz, H-10), 4.56 (1H, t, J = 5.4 Hz, H-20), 4.25 (1H, dd, J = 5.1, 3.9 Hz, H-30), 4.13 (1H, q, J = 3.9 Hz, H-40), 0 0 13 3.75 (1H, dd, J = 13.0, 2.7 Hz, H-5 ), 3.66 (1H, dd, J = 13.0, 3.9 Hz, H-5 ); C NMR (150 MHz, D2O) δ 151.3 (C-6), 147.5 (C-2), 145.9 (C-4), 139.0 (C-8), 116.7 (C-5), 86.1 (C-10), 83.4 (C-40), 71.6 (C-20), 68.2 (C-30), 59.1 (C-50); ESI-MS m/z 268.3 [M + H]+.

1 0 Guanosine (3): H NMR (600 MHz, DMSO-d6) δ 7.93 (1H, s, H-8), 5.69 (1H, d, J = 6.0 Hz, H-1 ), 4.39 (1H, t, J = 5.4 Hz, H-20), 4.08 (1H, t, J = 4.5 Hz, H-30), 3.86 (1H, q, J = 3.6 Hz, H-40), 3.61 (1H, dd, J = 12.0, 0 0 13 4.2 Hz, H-5 ), 3.52 (1H, dd, J = 12.0, 4.2 Hz, H-5 ); C NMR (150 MHz, DMSO-d6) δ 156.9 (C-6), 153.8 (C-2), 151.4 (C-4), 135.6 (C-8), 116.7 (C-5) 86.4 (C-10), 85.2 (C-40), 73.7 (C-20), 70.4 (C-30), 61.4 (C-50); ESI-MS m/z 284.3 [M + H]+. HPLC quantification of 1–3 in the callus extract was performed with a Waters HPLC system equipped with a Waters 1525 binary pump, Waters 2707 autosampler, and Waters 2998 photodiode array detector using Acclaim Polar Advantage II C18 (250 × 4.6 mm, 5 µm; Thermo Fisher Scientific). The eluent was as follows: 0.1% formic acid-water (A) and methanol (B), with 100%–95% of solvent A for 40 min at a flow rate of 1.0 mL/min under 254 nm. Each sample was injected in a volume of 10 µL. Total phenolic and flavonoid contents were determined, as described previously [36–38].

2.3. Antioxidant Activity of P. pyrifolia Callus Extract

2.3.1. DPPH Radical Scavenging Assay The DPPH radical scavenging activity of the callus extract was examined according to the method of Brand-Williams et al. [39]. Brieﬂy, 100 µL of aqueous solution of callus extract or ascorbic acid was mixed with 100 µL of DPPH in methanol (0.2 mM). The mixtures were then incubated in the dark for 30 min at 25 ◦C. The negative control and background test sample were prepared by adding 100 µL of DPPH solution to 100 µL of methanol and ascorbic acid solution or callus extract, respectively. The absorbance of the sample at 517 nm was then measured against a blank consisting of a mixture of 100 µL DPPH solution and 100 µL water. The radical scavenging activity was calculated using the following equation: Cosmetics 2018, 5, 71 5 of 25

Free radical scavenging activity (%) = [1 − (B − C)/A] × 100 (1) where A is the absorbance at 517 nm of the negative control, B represents the absorbance of the test sample at 517 nm, and C is the absorbance of the background test sample at 517 nm.

2.3.2. ABTS Radical Scavenging Assay The ABTS radical scavenging activity was evaluated using the method of Re et al. with minor modiﬁcations [40]. ABTS•+ solution was produced by mixing 7 mM ABTS with 2.45 mM potassium persulfate solution (1:1, v/v) at 25 ◦C in the dark for 12–16 h. Then, this solution was diluted with water to obtain an absorbance of 0.70 ± 0.05 at 734 nm and used as a negative control. An aliquot of 100 µL of the test sample solution was mixed with 3.9 mL of the diluted ABTS•+ solution. After incubation at room temperature for 30 min, the absorbance of the mixture was measured at 734 nm. The ABTS radical scavenging activity was calculated using Equation (1) with C as the absorbance of the test sample at 734 nm before incubation.

2.3.3. FRAP Assay The FRAP assay is a method for measuring the ability of antioxidants to reduce Fe3+ to Fe2+, resulting in the formation of a blue-colored complex of Fe2+ with TPTZ, which shows absorbance at 593 nm. The assay was carried out based on the method described by Pulido et al. with some modiﬁcations [41]. The FRAP reagent was prepared by mixing 2.5 mL of TPTZ solution (10 mM TPTZ in 40 mM HCl), 2.5 mL of FeCl3 solution (20 mM), and 45 mL of acetate buffer (300 mM at pH 3.6). Then, 3 mL of FRAP reagent was added to 100 µL of the sample solution. The mixture was incubated at 37 ◦C for 10 min, and the absorbance was measured at 593 nm relative to a blank (sample solution containing water instead of the callus extract). The antioxidant potential of the sample was determined based on a calibration curve prepared using FeSO4 at concentrations of 0–2000 µM.

2.3.4. Protein Glycation Assay The antiglycation effects of P. pyrifolia callus extract on collagen and elastin were evaluated using collagen and elastin glycation assay kits (Cosmo Bio Co., Ltd., Tokyo, Japan) according to the procedure described by the manufacturer. Briefly, all samples and standard solutions were kept at room temperature before analysis. For collagen glycation assay, 50 µL of collagen solution was aliquoted into 96-well black plates and incubated at 37 ◦C overnight to allow for gelation of the solution. To each well containing collagen gel was added 40 µL of different concentrations of aminoguanidine (antiglycation standard) or P. pyrifolia extract powder diluted in dilution buffer. Separately, elastin solution (50 µL) was added to each well of 96-well black plates followed by 40 µL of antiglycation standards and P. pyrifolia extract solution in dilution buffer. Then, glyceraldehyde solution (500 mM, 10 µL) was thoroughly mixed with each standard and sample in the wells, except for the sample blank. After immediately measuring fluorescence at 0 h using a fluorescence plate reader at an excitation wavelength of 370 nm and an emission wavelength of 440 nm, the plates were incubated at 37 ◦C for 24 h or 6 days under conditions of high humidity to induce collagen and elastin glycation, respectively. The formation of glycated collagen or elastin was then assessed by measuring the intensity of AGE-specific fluorescence. The inhibitory effect of glycation was calculated using the following equation based on the reduction of fluorescence intensity:

Inhibition of protein glycation (%) = (B − A)/A × 100 (2) where A represents the reduction in ﬂuorescence intensity of the test sample before and after incubation, and B is the reduction in ﬂuorescence intensity of the standard with 0 mM aminoguanidine before and after incubation. Cosmetics 2018, 5, 71 6 of 25

2.4. Antiaging Potential of a Callus Extract of P. pyrifolia

2.4.1. Cell Proliferation Assay In order to evaluate the effects of P. pyrifolia callus extract on keratinocyte (HaCaT) and fibroblast (CCD-986sk) cell proliferation, HaCaT or CCD-986sk cells were seeded at a density of 1 × 103 or 5 × 103 cells per well in 100 µL of Dulbecco’s modified Eagle’s medium (DMEM; Lonza, Zurich, Switzerland) containing 10% (v/v) fetal bovine serum (FBS; Thermo Fisher Scientific) and 1% (v/v) penicillin/streptomycin (Thermo Fisher Scientific) in each 96-well plate, and then incubated at 37 ◦C for 24 h. Next, the cells were treated with different concentrations of adenosine or P. pyrifolia callus extract in DMEM with 0.5% (v/v) FBS, alone or in combination, and incubated for a further 24 h. Cell viability was then evaluated by WST-1 assay (Roche Diagnostics, Mannheim, Germany) performed in accordance with the manufacturer’s instructions. Briefly, 10 µL of diluted WST-1 solution (5 mg/mL in phosphate-buffered saline [PBS], pH 7.4) was added to each well and incubated for 2 h. The absorbance was then measured at 450 nm and the percentage cell viability was calculated by comparing the values of treated cells to those of untreated cells.

2.4.2. Procollagen Synthesis Assay To determine whether the callus extract exerted antiwrinkle activity by promoting collagen production, the concentration of procollagen type I C-peptide synthesized by ﬁbroblast cells was evaluated as a biomarker of procollagen formation. Aliquots of 1 × 104 CCD-986sk cells in 100 µL of DMEM with 10% (v/v) FBS were seeded into each well of 96-well plates and then cultured until the cells reached 80% conﬂuence. After incubation in DMEM with 0.5% (v/v) FBS for 12 h, the cells were cultured in serum-free DMEM at 37 ◦C in the presence of various concentrations of callus extract alone or in combination with 0.015 µM of adenosine for a further 24 h. Next, the level of procollagen type I C-peptide in the culture medium was assessed using an enzyme-linked immunosorbent assay (ELISA) kit (Takara Bio, Shiga, Japan) according to the manufacturer’s instructions.

2.4.3. In Vitro Scratch Wound Recovery Assay To examine the promotion of skin regeneration by P. pyrifolia callus extract, HaCaT and CCD-986sk cells were seeded in Essen ImageLock 96-well plates (Essen Bioscience, Ann Arbor, MI, USA) at a density of 1 × 104 cells/well and 4 × 104 cells/well, respectively, and then cultured in complete medium at 37 ◦C for 48 h to form conﬂuent monolayers. After treating the cells with 5 µg/mL mitomycin C in culture medium for 1 h to block proliferation, a scratched wound area (cell-free zone) was formed in each well using a wound-maker. The cells were then washed twice with PBS (pH 7.4) and further incubated in 200 µL of DMEM with 0.5% (v/v) FBS and different concentrations of adenosine or P. pyrifolia callus extract alone or in combinations at 37 ◦C. Wound closure induced by cell migration was observed every 6 h using an IncuCyte Zoom microscope (Essen Bioscience) and the extent of wound recovery was automatically calculated using phase-contrast image analysis software.

2.5. Preparation and Characterization of P. pyrifolia Callus Extract-Loaded NLs P. pyrifolia callus extract-loaded NLs (PC-NLs) were produced by emulsification followed by microfluidization. Briefly, Lipoid P75-3 (2.5 g), Lipoid P100-3 (2.5 g), cholesterol (1 g), squalene (1 g), ethanol (1 g), and caprylic capryl triglyceride were mixed at 60 ◦C to form an oil phase. Separately, the P. pyrifolia callus extract (3 g) and glycerol (50 g) were dissolved in 34 g of water (aqueous phase). The oil phase was then slowly added to the aqueous solution and mixed at 3000 rpm for 10 min using a homogenizer (Primix Corp., Hyogo, Japan) to form a primary emulsion. Then, the primary emulsion was passed through a microfluidizer (LM20; Microfluidics Corp., Westwood, MA, USA) operating at 1000 bar (15 cycles) to prepare PC-NLs. Callus extract-loaded elastic NLs (PC-ENLs) were produced by the thin-film hydration method followed by microfluidization. Briefly, the organic phase containing Lipoid P75-3 (4.28 g), Lipoid Cosmetics 2018, 5, 71 7 of 25

P100-3 (4.28 g), and cholesterol (5.7 g) was dissolved in a mixture of chloroform and ethanol (4:1, v/v). Then, the organic solvent was completely removed using a rotary evaporator, forming a thin film in a round-bottomed flask. The aqueous phase containing the callus extract (1.85 g) and Tween 80 (1.07 g) in 11.14 mL of PBS (pH 7.4) was added to the flask, followed by hydration of the thin film with continuous sonication. After adding 33.42 mL of PBS (pH 7.4), the dispersion was then passed through a microfluidizer at a pressure of 1000 bar for 15 cycles to form nanosized elastic NLs containing callus extract. To evaluate the average encapsulation efficiency of P. pyrifolia callus extract in NLs or elastic NLs, 500 µL of the dispersion of NLs was centrifuged at 18,000 rpm for 1 h. The concentration of unincorporated adenosine in the supernatant was then determined by HPLC at 320 nm, as described above. In addition, the mean hydrodynamic diameter, polydispersity index (PDI), and zeta potential of the PC-NLs or PC-ENLs were assessed at 25 ◦C by dynamic light scattering (Malvern Zetasizer Nano ZS90; Malvern Instruments, Malvern, UK) after dilution in deionized water (1:20). The NLs were further diluted 1:100 in water, and drops of the diluted NLs were placed on copper grids. After removing excess liquid with filter paper, a drop of 2% (w/v) aqueous phosphotungstic acid was added to the grid for negative staining and the morphology of the NLs was examined by transmission electron microscopy (TEM, JEM-200; JEOL, Tokyo, Japan).

2.6. In Vitro Skin Permeability of P. pyrifolia Callus Extract-Loaded NLs The in vitro artificial skin permeabilities of P. pyrifolia callus extract, PC-NLs, or PC-ENLs were evaluated using a Skin PAMPA Explorer Test System (Pion, Billerica, MA, USA). Each well of the top (donor) compartment of a Skin PAMPA sandwich plate was hydrated with 200 µL of diluted hydration solution (pH 7.4) overnight. Before forming the sandwich, the bottom (acceptor) plate was filled with 200 µL of Prisma-HT buffer solution (pH 7.4). Then, 200 µL of each sample solution of P. pyrifolia callus extract, PC-NLs, or PC-ENLs diluted with Prisma-HT buffer solution (equivalent to 100 µg/mL adenosine) was loaded into the donor plate well and the resulting sandwich was incubated at room temperature. After 1, 3, 6, 9, 12, and 24 h, each PAMPA sandwich was separated, and 100 µL aliquots of both donor and acceptor solutions were collected. The adenosine from the permeated P. pyrifolia callus extract in the artificial skin permeation experiments was quantified using an HPLC system with UV detection, as described above. To evaluate the human skin permeability of the P. pyrifolia callus extract or PC-ENLs across an epidermal layer, human epidermis (Huskin; Hans Biomed Corp., Daejeon, Republic of Korea) was mounted with the SC facing upward on the receptor compartment of a Franz diffusion cell system containing 5 mL of PBS (pH 7.4). After the donor compartment was clamped in place, 500 µL of P. pyrifolia callus extract or PC-ENLs diluted in PBS (pH 7.4) (equivalent to 100 µg/mL adenosine) was loaded into the donor compartment (diffusion area: 0.785 cm2). The receptor compartments were stirred at 600 rpm and skin surfaces were maintained at 32 ◦C during the experiment. At 1, 3, 6, 9, 12, and 24 h after loading of the sample solutions, 200 µL of the receptor phase solution was withdrawn and filled with 200 µL of fresh PBS (pH 7.4). After filtering the samples through polyvinylidene difluoride (PVDF) membrane filters (pore size: 0.45 µm), permeated adenosine from the callus extract or PC-ENLs was assessed by HPLC at 254 nm, as described above.

2.7. Statistics The t test was used to compare two mean values of unpaired data, and one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test was used to compare three or more mean values of unpaired data. All data are expressed as means ± standard deviation, and p < 0.05 was taken to indicate statistical signiﬁcance. Cosmetics 2018, 5, 71 8 of 25

3. Results and Discussion

3.1. Preparation and Characterization of Callus Extract from P. pyrifolia The pale yellowish P. pyrifolia callus was successfully induced after transferring in vitro-derived young leaf explants into M&S medium with 2.0 mg/L picloram in comparison with those in 2 mg/mL of 2,4-dichlorophenoxy-acetic acid and 6-benzylaminopurine alone or in combination. Furthermore, plant cell proliferation at the site of explant injury as well as in suspension was maximally stimulated using picloram as a growth regulator in medium supplemented with vitamins, including myoinositol, pyridoxine hydrochloride, nicotinic acid, thiamine hydrochloride, and riboﬂavin. CosmeticsThe aqueous 2018, 5, x FOR extract PEER derived REVIEW from P. pyrifolia callus was screened using HPLC with a photodiode8 of 25 array detector (HPLC-PDA) to determine the chemical composition. Three compounds were detected andwere isolated detected from and the extract,isolated and from their the structures extract, and were their identified structures unambiguously were identified as uridine unambiguously (1), adenosine as uridine (1), adenosine (2), and guanosine (3) by spectroscopic analyses, including UV, NMR, and (2), and guanosine (3) by spectroscopic analyses, including UV, NMR, and mass spectrometry, as well mass spectrometry, as well as by comparison with standards (Figure 1). The contents of nucleosides as by comparison with standards (Figure1). The contents of nucleosides 1–3 in the extract were 1–3 in the extract were evaluated using calibration curves based on peak areas (Y) and evaluated using calibration curves based on peak areas (Y) and concentrations (X, µg/mL) in the concentrations (X, µg/mL) in the ranges of 10–1000 µg/mL. Each calibration curve showed good ranges of 10–1000 µg/mL. Each calibration curve showed good linear regression as follows: uridine linear regression as follows: uridine (1), Y = 19,494X − 19,998, r2 = 1.0000; adenosine (2), Y = 25,897X – 1 − r2 2 r2 3 ( ),41,736, Y = 19,494X r2 = 1.0000;19,998, guanosine= 1.0000;(3), Y = 25,009X adenosine − 8108.2, ( ), Y r =2 = 25,897X 1.0000. Therefore, – 41,736, th=e contents 1.0000; guanosine of the three ( ), 2 Y =ribonucleosides 25,009X − 8108.2, in ther callus= 1.0000. extract Therefore, were determined the contents to be of1620.15 the three ± 2.28 ribonucleosides (1), 1961.55 ± 6.98 in the(2), callusand extract1088.97 were ± 2.08 determined (3) µg/g. to be 1620.15 ± 2.28 (1), 1961.55 ± 6.98 (2), and 1088.97 ± 2.08 (3) µg/g.

(a)

(b)

(c)

FigureFigure 1. High-performance1. High-performance liquid liquid chromatography chromatography (HPLC)(HPLC) profiles proﬁles and and chemical chemical structures structures of ofthe the majormajor compounds compounds in inPyrus Pyrus pyrifolia pyrifoliacallus callus extract.extract. ( (aa)) HPLC HPLC chromatograms chromatograms of of three three standards, standards, uridineuridine (1 ),(1 adenosine), adenosine (2 (),2), and and guanosine guanosine ((33),), andand (b) aqueous aqueous extract extract from from P.P. pyrifolia pyrifolia calluscallus at at254 254 nm. nm. (c)( Structuresc) Structures of of compounds compounds and and their their ultravioletultraviolet (UV)(UV) spectra obtained obtained with with a aphotodiode photodiode array array detectordetector (210–400 (210–400 nm). nm).

Cosmetics 2018, 5, 71 9 of 25

Pyrimidine nucleoside uridine has been shown to rapidly restore the ocular surface in patients with dry eye by increasing hyaluronic acid synthesis, reducing degradative enzymes, such as matrix metalloproteinase-9, and enhancing the number of goblet cells [42]. Adenosine is a naturally occurring purine nucleoside formed by the breakdown of adenosine triphosphate, which is a member of an important class of bioactive molecules with antiwrinkle properties [43]. Therefore, adenosine has been applied in cosmetic preparations to improve facial wrinkles. The guanine-based purine, guanosine, exhibits beneficial effects on injured neural cells and can modulate several signaling pathways to provide neuroprotection. Furthermore, it can modulate glutamatergic metabolism, avoiding overactivation of glutamate receptors, and exerting antioxidant and anti-inflammatory activities [44]. Therefore, the callus extract was expected to possess biological activities related to skin regeneration. Phytochemicals, such as phenolic and flavonoid compounds, have been reported to possess significant antioxidant properties, which are crucial for antiaging skin care products conferring protection against UV radiation and inflammation [45]. However, the total content of phenolic compounds in the callus extract was 9.25 ± 0.14 mg/g of gallic acid equivalents, and flavonoids were not detected by the corresponding AlCl3 method, which has lower sensitivity for phenolics and flavonoids than the levels in the extracts of P. pyrifolia peel or flesh. In addition, the presence of major phenolics and flavonoids in the P. pyrifolia callus extract could not be clearly determined by HPLC-PDA experiments. This may have been due to environmental factors related to the light/dark incubation of callus and the level of picloram in M&S culture medium. Previous studies indicated that the production of phenolics was hindered when the callus was induced under dark conditions, and thereafter the total contents of phenolics and flavonoids were lower than those in the in vivo-derived plant extract [46]. Therefore, the light/dark incubation of cultures may play an important role in phenolic accumulation. Moreover, higher levels of picloram in the culture medium and extraction under aqueous conditions can reduce the total phenolic and flavonoid levels in the in vitro plant extract, which would further influence the antioxidant potential [31].

3.2. Antioxidant Activities of P. pyrifolia Callus Extract

3.2.1. Antioxidant Activities of P. pyrifolia Callus Extract The antioxidant activity of the P. pyrifolia callus extract was expressed as the ability to scavenge free radicals in DPPH and ABTS assays, and to reduce ferric ions in the FRAP test, because radical scavenging activities in the extracts of many plants, vegetables, and fruits have been reported to be linearly correlated with the total phenolic compound and total ﬂavonoid contents [47]. As shown in Figure2, the callus extract and ascorbic acid (positive control) exhibited dose-dependent radical scavenging activity. In the DPPH assay, the callus extract showed free radical scavenging activity of 76.9% ± 2.88% at 300 µg/mL, which was comparable to that of 44 µg/mL ascorbic acid (82.5% ± 3.63%). The IC50 value of the callus extract was 124 ± 6.64 µg/mL, which was equivalent to the value of 16.8 ± 1.81 µg/mL for ascorbic acid (Figure2a). However, the IC 50 of the callus extract in the ABTS test was 1236 ± 7.122 µg/mL, which was 6.84 times higher than that of ascorbic acid (181 ± 0.491 µg/mL) (Figure2b). The observed differences in the capacity of the callus extract to scavenge ABTS and DPPH radicals may have been mainly because, in these assays, the transfer of hydrogen and the transfer of electrons occur at different redox potentials and are dependent on the structures of the antioxidants [48]. The callus extract also showed dose-dependent ferric reducing power in the FRAP assay, which was based on the ability to reduce Fe3+ to Fe2+. At 300 µg/mL, the callus extract exhibited ferric reducing activity of 46.8% ± 3.02%, which was comparable to the value of 719 ± 61.8 µmoL Fe(II)/g for deionized water. The present ﬁndings obtained from three antioxidant assays (DPPH, ABTS, and FRAP) indicated that the callus extract had antioxidant properties related to the total phenolic content. However, antioxidant activity was lower than that reported previously for a pear extract; the DPPH and ABTS Cosmetics 2018, 5, 71 10 of 25 free radical scavenging activities of the ethanol extract of pear peel were reported to be 78.4% ± 0.5% and 78.4% ± 1.1%, respectively, at a concentration of 100 µg/mL [49]. Therefore, further studies are required to optimize the callus culture conditions and solvent system for extraction to enhance the contentsCosmetics 2018 of secondary, 5, x FOR PEER metabolites REVIEW with potent antioxidant activity. 10 of 25

100

Free radical-scavengingDPPHin activity (%) 0 0 1.76 8.8 17.6 44 88 25 50 100 150 300

Ascorbic acid P. pyrifolia callus extract μ Concentration ( g/mL) (a)

100

Free radical-scavenging activity in ABTSFree (%) radical-scavengingactivity 0 0 25 50 100 200 300 500 250 500 750 1000 1500 3000

Ascorbic acid P. pyrifolia callus extract μ Concentration ( g/mL) (b)

FigureFigure 2. Antioxidant2. Antioxidant activity ofactivity the aqueous of extractthe aqueous of P.pyrifolia extractcallus. (aof) 2,2-Diphenyl-2-picrylhydrazyl P. pyrifolia callus. (a) (DPPH)2,2-Diphenyl-2-picrylh and (b) 2,20-azinobis-(3-ethylbenzoline-6-sulfonate)ydrazyl (DPPH) and (b) 2,2′-azinobis-(3-ethylbenzoline-6-sulfonate) (ABTS) free radical scavenging activity. (ABTS) Valuesfree radical are the scavenging means ± standard activity. Values deviation are ( nthe= 6means per group). ± standard deviation (n = 6 per group).

3.2.2. Antiglycation Potential of P. pyrifolia Callus Extract Glycation of protein components of the ECM in the dermis severely damages skin elasticity by cross-linking the fibers, thereby reducing their elasticity. Non-cross-linked advanced glycation end products (AGEs), such as Nε-(carboxymethyl) lysine (CML), also accumulate in the epidermis as well as in collagen. The age-related accumulation of CML results in reduced skin elasticity and the

Cosmetics 2018, 5, 71 11 of 25

3.2.2. Antiglycation Potential of P. pyrifolia Callus Extract Glycation of protein components of the ECM in the dermis severely damages skin elasticity by cross-linking the fibers, thereby reducing their elasticity. Non-cross-linked advanced glycation end products (AGEs), such as Nε-(carboxymethyl) lysine (CML), also accumulate in the epidermis as well asCosmetics in collagen. 2018, 5, x TheFOR PEER age-related REVIEW accumulation of CML results in reduced skin elasticity11 andof 25 the development of a dull appearance and lack of firmness of the skin [50]. To evaluate the inhibitory development of a dull appearance and lack of firmness of the skin [50]. To evaluate the inhibitory activitiesactivities of P. pyrifolia of P. pyrifoliacallus extractcallus onextract collagen on collagen and elastin and glycation,elastin glycation, we measured we measured the total the fluorescent total AGEsfluorescent in collagen AGEs or elastin in collagen glycated or withelastin glyceraldehyde. glycated with glyceraldehyde. As shown in Figure As shown3a, aminoguanidine, in Figure 3a, one of theaminoguanidine, most promising one pharmacological of the most promising interventions pharmacological for inhibiting interventions glycation, for exhibited inhibiting 45.7% glycation,± 2.24% inhibitionexhibited of AGE 45.7% formation ± 2.24% inhibition in collagen of atAGE 741 formationµg/mL, in with collagen an IC at50 741of 727µg/mL,± 49.9 withµ ang/mL. IC50 of The 727 callus ± extract49.9 also µg/mL. inhibited The thecallus glycation extract also of collagen inhibited in the a dose-dependentglycation of collagen manner; in a dose-dependent at 5000 µg/mL, manner; the callus extractat exhibited 5000 µg/mL, 89.3% the ±callus2.70% extract inhibition exhibited of 89.3% AGE ± formation 2.70% inhibition in collagen. of AGE In formation addition, inafter collagen. treatment In with 3000addition,µg/mL after of treatment the callus with extract, 3000 the µg/mL magnitude of the callus of the extract, collagen the glycation magnitude inhibitory of the collagen effect was glycation inhibitory effect was 47.8% ± 3.18%, which was equivalent to that of 741 µg/mL 47.8% ± 3.18%, which was equivalent to that of 741 µg/mL aminoguanidine. aminoguanidine. Figure3b shows the effect of P. pyrifolia callus extract on total AGE formation in elastin over Figure 3b shows the effect of P. pyrifolia callus extract on total AGE formation in elastin over a a 6-day6-day incubation incubation period. period. The The fluorescence fluorescence intensityintensity of of elastin elastin incubated incubated with with glyceraldehyde glyceraldehyde increasedincreased significantly significantly by about by nine-foldabout nine-fold in comparison in comparison to that ofto elastinthat of alone, elastin indicating alone, indicating progressive formationprogressive of AGEs. formation When elastinof AGEs. glycation When elastin occurred glyc ination the presenceoccurred in of the 741 presenceµg/mL aminoguanidine,of 741 µg/mL the percentageaminoguanidine, inhibition the percentage of AGE formationinhibition of was AGE 57.4% formation± 0.451%, was 57.4% with ± 0.451%, an estimated with an estimated IC50 value of 602IC±50 2.72valueµ ofg/mL. 602 ± 2.72 The µg/mL. callus The extract callus alsoextract significantly also significantly reduced reduced the the formation formation ofof AGEs in in a concentration-dependenta concentration-dependent manner, manner, and and at 5000 at 5000µg/mL, µg/mL, the the callus callus extract decreased decreased AGE AGE formation formation by 82.7%by 82.7%± 4.59%. ± 4.59%. Upon Upon incubation incubation of of elastinelastin with with glyceraldehyde glyceraldehyde and and 3000 3000 µg/mLµg/mL callus callusextract, extract,the percentage inhibition of AGE formation was 51.3% ± 1.45%, which was comparable to the effect of the percentage inhibition of AGE formation was 51.3% ± 1.45%, which was comparable to the effect of 741 µg/mL aminoguanidine. 741 µg/mL aminoguanidine. Although the level of phenolic compounds in callus extract was low, positive glycation Althoughinhibitory the effects level on of collagen phenolic and compounds elastin, which in callus have extract crucialwas roles low, in maintaining positive glycation the structural inhibitory effectsintegrity on collagen and elasticity and elastin, of the which skin, were have observed crucial. roles However, in maintaining further cell-based the structural antioxidant integrity studies and elasticityare ofrequired the skin, to wereexamine observed. the possible However, antiaging further effect cell-based of the callus antioxidant extract studieson the areskin required via its to examineinhibition the possible of ROS-induced antiaging effectoxidative of the damage callus extractto cells, on which the skin leads via to its lipid inhibition peroxide of ROS-inducedformation, oxidativemitochondrial damage to and cells, DNA which damage, leads and to alterations lipid peroxide in protein formation, and gene mitochondrial function [9]. and DNA damage, and alterations in protein and gene function [9].

100

Inhibition of collagen glycation (%) glycation collagen of Inhibition 10

0 0 59.3 296 741 2500 3000 4000 5000

Aminoguanidine P. pyrifolia callus extract μ Concentration ( g/mL) (a)

Figure 3. Cont.

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20 Inhibition of elastin glycation (%) glycation of elastin Inhibition 10

0 0 59.3 296 741 2500 3000 4000 5000

Aminoguanidine P. pyrifolia callus extract μ Concentration ( g/mL) (b)

FigureFigure 3. In vitro 3. In antiglycationvitro antiglycation effects effects of P. of pyrifolia P. pyrifoliacallus callus extract. extract. Inhibition Inhibition of of advanced advanced glycation glycationend productend (AGE) product formation (AGE) formation in (a) collagen in (a) andcollagen (b) elastinand (b) via elastin glyceraldehyde via glyceraldehyde in the absencein the absence or presence or presence of the indicated concentrations of aminoguanidine or callus extract. Data are the means ± of the indicated concentrations of aminoguanidine or callus extract. Data are the means ± standard standard deviation percentages of the control (n = 6 per group). deviation percentages of the control (n = 6 per group).

3.3. Skin3.3. Regeneration Skin Regeneration Activities Activities of P. of pyrifolia P. pyrifolia Callus Callus Extract Extract 3.3.1. Effects of Callus Extract and Combination of Callus Extract and Adenosine on Keratinocyte 3.3.1. Effects of Callus Extract and Combination of Callus Extract and Adenosine on Keratinocyte and Fibroblastand Fibroblast Cell Proliferation Cell Proliferation In vitro cell viability assays were performed using HaCaT and CCD-986sk cells to determine the In vitro cell viability assays were performed using HaCaT and CCD-986sk cells to determine effects of P. pyrifolia callus extract alone and in combination with adenosine on cell proliferation. As the effectsshown of inP. Figure pyrifolia 4a,callus adenosine extract and aloneP. pyrifolia and callus in combination extract exhibited with dose-dependent adenosine oncell stimulation proliferation. of As shownHaCaT in Figure cell proliferation;4a, adenosine 0.015 and µMP. pyrifoliaadenosinecallus significantly extract exhibitedincreased dose-dependentcell proliferation stimulationby 110% ± of HaCaT2.51% cell proliferation; and cells treated 0.015 withµ M15 adenosineµM adenosine significantly showed 49% increased more proliferation cell proliferation than the by controls. 110% ± The2.51% and cellsrelative treated proliferation with 15 µ Mof cells adenosine treated showed with 1.36 49% µg/mL more P. proliferation pyrifolia callus than extract the was controls. 123% The± 3.68%, relative proliferationwhich was of cells equivalent treated to with that of 1.36 cellsµg/mL treatedP. with pyrifolia 1.5 µMcallus adenosine extract (124% was 123%± 3.71%).± 3.68%,At 13.6 whichµg/mL, was equivalentP. pyrifolia to that callus of cells extract treated induced with a 1.531%µ increaseM adenosine in HaCaT (124% cell± proliferation3.71%). At compared 13.6 µg/mL, to controls,P. pyrifolia callus extractindicating induced that P. apyrifolia 31% increase callus extract in HaCaT alone celleffectively proliferation stimulated compared HaCaT cell to controls, growth. Cells indicating treated that simultaneously with adenosine (0.015 µM) and P. pyrifolia callus extract (0.0136 µg/mL) showed P. pyrifolia callus extract alone effectively stimulated HaCaT cell growth. Cells treated simultaneously significantly increased proliferation (172% ± 12.6%), which was 1.28- and 1.87-fold higher than those with adenosine (0.015 µM) and P. pyrifolia callus extract (0.0136 µg/mL) showed significantly increased seen with adenosine and P. pyrifolia callus extract alone, respectively (Figure 4b). ± proliferationSimilarly, (172% the12.6%), proliferation which wasof CCD-986sk 1.28- and 1.87-foldcells also higherincreased than with those increasing seen with adenosine adenosine and P.concentration, pyrifolia callus and extract the relative alone, respectivelycell proliferation (Figure at 154 b).µM adenosine was 70% higher than that of Similarly,controls. On the the proliferation other hand, fibroblast of CCD-986sk cell growth cells was increased also increased to 156% with± 5.42% increasing at 0.0136 µg/mL adenosine of concentration,P. pyrifolia and callus the extract, relative which cell was proliferation 11% higher at than 15 µ thatM adenosine seen with 0.15 was µM 70% adenosine, higher than but thatcell of controls.proliferation On the other was hand,not significantly fibroblast cellincreased growth at wasP. pyrifolia increased callus to 156%extract± concentrations5.42% at 0.0136 >0.0136µg/mL of P. pyrifoliaµg/mL (Figurecallus extract,4c). Combined which treatment was 11% with higher adenosine than thatand P. seen pyrifolia with callus 0.15 extractµM adenosine, also resulted but in cell proliferationa greater was cell not proliferative significantly effect increased than either at agentP. pyrifolia alone (Figurecallus extract4d). Additionally, concentrations the proliferation >0.0136 µ g/mLof human fibroblasts was maximized by treatment with 0.0136 µg/mL of P. pyrifolia callus extract (Figure4c). Combined treatment with adenosine and P. pyrifolia callus extract also resulted in a greater combined with 0.015 µM adenosine, and the relative cell proliferation was 12% and 5% greater than cell proliferative effect than either agent alone (Figure4d). Additionally, the proliferation of human those seen with adenosine (0.015 µM) or P. pyrifolia callus extract (0.0136 µg/mL), respectively. fibroblasts was maximized by treatment with 0.0136 µg/mL of P. pyrifolia callus extract combined with

0.015 µM adenosine, and the relative cell proliferation was 12% and 5% greater than those seen with adenosine (0.015 µM) or P. pyrifolia callus extract (0.0136 µg/mL), respectively. Cosmetics 2018, 5, 71 13 of 25 Cosmetics 2018, 5, x FOR PEER REVIEW 13 of 25

The strong cell proliferation activity of P. pyrifolia callus extract on keratinocytes and fibroblasts The strong cell proliferation activity of P. pyrifolia callus extract on keratinocytes and ﬁbroblasts may be closely related to the presence of uridine (1), adenosine (2), and guanosine (3), suggesting may be closely related to the presence of uridine (1), adenosine (2), and guanosine (3), suggesting that that the callus extract may be a potent rejuvenator when included along with other antiaging the callus extract may be a potent rejuvenator when included along with other antiaging ingredients, ingredients, such as adenosine, in cosmetic formulations to improve undesirable wrinkles. such as adenosine, in cosmetic formulations to improve undesirable wrinkles.

180

160 *** 140 *** *** *** 120 ** ** * 100 *

Relative HaCaT cellproliferation (%) 20

0 0 0.015 0.15 1.5 15 0 0.0136 0.136 1.36 13.6

Adenosine (μM) P. pyrifolia callus extract (μg/mL) Concentration (a)

200 ### μ Adenosine (0.015 M) *** ### 180 ### *** ### *** 160 ***

140 ***

120

100

Relative HaCaT cell proliferation(%) 20

0 Control 0 0.00136 0.0136 0.136 1.36

P. pyrifolia callus extract (μg/mL)

(b)

Figure 4. Cont.

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200

180 *** *** 160 *** *** 140 *** *** 120 * 100 ** 80

20 Relative CCD-986sk cell proliferation (%) proliferation CCD-986sk cell Relative 0 0 0.015 0.15 1.5 15 0 0.00136 0.0136 0.136 1.36

Adenosine (μM) P. pyrifolia callus extract (μg/mL) Concentration (c) 180 Adenosine (0.015 μM) 160 ### ### *** *** 140 *** *** *** 120

100

20 Relative CCD-986sk cell proliferation (%) proliferation cell CCD-986sk Relative 0 Control 0 0.00136 0.0136 0.136 1.36

μ P. pyrifolia callus extract ( g/mL) (d)

FigureFigure 4. 4.Relative Relative HaCaT HaCaT cell cell proliferation proliferation after after treatment treatment with with ( a(a))P. P. pyrifolia pyrifoliacallus callus extract extract alone alone or or (b(b)) in in combination combination withwith adenosineadenosine (0.015 µM).µM). CCD CCD-986sk-986sk cell cell proliferation proliferation after after treatment treatment with with (c) (cP.) P.pyrifolia pyrifolia calluscallus extract extract alone alone or (d or) in (d combination) in combination with adenosine with adenosine (0.015 µM). (0.015 ValuesµM). Valuesare means are ± meansstandard± standard deviation deviation (n = 6 per (n group).= 6 per * group). p < 0.05, * p**< p 0.05,< 0.01, ** p***< p 0.01, < 0.001 *** pcompared< 0.001 compared to untreated to untreatedcontrols. controls.### p < 0.001 ### comparedp < 0.001 compared to 0.015 µM to 0.015adenosineµM adenosine alone. alone.

3.3.2.3.3.2. Procollagen Procollagen Synthesis Synthesis Effects Effects of of Treatment Treatment with withP. P. pyrifolia pyrifoliaCallus Callus Extract Extract Alone Alone and and in in CombinationCombination with with Adenosine Adenosine Collagen,Collagen, oneone ofof thethe majormajor proteinsproteins inin ECM,ECM, isis the the main main building buildingblock blockof ofconnective connective tissue,tissue, hair,hair, and and nails,nails, andand isis responsible for maintaining maintaining th thee elasticity, elasticity, strength, strength, and and flexibility ﬂexibility of ofskin. skin. In Inaddition, addition, adenosine adenosine has has been been shown shown to to modulate modulate ce cellularllular and organ functionfunction viavia occupancyoccupancy of of four four speciﬁcspecific cell cell surface surface receptors receptors (A1, (A1, A2A, A2A, A2B, A2B, and and A3), A3), all all of of which which are are members members of of the the large large family family ofof 7-transmembrane 7-transmembrane spanning spanning and and heterotrimeric heterotrimeric G G protein-associated protein-associated receptorsreceptors [51[51].]. InIn particular,particular, the activation of adenosine A2A receptors promoted collagen synthesis of human fibroblast cells [52]. Therefore, topical treatment with adenosine increased the number of myofibroblasts, which are

Cosmetics 2018, 5, 71 15 of 25

Cosmetics 2018, 5, x FOR PEER REVIEW 15 of 25 theresponsible activation for of adenosinethe contraction A2A receptors of granulation promoted tiss collagenue during synthesis tissue ofregeneration, human fibroblast with increasing cells [52]. Therefore,concentration. topical Therefore, treatment we with examined adenosine the effects increased of callus the number extract ofalone myofibroblasts, or in combination which with are responsibleadenosine on for collagen the contraction production of granulationby human fibrobla tissuests during by measuring tissue regeneration, the level of procollagen with increasing type I concentration.C-peptide. As Therefore,shown in Figure we examined 5, the biosynthesis the effects ofof callusprocollage extractn type alone I C-peptide or in combination by CCD-986sk with adenosinecells was onpromoted collagen productionby the extract by humanin a dose-dependent fibroblasts by measuring manner and the levelsignificantly of procollagen increased type by I C-peptide.treatment Aswith shown 0.0136–1.36 in Figure µg/mL5, the of biosynthesis P. pyrifolia ofcallus procollagen extract in type comparison I C-peptide with by the CCD-986sk control group; cells wascells promoted treated with by the 1.36 extract µg/mL in of a P. dose-dependent pyrifolia callus mannerextract showed and significantly 1.60- and 1.43-fold increased increases by treatment in the µ withlevels 0.0136–1.36 of procollageng/mL type of I C-peptide,P. pyrifolia respectively,callus extract compared in comparison to control with cells the and control those group; treated cells with µ treatedadenosine with alone 1.36 (0.015g/mL µM). of P.In pyrifoliaaddition,callus the synthe extractsis showedof procollagen 1.60- and type 1.43-fold I C-peptide increases was enhanced in the levelsby adding of procollagen P. pyrifolia typecallus I extract C-peptide, to adenosine respectively, (0.015 compared µM); the level to control of procollagen cells and type those I C-peptide treated µ withproduction adenosine after alone treatment (0.015 withM). Ina combination addition, the of synthesis P. pyrifolia of procollagencallus extract type (0.0136 I C-peptide µg/mL) was and µ enhancedadenosine by (0.015 adding µM)P. pyrifoliawas 1.51-,callus 1.11-, extract and 1.35-fold to adenosine greater (0.015 than M);after the treatment level of procollagenwith control, type callus I µ C-peptideextract alone production (0.0136 µg/mL), after treatment and adenosine with a combination alone (0.015of µM),P. pyrifolia respectively.callus extractThese results (0.0136 confirmedg/mL) µ andtheadenosine fibrogenic (0.015 role of M)the was callus 1.51-, extract 1.11-, alone and 1.35-foldand in combination greater than with after adenosine treatment withon the control, skin. callus extract alone (0.0136 µg/mL), and adenosine alone (0.015 µM), respectively. These results confirmed the fibrogenic role of the callus extract alone and in combination with adenosine on the skin.

160 P. pyrifolia callus extract alone P. pyrifolia callus extract + Adenosine (0.015 μM) 140

# # *** *** 120 *** # ## *** *** *** *** 100 # 80

20 Procollagen Type I C-peptideProcollagen (ng/mL) Type

0 0 0.00136 0.0136 0.136 1.36 μ P. pyrifolia callus extract ( g/mL) FigureFigure 5. 5.Procollagen Procollagen type type I C-peptideI C-peptide synthesis synthesis by by CCD-986sk CCD-986sk cells cells after after treatment treatment with withP. P. pyrifolia pyrifolia calluscallus extract extract alone alone or inor combination in combination with adenosinewith aden (0.015osine µ(0.015M) for µM) 24 h. for Values 24 h. are Values means are± standard means ± deviationstandard ( ndeviation= 6 per group). (n = 6 per *** pgroup).< 0.001 *** compared p < 0.001 to compared untreated to control. untreated # p < control. 0.05, ## #p p< <0.01 0.05, compared ## p < 0.01 tocompared the same concentrationto the same concentration of P. pyrifolia ofcallus P. pyrifolia extract callus alone. extract alone.

3.3.3. Effects of P. pyrifolia Callus Extract Alone and in Combination with Adenosine on In Vitro 3.3.3. Effects of P. pyrifolia Callus Extract Alone and in Combination with Adenosine on In Vitro Scratch Wound Recovery Scratch Wound Recovery Next, we examined the dose-dependent effects of P. pyrifolia callus extract alone and in Next, we examined the dose-dependent effects of P. pyrifolia callus extract alone and in combination with adenosine on HaCaT cell proliferation/migration using a wound healing assay. combination with adenosine on HaCaT cell proliferation/migration using a wound healing assay. Both P. pyrifolia callus extract and adenosine alone promoted relative wound recovery in a dose- Both P. pyrifolia callus extract and adenosine alone promoted relative wound recovery in a dose- and and time-dependent manner. Treatment with P. pyrifolia callus extract alone (1.36 µg/mL) for 9 h time-dependent manner. Treatment with P. pyrifolia callus extract alone (1.36 µg/mL) for 9 h signiﬁcantly accelerated wound closure of keratinocytes, with a value of 68.5% ± 2.18%, which was significantly accelerated wound closure of keratinocytes, with a value of 68.5% ± 2.18%, which was higher than those for control treatment and treatment with 0.15 µg/mL adenosine alone. Similarly, higher than those for control treatment and treatment with 0.15 µg/mL adenosine alone. Similarly, adenosine also induced a dose-dependent increase in HaCaT cell proliferation/migration up to 15 µM, adenosine also induced a dose-dependent increase in HaCaT cell proliferation/migration up to 15 µM, and the relative wound recovery at 1.5 µM adenosine was equivalent to that of 1.36 µg/mL of callus and the relative wound recovery at 1.5 µM adenosine was equivalent to that of 1.36 µg/mL of callus extract (Figure6a). The process of wound closure, driven by the proliferation/migration of HaCaT extract (Figure 6a). The process of wound closure, driven by the proliferation/migration of HaCaT cells, is illustrated by representative images in Figure 6b,c. The relative wound recovery at 9 h after the combined treatment with P. pyrifolia callus extract (0.0136 µg/mL) and adenosine (0.015 µM) was

Cosmetics 2018, 5, 71 16 of 25

cells,Cosmetics is illustrated 2018, 5, x FOR by PEER representative REVIEW images in Figure6b,c. The relative wound recovery at 9 h16 after of 25 the combined treatment with P. pyrifolia callus extract (0.0136 µg/mL) and adenosine (0.015 µM) was 72.8%72.8%± ± 5.07%,5.07%, whichwhich waswas 21%21% greatergreater thanthan thosethose seenseen withwith thethe samesame concentrationsconcentrations ofofP. P. pyrifolia pyrifolia calluscallus extract extract (60.0% (60.0%± ±3.76%) 3.76%) oror adenosineadenosine (60.4% (60.4%± ± 2.04%) alonealone (Figure(Figure6 6b).b). Although Although the the wounded wounded areaarea in in the the combined combined treatment treatment group group was was recovered recovered completely completely at at 12 12 h h (93.7% (93.7%± ±5.46%), 5.46%), sizeable sizeable woundwound area area gaps gaps in in the the monolayer monolayer were were still still present present in in the the control control as as well well as as in in the the cells cells treated treated with with P.P. pyrifolia pyrifoliacallus callus extract extract or adenosineor adenosine alone. alone. At 12 At h after 12 h treatment after treatment with serum-free with serum-free medium (control),medium P.(control), pyrifolia P.callus pyrifolia extract, callus or extract, adenosine, or adenosine, the relative the wound relative recovery wound recovery values were values 85.2% were± 85.2%3.89%, ± 82.2%3.89%,± 82.2%4.37%, ± and4.37%, 89.0% and± 89.0%5.89%, ± 5.89%, respectively. respectively.

100

90 *** 80 *** 70 *** *** *** *** 60 * *** *** 50

Relative wound recovery of HaCaTcells at 9h (%) 0 0.015 0.15 1.5 15 150 0 0.0136 0.136 1.36 13.6 136

Adenosine (μM) P. pyrifolia callus extract (μg/mL) (a)

Control Adenosine (0.015 μM) 110 P. pyrifolia callus extract (0.0136 μg/mL) P. pyrifolia callus extract (0.0136 μg/mL) + Adenosine (0.015 μM) 100

90 $$$ ### 80 *** 70 * 60 # *** * 50

Relative wound of HaCaT cells (%) recovery 0

0123456789101112 Time (h)

(b)

Figure 6. Cont.

Cosmetics 2018, 5, 71 17 of 25 Cosmetics 2018, 5, x FOR PEER REVIEW 17 of 25

(c)

Figure 6. RelativeRelative scratch scratch wound wound recovery recovery of of HaCaT HaCaT cells cells after after incubation incubation with with adenosine adenosine and and P. pyrifoliaP. pyrifolia callus callus extract.extract. (a) (Relativea) Relative scratch scratch wound wound recovery recovery of ofHaCaT HaCaT cells cells after after 9 h. 9 ( h.b) (Timeb) Time course course of relativeof relative scratch scratch wound wound recovery recovery area area of of HaCaT HaCaT cells. cells. (c ()c )Representative Representative microscopic microscopic images images of scratch woundswounds afterafter cellscells were were treated treated with with serum-free serum-free medium medium (control), (control), adenosine adenosine (0.015 (0.015µM), µM), and andP. pyrifolia P. pyrifoliacallus callus extract extract (0.0136 (0.0136µg/mL) µg/mL) alone or alone in combination or in combination with adenosine with adenosine (0.015 µM). (0.015 Values µM). are Valuesmeans ±arestandard means ± deviationstandard deviation (n = 6). * p (n< = 0.05, 6). * ***p

In vitrovitroscratch scratch wound wound recovery recovery assay assay with with human human fibroblast fibroblast cells yielded cells similaryielded results similar (Figure results7). (FigureTreatment 7). ofTreatment fibroblasts of with fibroblastsP. pyrifolia withcallus P. pyrifolia extract callus or adenosine extract aloneor adenosine led to rapid alone scratch led to wound rapid scratchclosure wound of CCD-986sk closure cellof CCD-986sk monolayers. cell Therefore, monolayers the. recoveryTherefore, rates the afterrecovery 36 h rates of treatment after 36 withh of treatmentP. pyrifolia withcallus P. extractpyrifolia (0.0136 callus extractµg/mL) (0.0136 or adenosine µg/mL) or (0.015 adenosineµM) alone (0.015 were µM) 22% alone and were 33% 22% higher and 33%than higher that of serum-freethan that of medium serum-free control medium (56.9 ± control7.31%), (56.9 respectively ± 7.31%), (Figure respectively7a). In addition, (Figure at7a). 36 In h addition,after treatment at 36 h with after 1.36 treatmentµg/mL with of P. pyrifolia1.36 µg/mLcallus of extractP. pyrifolia or 1.5 callusµM adenosineextract or 1.5 alone, µM theadenosine wound alone,closure the area wound had increased closure area by 38% had and increased 48% compared by 38% toand the 48% control compared group, respectively,to the control but group, there respectively,were no obvious but improvements there were no in scratchobvious wound improvements recovery when in scra thetch concentrations wound recovery of callus when extract the or concentrationsadenosine were increasedof callus aboveextract 1.36 orµ g/mLadenosin ande 1.5wereµM, increased respectively. above In addition, 1.36 µg/mL combined and treatment1.5 µM, respectively.with P. pyrifolia In callusaddition, extract combined (0.0136 treatmentµg/mL) andwith adenosine P. pyrifolia (0.015 callusµ M)extract significantly (0.0136 µg/mL) accelerated and adenosinewound closure (0.015 in comparisonµM) significantly to the control.accelerated A synergistic wound effectclosure on in wound comparison closure wasto the observed control. after A synergistictreatment with effectP. pyrifoliaon woundcallus closure extract was (0.0136 observedµg/mL) after along treatment with adenosine with P. pyrifolia (0.015 µcallusM), and extract their (0.0136wound µg/mL) recovery along rates werewith adenosine maximally (0.015 accelerated µM), byand 31% their and wound 18% compared recovery torates each were material maximally alone, acceleratedrespectively, by at 31% 24 h and after 18% treatment compared (Figure to each7b,c). materi Thus,al almost alone, complete respectively, wound at 24 closure h after at treatment 48 h was (Figureobserved 7b,c). with Thus, combined almostP. pyrifoliacompletecallus wound extract closure (0.0136 at 48µ g/mL)h was observed and adenosine with (0.015combinedµM) P. treatment, pyrifolia callusresulting extract in relative (0.0136 wound µg/mL) recovery and adenosine of 92.1% ±(0.3.14%,015 µM) which treatment, was 1.20-, resulting 1.08-, and in 1.10-foldrelative wound higher recoverythan the recoveryof 92.1% rates ± 3.14%, of control which cells was or 1.20-, cells treated1.08-, and with 1.10-fold the same higher concentrations than the of recoveryP. pyrifolia ratescallus of controlextract (85.2%cells or± cells4.90%) treated and wi adenosineth the same (84.0% concentrations± 6.91%) alone, of P. respectively.pyrifolia callus extract (85.2% ± 4.90%) and adenosine (84.0% ± 6.91%) alone, respectively.

Cosmetics 2018, 5, 71 18 of 25

Cosmetics 2018, 5, x FOR PEER REVIEW 18 of 25 The results presented here support the enhancement of skin regeneration by callus extract via its majorThe bioactive results presented nucleosides, here includingsupport the uridine, enhancemen adenosine,t of skin and regeneration guanosine. by callus Uridine extract ointment via its major bioactive nucleosides, including uridine, adenosine, and guanosine. Uridine ointment was was reported to ameliorate 5-fluorouracil (FU)-induced skin toxicity as well as to protect against reported to ameliorate 5-fluorouracil (FU)-induced skin toxicity as well as to protect against 5-FU-induced keratinocyte damage without influencing the anticancer efficacy of 5-FU [53]. In addition, 5-FU-induced keratinocyte damage without influencing the anticancer efficacy of 5-FU [53]. In adenosine A2A receptors are expressed on most cell types involved in wound healing, including addition, adenosine A2A receptors are expressed on most cell types involved in wound healing, macrophages, fibroblasts, and microvascular endothelial cells. Previous studies indicated that including macrophages, fibroblasts, and microvascular endothelial cells. Previous studies indicated boththat adenosine both adenosine A2A andA2A A2Band A2B receptors receptors contributed contributed to to enhanced enhancedfibroblast fibroblast and endothelial endothelial cell cell migrationmigration [54 ].[54]. Furthermore, Furthermore, adenosine adenosine can promotecan prom angiogenesisote angiogenesis by inhibiting by inhibiting endothelial endothelial secretion of thesecretion antiangiogenic of the antiangiogenic factor, thrombospondin-1, factor, thrombospo stimulatingndin-1, stimulating the production the production of vascular of endothelial vascular growthendothelial factor (VEGF)growth byfactor macrophages (VEGF) byvia macrophages activation ofvia adenosine activation A2A of adenosine receptors, A2A and receptors, inducing VEGFand releaseinducing by retinal VEGF endothelial release by retinal cells through endothelial A2B cells receptors through [51 A2B,55]. receptors [51,55].

100 *** 90 *** *** *** *** *** *** 80 ** *** * 70

0 0 0.015 0.15 1.5 15 150 0 0.0136 0.136 1.36 13.6 136 Relative woundRelative of CCD-986sk at cellsrecovery 36 h (%) μ μ Adenosine ( M) P. pyrifolia callus extract ( g/mL) (a)

Control Adenosine (0.015 μM) 110 P. pyrifolia callus extract (0.0136 μg/mL) P. pyrifolia callus extract (0.0136 μg/mL) + Adenosine (0.015 μM) 100 # 90 $$ *** *** * 80 $$$ *** # 70 *** *

60 **

Relative woundCCD-986sk of recovery (%) cells 0 4 8 12 16 20 24 28 32 36 40 44 48 Time (h)

(b)

Figure 7. Cont.

Cosmetics 2018, 5, 71 19 of 25

Cosmetics 2018, 5, x FOR PEER REVIEW 19 of 25

(c)

FigureFigure 7. 7. RelativeRelative scratch scratch wound wound recovery recovery of of CCD-986s CCD-986skk cells cells after after incubation incubation with with adenosine adenosine and and P. pyrifoliaP. pyrifolia calluscallus extract. extract. (a) ( aRelative) Relative scratch scratch wound wound recovery recovery of of CCD-986sk CCD-986sk cells cells after after 36 36 h. h. (b (b) )Time Time coursecourse of of relative relative scratch scratch wound wound re recoverycovery area area of CCD-986sk cells. ( (cc)) Representative Representative microscopic microscopic imagesimages of of scratch scratch wounds wounds after after treatment treatment with with serum-free serum-free medium medium (control), (control), adenosine adenosine (0.015 (0.015 µM),µM), oror P. pyrifoliapyrifoliacallus callus extract extract (0.0136 (0.0136µg/mL) µg/mL) alone alone or inorcombination in combination with with adenosine adenosine (0.015 (0.015µM). ValuesµM). Valuesare means are ±meansstandard ± standard deviation deviation (n = 6). ( *np =< 6). 0.05, * p** < p0.05,< 0.01, ** p *** < p0.01,< 0.001 *** p compared < 0.001 compared to the untreated to the untreatedcontrol. # control.p < 0.05 # compared p < 0.05 compared to adenosine to aloneadenosine (0.015 aloneµM). (0.015 $$ p < µM). 0.01, $$ $$$ pp << 0.01, 0.001 $$$ compared p < 0.001 to comparedP. pyrifolia tocallus P. pyrifolia extract callus alone extract (0.0136 aloneµg/mL). (0.0136 Scale µg/mL). bar in (Scalec): 300 barµm. in (c): 300 µm.

Therefore,Therefore, the the overall overall results results suggest suggest that that topi topicalcal application application of of callus callus extract extract alone alone and and in in combinationcombination with with adenosine adenosine has has the the potential potential to to ac acceleratecelerate the the regeneration regeneration of of damaged damaged skin skin via via stimulationstimulation of of keratinocyte keratinocyte and and fibroblast ﬁbroblast cell proliferation/migration, activating activating collagen collagen synthesis synthesis andand stabilizing stabilizing the the epithelial epithelial barrier. barrier.

3.4.3.4. Characterization Characterization and and Skin Skin Permeability Permeability of of P. P. pyrifolia Callus Extract-Loaded NLs

3.4.1. Characterization of NLs Containing P. pyrifolia Callus Extract 3.4.1. Characterization of NLs Containing P. pyrifolia Callus Extract To increase the skin permeability of P. pyrifolia callus extract, extract-loaded NLs were prepared To increase the skin permeability of P. pyrifolia callus extract, extract-loaded NLs were prepared by emulsification or reverse-phase evaporation followed by microfluidization. All liposomes exhibited by emulsification or reverse-phase evaporation followed by microfluidization. All liposomes ~80% mean entrapment efficacy of callus extract. The mean particle size of the callus extract-loaded exhibited ~80% mean entrapment efficacy of callus extract. The mean particle size of the callus liposomes produced by nano-emulsification (PC-NLs) was 96.2 ± 0.6 nm, with a narrow size range extract-loaded liposomes produced by nano-emulsification (PC-NLs) was 96.2 ± 0.6 nm, with a distribution (PDI = 0.277 ± 0.009). The zeta potential was −67.2 ± 1.40 mV. The elastic NLs narrow size range distribution (PDI = 0.277 ± 0.009). The zeta potential was −67.2 ± 1.40 mV. The incorporating callus extract (PC-ENLs) produced by thin-film hydration had larger vesicles than elastic NLs incorporating callus extract (PC-ENLs) produced by thin-film hydration had larger vesiclesPC-NLs, than which PC-NLs, may havewhich been may producedhave been by produced the incorporation by the incorporation of Tween 80of Tween as an edge80 as activator.an edge ± ± activator.The average The size average and PDIsize ofand PC-ENLs PDI of PC-ENLs were 210 we5.8re 210 nm and± 5.8 0.293 nm and0.021, 0.293 respectively,± 0.021, respectively, and they − ± andalso they had also a negative had a negative surface charge, surface with charge, a zeta with potential a zeta potential of 43.7 of −0.89043.7 ± mV. 0.890 TEM mV. revealed TEM revealed that all thatliposomes all liposomes had nano-sized had nano-sized globular vesicles,globular andvesicl theires, sizesand weretheir similarsizes were to the similar results to obtained the results with obtaineda particle with size a analyzer particle (Figuresize analyzer8). TEM (Figure images 8). ofTEM PC-NLs images showed of PC-NLs more showed regular more and well-defined regular and well-defined spherical liposomes than PC-ENLs. Most PC-NL vesicles were elliptical in shape with a large interior aqueous phase and a flexible lipid layer.

Cosmetics 2018, 5, 71 20 of 25 spherical liposomes than PC-ENLs. Most PC-NL vesicles were elliptical in shape with a large interior aqueous phase and a ﬂexible lipid layer. Cosmetics 2018, 5, x FOR PEER REVIEW 20 of 25

(a) (b)

FigureFigure 8.8. Representative transmission electron micrographs of (aa)) P.P. pyrifoliapyrifolia calluscallus extract-loadedextract-loaded nanoliposomesnanoliposomes (NLs) producedproduced byby nano-emulsiﬁcationnano-emulsification (PC-NLs)(PC-NLs) andand elasticelastic NLsNLs containingcontaining calluscallus extractextract (PC-ENLs).(PC-ENLs). ScaleScale barsbars inin ((aa,,bb):): 200200 nmnm andand 0.50.5 µµm,m, respectively.respectively.

3.4.2.3.4.2. In Vitro SkinSkin PermeabilityPermeability ofof NLsNLs ContainingContaining P.P. pyrifolia pyrifoliaCallus Callus Extract Extract TheThe artificialartificial skinskin permeabilitypermeability of P.P. pyrifoliapyrifolia calluscallus extractextract waswas significantlysignificantly increasedincreased byby encapsulationencapsulation into NLs (Figure(Figure9 9).). ComparedCompared toto thethe skinskin permeationpermeation fromfrom P. pyrifoliapyrifolia calluscallus extractextract solution,solution, thethe permeatedpermeated concentrationconcentration values for adenosineadenosine in thethe calluscallus extractextract fromfrom PC-NLsPC-NLs andand PC-ENLsPC-ENLs were increased by by 1.25- 1.25- and and 1.75-fold, 1.75-fold, resp respectively,ectively, atat 24 24 h h(Figure (Figure 9a).9a). In Inaddition, addition, the theeffective effective permeability permeability of adenosine of adenosine from fromPC-ENLs PC-ENLs prepared prepared as elastic as elasticNLs was NLs significantly was significantly greater greaterthan those than ofthose PC-NLs of PC-NLsand callus and extract callus solution extract, solution,and the maximum and the maximum skin permeability skin permeability of adenosine of adenosinefrom PC-ENLs from PC-ENLswas 1.63- was and 1.63- 2.28-fold and 2.28-fold greater greater than thanthose those of PC-NLs of PC-NLs and and callus callus extract extract alone, ± × −6 ± × −6 ± × −6 respectivelyrespectively ([0.54([0.54 ± 0.04] × 1010−6 vs.vs. [0.33 [0.33 ± 0.06]0.06] × 10−106 vs. [0.24vs. [0.24 ± 0.07]0.07] × 10−6 cm/s,10 respectively).cm/s, respectively). ElasticElastic NLsNLs areare preparedprepared byby incorporatingincorporating aa single-chainsingle-chain surfactantsurfactant as an edgeedge activator,activator, whichwhich decreasesdecreases thethe transitiontransition temperaturetemperature andand destabilizesdestabilizes thethe lipidlipid bilayersbilayers ofof liposomes,liposomes, followedfollowed byby increasingincreasing theirtheir deformabilitydeformability andand elasticity,elasticity, therebythereby facilitatingfacilitating skinskin penetrationpenetration ofof liposomesliposomes byby squeezingsqueezing themthem acrossacross microlamellarmicrolamellar spacesspaces amongamong keratinocyteskeratinocytes to penetratepenetrate acrossacross thethe skinskin layerlayer asas wellwell asas throughthrough veryvery tinytiny porespores inin thethe skinskin [[5566].]. Therefore, enhancedenhanced skinskin permeationpermeation ofof PC-ENLsPC-ENLs waswas alsoalso confirmed confirmed using using an an excised excised human human epidermal epidermal layer. layer. Incorporation Incorporation of P. pyrifoliaof P. pyrifoliacallus extract callus intoextract elastic into NLs elastic significantly NLs significantly improved theimproved skin penetration the skin of penetration the callus extract of the in comparisoncallus extract with in calluscomparison extract solutionwith callus (Figure extract9b). Aftersolution a 24-h (Figur incubation,e 9b). theAfter cumulative a 24-h incubation, penetrated concentrationthe cumulative of adenosinepenetrated fromconcentration the PC-ENL of adenosine formulation from was the 24.7-fold PC-ENL higher formulation than that was of 24.7-foldP. pyrifolia highercallus than extract that alone.of P. pyrifolia Thus, the callus skin extract flux of alone.P. pyrifolia Thus,callus the wasskin increasedflux of P. bypyrifolia 1598% callus after encapsulationwas increased into by elastic1598% ± ± µ 2 NEsafter (3.76encapsulation1.71 vs. into 0.222 elastic0.326 NEs g/cm(3.76 ±/h, 1.71 respectively). vs. 0.222 ± 0.326 µg/cm2/h, respectively). TheThe enhancedenhanced permeabilitypermeability of adenosineadenosine from thethe calluscallus extractextract couldcould bebe attributedattributed toto thethe synergisticsynergistic effectseffects of of elastic elastic NLs NLs acting acting as a carrier.as a carrier. Topically Topically applied applied elastic NLs elastic have NLs been have suggested been tosuggested interact withto interact the skin, with resulting the skin, in resulting several sequentialin several events.sequential Initially, events. water Initially, in the water dispersion in the immediatelydispersion immediately evaporates, whichevaporates increases, which the increases concentration the concentration gradient of NLs gradient through of the NLs skin, through followed the byskin, establishing followed by hydration establishing gradients hydration along gradients the skin. Thus,along waterthe skin. content Thus, in water the upper content layer in the increases upper inlayer comparison increases to in the comparison inner viable to epidermis the inner layer, viable which epidermis forms transepidermallayer, which forms hydration transepidermal gradients. Thereafter,hydration gradients. the hydration Thereafter, gradient the ashydration well as watergradient affinity as well and as highwater elasticity affinity and of elastic high elasticity NLs act asof drivingelastic NLs forces act toas pulldriving the forces liposomes to pull toward the liposomes the inner toward layer ofthe the inner skin layer until of they the skin have until reached they thehave water-rich reached the viable water-rich epidermis. viable After epidermis. they have Afte permeatedr they have across permeated the SC across barrier the by SC diffusion barrier via by interclusterdiffusion via and intercluster intercorneocyte and intercorneocyte pathways, the pull pa mechanismthways, the is pull then mechanis changed tom pushis then mechanism changed onto thepush liposomes mechanism [56, 57on]. the Furthermore, liposomes Subongkot[56,57]. Furtherm et al. reportedore, Subongkot that elastic et al. NLs reported were distinctly that elastic present NLs were distinctly present in the hair follicular region, suggesting that the transfollicular pathway may also play an important role in the release of encapsulated active ingredients, compared to the inter- or trans-cellular route of the nonfollicular region [58]. Thus, P. pyrifolia callus extract-loaded elastic NLs may be useful as a potent skin rejuvenating cosmetic preparation exerting an antiaging activity on the skin.

Cosmetics 2018, 5, 71 21 of 25 in the hair follicular region, suggesting that the transfollicular pathway may also play an important role in the release of encapsulated active ingredients, compared to the inter- or trans-cellular route of the nonfollicular region [58]. Thus, P. pyrifolia callus extract-loaded elastic NLs may be useful as a potentCosmetics skin 2018 rejuvenating, 5, x FOR PEER cosmeticREVIEW preparation exerting an antiaging activity on the skin. 21 of 25

40 P. pyrifolia callus extract PC-NLs PC-ENLs g/mL) 35 ### μ *** 30

25 *

0 Permeatedadenosine concentration(

0 3 6 9 12 15 18 21 24 Time (h)

(a)

30 P. pyrifolia callus extract PC-ENLs g/mL)

μ 25

20 ***

0 Permeatedadenosine concentration (

0 3 6 9 12 15 18 21 24 Time (h)

(b)

FigureFigure 9.9. InIn vitrovitro permeation of P. pyrifoliapyrifolia calluscallus extract-loadedextract-loaded nanoliposomesnanoliposomes (NLs)(NLs) acrossacross artiﬁcialartificial skinskin (Skin(Skin PAMPA)PAMPA) andand humanhuman epidermalepidermal layer.layer. TimeTime coursecourse ofof changeschanges inin cumulativecumulative permeatedpermeated concentrationsconcentrations ofof adenosineadenosine fromfrom thethe aqueousaqueous solutionsolution ofof calluscallus extractextract oror calluscallus extract-loadedextract-loaded NLsNLs (PC-NLs)(PC-NLs) oror elasticelastic NLsNLs (PC-ENLs)(PC-ENLs) throughthrough ((a)) SkinSkin PAMPAPAMPA andand ((bb)) humanhuman epidermis.epidermis. ValuesValues areare meansmeans ±± standard deviation ( n = 6 per group). * p < 0.05, *** p < 0.001 compared to P. pyrifoliapyrifolia calluscallus extract.extract. ###### p < 0.001 compared to PC-NLs.

Cosmetics 2018, 5, 71 22 of 25

4. Conclusions An aqueous extract was prepared using callus from in vitro-derived leaves of P. pyrifolia grown on M&S medium with picloram as a growth regulator. The total phenolic content in the callus extract was 9.25 ± 0.41 gallic acid equivalent/g dried callus extract and the DPPH free radical scavenging activity of the callus extract (300 µg/mL) was equivalent to that of 44 µg/mL ascorbic acid (82.5% ± 3.63%). The major biological components in the callus extract were identified as uridine (1), adenosine (2), and guanosine (3), and the IC50 values for inhibition of AGE formation from collagen and elastin were 602 ± 2.72 and 3037 ± 102.5 µg/mL, respectively. The extract significantly promoted keratinocyte and fibroblast proliferation in a dose-dependent manner, and fibroblasts treated with 1.36 µg/mL extract exhibited 1.60- and 1.43-fold increases in the levels of procollagen type I C-peptide compared to controls and cells treated with adenosine (0.015 µM), respectively. The in vitro wound recovery rates of keratinocytes and fibroblasts were also 75% and 38% greater, respectively, than those of serum-free controls at 9 h and 36 h after extract treatment (1.36 µg/mL). Moreover, the combination of extract and adenosine significantly enhanced both cell proliferation/migration and procollagen synthesis. The extract flux across the human epidermis was increased by 1598% after incorporation into elastic NLs. These observations suggest that elastic NLs loaded with P. pyrifolia callus extract have potential use as skin rejuvenators and antiaging ingredients in cosmetic formulations.

Author Contributions: Conceptualization, H.J.K. and J.W.P.; methodology, H.J.K.; validation, D.E.P. and D.A.; formal analysis, R.P. and V.K.P.; investigation, H.J.K. and J.W.P.; resources, H.J.K. and J.W.P.; data curation, D.E.P. and D.A.; writing—original draft preparation, D.E.P. and D.A.; writing—review and editing, H.J.K. and J.W.P.; visualization, H.J.K.; supervision, J.W.P.; project administration, H.J.K. and J.W.P.; funding acquisition, J.W.P. Funding: This research was supported by the Basic Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (NRF-2017R1D1A1B03032283). Acknowledgments: We thank BIO-FD&C Co. Ltd. (Hwasun-gun, Jeonnam, Republic of Korea) for their technical support in this research. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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