Antioxidant, Anti-Inflammatory and Attenuating Intracellular Reactive Oxygen Species Activities of Nicotiana tabacum var. Virginia Leaf Extract Phytosomes and Shape Memory Gel Formulation

Oxidative stress is one of the major causes of skin aging. In this study, the shape memory gels containing phytosomes were developed as a delivery system for Nicotiana tabacum var. Virginia fresh (VFL) and dry (VDL) leaf extracts. The extracts were loaded in the phytosomes by a solvent displacement method. The physical and chemical characteristics and stability of phytosomes were evaluated by dynamic light scattering and phytochemistry, respectively. The in vitro antioxidant activity and intracellular reactive oxygen species reduction of phytosomes and/or extracts were investigated by the DPPH and ABTS radical scavenging assays, FRAP assay, and DCFH-DA fluorescent probe. The cytotoxicity and anti-inflammatory activity of VDL and VFL phytosomes were studied by an MTT and a nitric oxide assay, respectively. Here, we first reported the total phenolic content in the dry leaf extract of N. tabacum var. Virginia was significantly greater than that of the fresh leaf extract. The HPLC analysis results revealed that VDL and VFL extracts contained 4.94 ± 0.04 and 3.13 ± 0.01 µg/mL of chlorogenic acid and 0.89 ± 0.00 and 0.24 ± 0.00 µg/mL of rutin, respectively. The phytosomes of the VDL and VFL extracts displayed stable size, polydispersity index, zeta potential values, and good chemical stability. VDL and VDL phytosomes showed higher phenolic and flavonoid contents which showed stronger DPPH and ABTS radical scavenging effects and reduced the intracellular ROS. The results suggested that the phenolic compounds are the main factor in their antioxidant activity. Both VDL and VFL phytosomes inhibited nitric oxide production induced by LPS, suggesting the anti-inflammatory activity of the phytosomes. The shape memory gel containing VDL and VFL phytosomes had good physical stability in terms of pH and viscosity. The VDL and VFL phytosomes dispersed in the shape memory gels can be considered as a promising therapeutic delivery system for protecting the skin from oxidation and reactive oxygen species.


Introduction
Skin aging can be caused by internal and external factors [1]. The internal factors include chronological aging and hormonal deficiency, resulting in the deterioration of tissues in the dermis and epidermis. The decrease in the number of fibroblasts synthesizing collagen, elastin fiber, and glycosaminoglycan leads to an increase in laxity and wrinkles [2]. phytosomes feasible for topical application. The method to prepare the shape memory gel containing phytosome developed in this study was simple and was achievable for scaling up commercially.

Results and Discussion
2.1. Physical Characterization and Colloidal Stability of VDL and VFL Phytosomes, Phytosomes w/o Chloresterol, Phytosomes w/o Poloxamer, and Nanoparticles The average particle size and stability of the VDL and VFL phytosomes with and without cholesterol or poloxamer, and VDL and VFL nanoparticles are shown in Figure 1. The size of the VDL phytosome with cholesterol, VDL phytosome without cholesterol, VDL phytosome without poloxamer, and VDL nanoparticles were 198.17 ± 6.63 nm, 248.63 ± 1.33 nm, 311.00 ± 5.21 nm, and 249.30 ± 1.56 nm, respectively. The size of the VFL phytosome with cholesterol, VFL phytosome without cholesterol, VFL phytosome without poloxamer, and VFL nanoparticles shown in Figure 2 were 163.40 ± 0.26 nm, 203.83 ± 1.44 nm, 191.43 ± 1.19 nm, and 178.67 ± 0.82 nm, respectively. The results showed that the particle size significantly increased in the absence of cholesterol in both VDL and VFL phytosome formulations. The importance of cholesterol in liposomes was reported. The solidity of the liposomes depended on the content of cholesterol. Numerous studies used cholesterol as a stabilizer of liposomes because it helped the packing of phospholipid molecules, reduced bilayer permeability, prevented liposome aggregation, and increased the rigidity and resistance to shear stress of the lipid bilayer [21]. Here, 0.9% w/w of cholesterol was used and was considered optimal for VDL and VFL phytosome formulations. In the absence of 0.1% w/v poloxamer 407, the VDL and VFL phytosomes were also significantly larger than the phytosomes stabilized with poloxamer. Poloxamer was assumed to be attached to the phytosome by adsorp-tion. Minnelli et al. showed that combining poloxamer 407 with liposome increased liposome stability by hindering liposome aggregation, shielding the colloidal surface with the hydrophilic portions of the polymer, and decreasing the fusion of phosphatidylcholine multilamellar vesicles [22]. The size of the phytosomes significantly increased when samples were stored at 4 • C. Generally, the size of the liposome/phytosome is influenced by the elasticity modulus of the lipid bilayer, which depends on the temperature below the phase transition. Most membrane compositions form larger liposomes/phytosomes close to or below the gel-to-liquid crystalline phase transition temperature, where the membrane elasticity modulus is much larger [23]. The colloidal stability study results suggested that VDL and VFL phytosomes and nanoparticles should be stored at 30 • C to maintain the size of the particles. Interestingly, the effects of membrane stiffness of phytosomes were not found to significantly affect the size of VFL phytosomes when stored at 4 • C. This result suggested that the stability of phytosomes was mostly dependent on the zeta potential values of the phytosomes, which defeated the effect of the membrane elasticity [24]. The polydispersity index of VDL phytosome with cholesterol, VDL phytosome without cholesterol, VDL phytosome without poloxamer, and VDL nanoparticles were 0.136 ± 0.019, 0.128 ± 0.024, 0.169 ± 0.040, and 0.164 ± 0.027, respectively ( Figure 3). The polydispersity index of VFL phytosome with cholesterol, VFL phytosome without cholesterol, VFL phytosome without poloxamer, and VFL nanoparticles were 0.174 ± 0.016, 0.234 ± 0.017, 0.189 ± 0.035, and 0.098 ± 0.015, respectively ( Figure 4).  The phytosome and nanoparticle size distribution can be articulated through the polydispersity index value. The nanoparticles with PDI values less than 0.3 are considered monodisperse, while a PDI value higher than 0.7 indicates a polydisperse system [25]. All VDL and VFL phytosomes and nanoparticles had PDI values less than 0.2, suggesting the uniformity of a sample based on the size. Heterogenicity can occur due to phytosome or nanoparticle aggregation. The polydispersity index of VDL phytosomes without poloxamer significantly increased to be higher than 0.4 when the phytosomes were stored at 4 • C, 30 • C, and 45 • C. VDL phytosomes containing poloxamers with and without cholesterol showed higher PDI values when they were kept at 4 • C. These results might be due to the aggregation of phytosomes at low temperatures. The poloxamer increased colloidal stability by forming hydrophilic layers of polyoxyethylene oxide at the surface of phytosomes and nanoparticles, hence preventing particle aggregation attributed to van der Waals forces between phytosomes and nanoparticles [26].
Zeta potential results showed that all VDL and VFL formulations exhibited a negative charge with values ranging from 22.57 ± 0.23 mV to −40.07 ± 0.86 mV, and −44.23 ± 01.86 mV to 76.93 ± 1.71 mV, respectively. The negative charge value was due to the presence of phosphate and carbonyl groups of phosphatidylcholines. Compared with phytosomes coated with poloxamer, a more negative charge of phytosomes and nanoparticles was observed with phytosomes prepared without poloxamer. This could be because poloxamer adsorption on the phytosome or nanoparticle surface forms a coating layer, shielding the negative surface charge and shifting the plane of shear away from the particle surface. Overall, phytosomes containing cholesterol and poloxamer encapsulating VDL and VFL were the best formulation with optimal size, PDI, and zeta potential values. Both formulations were recommended to store at 30 • C.
Phytosomes of herbal extracts have been developed and characterized. Tiwari et al. developed herbal extract-loaded phytosomes. They showed that the encapsulation of herbal extract in the phytosomes did not change the chemical structure due to an FTIR analysis [27]. Direito et al. reported that the size of phytosomes depends on the amount of lipid composition in the formulation. It has been reported that increasing phospholipids in the phytosome increased the tendency of agglomeration [28]. The surface charge expressed as the zeta potential is an important physicochemical parameter that influences the stability of nanosuspensions which may also influence the biodistribution, pharmacokinetics, cellular affinity, and drug internalization [29]. When compared to a positive surface charge, the negative zeta potential is generally associated with higher biocompatibility [30,31].

Total Phenolic and Flavonoid Contents of VDL and VFL Phytosomes, Phytosomes w/o Cholesterol, Phytosomes w/o Poloxamer, and Nanoparticles
The total phenolic content in the VDL and VFL ethanolic extracts using the Folin-Ciocalteu reagent was expressed in terms of the gallic acid equivalent. The standard curves plotted between the absorbance, and gallic acid reacted with the Folin-Ciocalteau reagent are shown in Figure S1A. The total phenolic contents in the VDL and VFL extracts were 265 ± 0.67 and 258.56 ± 0.81 mg gallic acid equivalent/mL crude extract, respectively. The phytosomes of VDL and VFL contained 260.35 ± 2.29 and 238.54 ± 1.86 mg gallic acid equivalent/mL ( Figure 7A). Here, we first reported the total phenolic content in the dry leaf extract of N. tabacum var. Virginia was significantly greater than that of the fresh leaf extract. The standard curves of quercetin and EGCG reacted with the aluminum chloride reagent were presented in Figures S1B and 1C, respectively. The total flavonoid contents in the VDL and VFL extracts were 3620. 52 Figure 7B). The results suggested that the VDL extract and phytosome contained a larger amount of flavonoids compared with the VFL extract and phytosome. The component of VDL phytosomes did not affect the encapsulation of the total phenolic and total flavonoids, but the encapsulation of the total phenolic and total flavonoids of VFL was affected by the VFL phytosome formulation. The stability of the total phenolic and total flavonoids in VDL and VFL phytosomes was temperature dependent. The total phenolic and flavonoid contents significantly reduced when the phytosomes were stored at 45 • C ( Figure 7C). The total flavonoids were prone to degrade more easily compared with total phenolic compounds encapsulated in the phytosomes ( Figure 7D).
The results revealed that both the VDL extract and phytosomes had higher DPPH and ABTS radical scavenging activity and ferric reducing power than the VFL extract and phytosomes. Our results indicated that the VDL extract and VDL phytosomes demonstrated stronger DPPH and ABTS radical scavenging effects, probably because they contained higher amounts of phenolic and flavonoid contents, compared with VFL and VFL phytosomes. These results suggested that phenolic compounds are the main factor in their antioxidant activity.

Correlation between the Total Phenolic and Total Flavonoid Content and Antioxidant Activities
The correlation between the phenolic and flavonoid contents and antioxidant activities was evaluated by the Pearson's correlation test shown in Table 1. The total phenolic and total flavonoids in the VDL phytosomes, VFL extract, and VFL phytosomes showed a very high correlation with all three antioxidant activities. The total flavonoids in the VDL extract showed a high correlation with the DPPH free radical scavenging activity. These results supported the fact that the phenolic and flavonoids in the VDL and VFL extracts and phytosomes could scavenge free radicals and reduce ferric ions.
The total phenolic content and total flavonoid contents reported by Yati et al. were lower than our findings [32]. This might be due to the fact that the solvent used for polyphenol extraction is different. The higher effectiveness of ethanol in extracting phenolic compounds and flavonoids was shown in previous studies [33][34][35][36]. The ethanol extracts also exhibited a higher activity than the aqueous extract [33][34][35]. The extracts containing more polyphenols had higher antioxidant activity. Therefore, the 95% ethanol solvent might be more suitable to extract phenolic and flavonoids from Nicotiana tabacum var. Virginia.

HPLC Analysis of VDL and VFL Extracts
Chlorogenic acid and rutin have been reported as the main phenolic compounds in tobacco leaves. Chen et al. have shown that chlorogenic acid and rutin concentration were the highest among the other polyphenol compounds in tobacco leaves [37]. A reversephased HPLC was applied to analyze polyphenols in the VDL and VFL extracts. The HPLC profiles of the standard chlorogenic acid and rutin are presented in Figure 9. Chlorogenic acid and rutin were detected with high concentrations in the VDL and VFL extracts. The VDL and VFL extracts contained 4.94 ± 0.04 and 3.13 ± 0.01 µg/mL of chlorogenic acid, respectively, and 0.89 ± 0.00 and 0.24 ± 0.00 µg/mL of rutin, respectively. According to the literature, the peak at a retention time of 9.5 min might be assigned for nicotine [38]. At a retention time of 10.87 min and 11.13 min, the peaks might be identified as isomers of chlorogenic acid (3-caffoylquinic acid), such as 4-caffoylquinic acid and 5-caffoylquinic acid, respectively [39].

Effects of VDL and VFL Extracts and Phytosomes on Keratinocyte Cell Viability
The IC 50 values of the VDL extract, VFL extract, VDL phytosomes, and VFL phytosomes after exposure to HaCaT cells for 24 h were 1137 µg/mL, 164.8 µg/mL, 15,741 µg/mL, and 910.5 µg/mL, respectively ( Figure 10A). The IC 50 values of the VDL extract, VFL extract, VDL phytosomes, and VFL phytosomes increased upon exposure with HaCaT cells for 48 h to 1222 µg/mL, 166 µg/mL, 28,671 µg/mL, and 916.4 µg/mL, respectively ( Figure 10B). After 72 h, the IC 50 values of the VDL extract, VFL extract, VDL phytosomes, and VFL phytosomes were 10,326 µg/mL, 1528 µg/mL, 139,737 µg/mL, and 7805 µg/mL, respectively ( Figure 10C). The IC 50 values of the VFL extract and VFL phytosomes were less than that of the VDL extract and VDL phytosomes, indicating the higher cytotoxicity against human keratinocyte cells. The IC 50 values of all extracts and phytosomes increased with the incubation time, suggesting that VDL and VFL extracts and their phytosomes were safe to apply on skin. Phytosomes had higher IC 50 values compared to the extract, indicating that they can protect cells from cytotoxic agents in the extracts.

Reactive Oxygen Species' Levels in HaCaT Cells Exposed to VDL and VFL Phytosomes
Intracellular ROS were assayed using fluorescent probe DCFH-DA, which can cross cell membranes and oxidize to a fluorescent DCF by intracellular ROS. Compared with the hydrogen peroxide-induced HaCaT cells, the fluorescent intensity of cells treated with VDL and VFL phytosomes was significantly decreased, indicating that the intracellular ROS level was significantly decreased (Figure 11). The results suggested that VDL and VFL phytosomes reduced the intracellular ROS, probably due to the total phenolic and total flavonoid compounds acting as an antioxidant in the extract. Free radicals and other ROS such as the oxygen singlet free radical, hydroxyl radical, peroxyl radical, and nitric oxide free radical are continuously formed at low concentrations during normal essential metabolic processes [40]. The natural antioxidant system regulates the amount of these free radical species to maintain redox hemostasis [41]. Polyphenols are strong antioxidants that can neutralize free radicals by donating an electron or hydrogen atom to a free radical through the H-atom transfer mechanism [42]. Tobacco leaves contain significant concentrations of polyphenols and carotenoids, which are important naturally occurring antioxidants [43,44]. Although we are the first to show the intracellular ROS suppression of N. tabacum leaves, the antioxidant activities of N. tabacum leaves were expressed in several studies by scavenging activities on hydroxyl, superoxide anion, DPPH and ABTS radicals, ferric thiocyanate forming complex, and reducing power [32,45,46]. These results supported the intracellular ROS inhibition of N. tabacum leaves firstly presented in our study.

Inhibition of LPS-Induced Nitric Oxide Production
The effects of VDL and VFL phytosomes on RAW264.7 cell viability are shown in Figure 12A. The viability of RAW264.7 cells treated with 7.8-500 µg/mL was not significantly reduced, indicating that phytosomes at this concentration range showed no cytotoxic effect on RAW264.7 cells. Natural polyphenols have demonstrated anti-inflammatory activity in vitro and in vivo, highlighting their therapeutic applications in a variety of diseases. Numerous studies have shown the anti-inflammatory and immune modulation activities of polyphenols [15,21]. The ability of these natural compounds to modulate the expression of several pro-inflammatory genes such as multiple cytokines, lipoxygenase, nitric oxide synthases, and cyclooxygenase, in addition to their antioxidant properties such as ROS scavenging, helps to regulate inflammatory signaling [22,23]. Thus, VDL and VFL phytosomes at 15.6-500 µg/mL were further used to study the anti-inflammatory effect of the phytosomes by inhibiting nitric oxide production. RAW264.7 cells were stimulated with LPS with or without co-treatment with VDL and VFL phytosomes. As shown in Figure 12B, both VDL and VFL phytosomes inhibited the NO production induced by LPS. The VFL phytosome inhibited LPS-induced NO production in RAW264.7 cells to a greater extent than the VDL phytosome. The viability of RAW264.7 cells treated with LPS and samples were not changed during the experiment ( Figure 12C). A previous study on the phytoconstituent isolated from N. tabacum demonstrated significant inhibition of COX-2 by the downregulation of COX-2 mRNA [47]. Therefore, our data suggested that phytosomes that incorporated N. tabacum Virginia leaf extract can be considered as a potential candidate for the mitigation of dermal inflammations.

Characterization of Shape Memory Gel Containing VDL and VFL Phytosomes
The physical stability of the shape memory gel containing VDL and VFL phytosomes was confirmed by the maintained appearance, viscosity, and pH. The appearance of gels is shown in Figure S2. Figure 13 exhibits the change in viscosity for gels containing 0.1% w/w VDL and VFL phytosomes as a function of shear rate. All samples showed a similar trend of change in viscosity. Initially, the gel showed high viscosity, followed by a gradual decrease in viscosity as the shear rate was applied, and followed by a plateau region. These results suggested that the gel exhibited pseudoplastic rheology behavior. The viscosity of gels containing VDL and VFL phytosomes was not significantly changed after the six heatingcooling cycles of the accelerated stability study (Figure 13A), and after 1 month-storage at 4 • C, 30 • C, and 45 • C ( Figure 13B,C). The pH of VDL and VFL phytosome gels was not changed during the accelerated and long-term (1-month) stability studies ( Figure 14).  The sustained release behavior of the total phenolic content from VDL and VFL extracts, phytosomes, and gels was observed ( Figure 15). The VDL extract, VDL phytosome, and VDL gel released the total phenolic content up to 73.76 ± 3.73%, 66.98 ± 5.33%, and 38.04 ± 2.59%, respectively, within 24 h. The VFL extract, VFL phytosome, and VFL gel released 13.92 ± 2.26%, 55.78 ± 3.86%, and 21.44 ± 5.81% of total phenolic content, respectively, in 24 h. The results suggested that the VDL extract contained a higher amount of hydrophilic polyphenols compared with the VFL extract, hence releasing the phenolic compounds at a higher rate. The VDL extract entrapped in the phytosomes gradually released total phenolic compounds from the phytosomes. The shape memory gel released phenolic compounds to a lesser extent compared with VDL extract and phytosomes. Interestingly, the VFL phytosome released the phenolic compounds faster than that of the extract and the gel. These results indicated that phytosomes enhanced the water solubility of the VFL extract. Alshahrani et al. showed that phytosomes loaded with Cuscuta reflexa extract released 96.3 ± 3.7% of the polyphenol and flavonoids phytoconstituents from phytosomes in 12 h, compared to 49.3 ± 2.5% in the plain extract. Therefore, the phytosomal nanocarriers have the potential to increase the bioavailability of the extract [48]. The release of the phenolic compounds in the gel-loading phytosomes occurred in several steps. The release was initiated by the penetration of the PBS medium into the gel, which created pores and degraded polymers. Then, the phenolic compounds diffused from the gel matrix to the medium, followed by the dissolution of the phenolic compounds in the medium [49]. Shape memory gel is one of the new materials that can fulfill drug delivery and cosmetic application. Polyurethane-62 is a copolymer comprised, in part, of the carbamate (i.e., urethane) linkages that can form hydrogen bonds to yield its high mechanical strength. Combining hydrophobic polyurethane with butylene glycol generates a swellable copolymer network that is robust and durable [50]. Polyurethane-butylene glycol serves as a shape memory polymer. This stimuli-responsive material can memorize its original shape, which occurs during the gelation process when an appropriate stimulus is applied. Biocompatibility and cytotoxicity of the shape memory polymer are crucial concerns for drug delivery. It was suggested that polyurethane could be used for implanted medical devices with shape memory requirements. Peng et al. reported that the polyurethane grafted with poly-lactic acid had biocompatibility comparable to pure PLA [51]. Polyurethane-62 has been widely used in cosmetics, including moisturizers, sunscreens, serums, and water gel lotions. The Australian Industrial Chemicals Introduction Scheme (AICIS) determined that polyurethane-62 was not considered to pose an unreasonable risk to the health of workers and the public [52]. Polyurethan-62 is a high molecular weight polymer. The molecular weight is approximately 100,000 Da, which is not expected to penetrate the skin.

Conclusions
The phytosomes composed of phosphatidyl choline and cholesterol coated with poloxamer were successfully developed and suitable for the delivery of N. tabacum var. Virginia fresh and dry leaf extract to the skin. The phytosomes with different formulars were fabricated and the physical stability was compared. Both phosphatidyl choline and cholesterol played an important role in the physical stability of the phytosomes but did not affect the encapsulation efficiency of the phenolic compounds. VDL and VDL phytosomes had higher phenolic and flavonoid contents and displayed stronger DPPH and ABTS radical scavenging effects, suggesting that phenolic compounds are the main factor in their antioxidant activity. VDL and VFL phytosomes reduced the intracellular ROS and inhibited the NO production induced by LPS. The viscosity and pH of gels containing VDL and VFL phytosomes were not significantly changed after the six heating-cooling cycles of the accelerated stability study and after 1 month storage at 4 • C, 30 • C, and 45 • C.

Preparation of VDL and VFL Phytosomes and Nanoparticles
Tobacco leaf extract (VDL or VFL) in 95% ethanol (0.5 g/mL, 400 µL) was mixed with phosphatidylcholine (30 mg in 1900 µL 95% ethanol). Cholesterol (2 mg) in acetone solution (200 µL) was added to the above solution and mixed thoroughly. Then, the mixture was added dropwise into 15 mL of 0.1% w/v poloxamer 407 at a rate of 1 mL/h with a stirring speed of 700 rpm. The obtained phytosome was named "VDL phytosome" or "VFL phytosome" [53]. "VDL phytosomes (w/o cholesterol)" and "VFL phytosomes (w/o cholesterol)" were prepared by the following method, where cholesterol was not included. Tobacco leaf extract (VDL or VFL) in 95% ethanol (0.5 g/mL, 400 µL) was mixed with phosphatidylcholine (30 mg in 1900 µL 95% ethanol). The mixture was added dropwise into 15 mL of 0.1% w/v poloxamer 407 at a rate of 1 mL/h with a stirring speed of 700 rpm. To prepare "VDL phytosome (w/o poloxamer)" and "VFL phytosome (w/o poloxamer)", mixtures of VDL or VFL (0.5 g/mL, 400 µL) with phosphatidylcholine (30 mg in 1900 µL), 95% ethanol, and cholesterol (2 mg in 200 µL acetone) were infused into 15 mL of de-ionized water at a rate of 1 mL/h with a stirring speed of 700 rpm. "VDL nanoparticles" and "VFL nanoparticles" were prepared by the infusion of VDL or VFL extracts (0.5 g/mL, 400 µL) into 0.1% poloxamer 407 solution at a rate of 1 mL/h with a stirring speed of 700 rpm [54][55][56]. The obtained VDL and VFL phytosomes and nanoparticles were washed three times with de-ionized water and characterized.

Characterization and Stability Study of VDL and VFL Phytosomes and VDL and VFL Nanoparticles
The freshly prepared VDL and VFL phytosomes or nanoparticles were measured for the size, PDI, and zeta potential values using the Zetasizer (Malvern Instruments, Worcestershire, UK). The colloidal stability of VDL and VFL phytosomes or nanoparticles was studied by storing phytosomes or nanoparticles at 4 • C, 30 • C, and 45 • C for 0.5, 1, 2, and 3 months. At the end of the incubation time, the particle size, PDI, and zeta potential of VDL and VFL phytosomes and nanoparticles were analyzed.

Quantitative Analysis of Total Phenolic Compounds in VDL and VFL Phytosomes
VDL and VFL extracts and phytosomes and gallic acid standard solution (3.9-125 µg/mL) were placed in a 96-well plate (50 µL/well). A Folin-Ciocalteau reagent (10% v/v, 100 µL) was added to the wells, mixed well, and incubated for 4 min at room temperature. Then, the sodium carbonate solution (10% w/v, 50 µL) was added to the mixture and incubated in the dark for 60 min at room temperature [57]. The absorbance was measured at a wavelength of 765 nm using a UV-Vis spectrophotometer microplate reader. The total phenolic contents in phytosomes were calculated by constructing a standard curve between the absorbance and the concentration of the gallic acid standard solution. The total phenolic content was expressed as the gallic acid equivalent (GAE).

Quantitative Analysis of Total Flavonoid Content in VDL and VFL Phytosomes
The VDL and VFL extracts and phytosomes, quercetin standard solution (7.8-500 µg/mL), and EGCG standard solution (7.8-1000 µg/mL) were placed in a 96-well plate (50 µL/well). Sodium nitrate (5% w/v, 30 µL) was added to the wells, mixed well, and incubated for 5 min at room temperature. Then, the aluminum chloride solution (2% w/v, 50 µL) was added to the mixture and incubated for 6 min at room temperature [57]. Sodium hydroxide (1 N, 50 µL) was added to the wells and incubated for another 10 min at room temperature. The absorbance was measured at a wavelength of 510 nm using a UV-Vis spectrophotometer microplate reader. The total flavonoid contents in phytosomes were calculated by constructing a standard curve between the absorbance and the concentration of quercetin or the EGCG standard solution. The total phenolic content was expressed as the quercetin equivalent or EGCG equivalent.

Chemical Stability Study of VDL and VFL Phytosomes
The chemical stability of VDL and VFL phytosomes stored at 4 • C, 30 • C, and 45 • C for 3 months was investigated by a quantitative analysis of total phenolic and total flavonoid contents in VDL and VFL phytosomes.

HPLC Analysis of Bioactive Compounds in VDL and VFL Extracts
HPLC separation was achieved on the HPLC (Agilent, Santa Clara, CA, USA) equipped with the 1260 Infinity II quaternary pump, 1260 Infinity II autosampler, 1260 Infinity II multi-column thermostat, and 1260 Infinity II PDA detector. The separation was completed in an ACE 5 C18-AR column (4.6 × 250 mm i.d., 4.6 mm) with a C 18 guard column. The mobile phases were (B) acetonitrile and (C) 0.085% phosphoric acid in water using the following gradient elution: 10% B in C to 30% B in C for 15 min; 10% B for 5 min before each analysis, and the flow rate was set at 0.7 mL/min with the controlled temperature at 25 • C. The UV detector was set at the wavelength of 326 nm for the chlorogenic acid analysis and 356 nm for the rutin analysis, and the injection volume was 10 µL for every sample and reference standard [38,39].

Cell Culture
HaCaT and RAW264.7 cell lines were obtained from Dr. Kanokwan Kiettisin and Dr. Natthachai Duangnin, respectively. Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) with high glucose and supplemented with 10% FBS and 1% penicillinstreptomycin. Cells were incubated in 5% CO 2 at 37 • C for growing. Cells were subcultured by incubating with 0.25% trypsin-EDTA every three days.

Cytotoxicity Assay of VDL and VFL Phytosomes against HaCaT Keratinocyte Cells
In 96-well plates, HaCaT cells were seeded in a culture medium at 8 × 10 3 cells/well. Prior to an MTT assay, cells were allowed to adhere for 24 h. VDL and VFL extracts and VDL and VFL phytosomes were added to the cells and were incubated in 5% CO 2 at 37 • C for 24 h. After treatment for 24, 48, and 72 h, an MTT solution in the culture medium (0.5 mg/mL) was added to each well and incubated for 2 h at 37 • C [58]. The formazan products resulting from the viable cells' metabolism were dissolved in DMSO (100 µL/well). The absorbances were measured at 550 nm. The IC 50 values were calculated using GraphPad Prism v.7.0 (La Jolla, CA, USA). Cell viability was calculated using the following equation. HaCaT cells were trypsinized and added into the microcentrifuge tubes (1 × 10 6 cells/mL). Cells were co-incubated for 2 h at 37 • C with 20 mM hydrogen peroxide and VDL or VFL phytosomes at different concentrations. Cells were washed three times with PBS, pH 7.4, followed by incubation with 10 µM DCFH-DA at 37 • C for 30 min [59]. The fluorescent intensity was detected by flow cytometry (Accuri, BD, Franklin Lakes, NJ, USA). For each sample, 10,000 events were recorded.

In Vitro Anti-Inflammatory Assay of VDL and VFL Phytosomes
The viability of RAW264.7 cells after the 24 h incubation with VDL and VFL phytosomes (7.8-1000 µg/mL) was tested by the MTT assay. The anti-inflammatory effect of VDL and VFL phytosomes was determined from nitric oxide secretion against macrophage cells. RAW 264.7 cells (1 × 10 4 cells/well) were incubated with LPS (50 ng/mL) in the presence or absence of VDL and VFL phytosomes (15.6-500 µg/mL) for 18 h at 5% CO 2 , 37 • C. After incubation, an equal medium volume was mixed with the Griess reagent, consisting of 20 mg/mL sulfanilamide and 1 mg/mL N-(1-naphthylethylenediamine in 5% phosphoric acid at a 1:1 ratio [60,61]. The absorbance of the cell supernatant was recorded at 540 nm to quantify the nitrite levels using a UV-Vis spectrophotometer microplate reader (Spectramax M3, Molecular Devices, San Jose, CA, USA) equipped with SoftMax ® Pro 7 software. The amount of nitrite was calculated from the sodium nitrite standard curve. The treated cells were then tested for cell viability using an MTT assay. The medium was replaced with 0.5 mg/mL MTT reagent (100 µL/well) and incubated for 2 h. The formazan product was measured at 550 nm using a UV-Vis spectrophotometer microplate reader (Spectramax M3, Molecular Devices, San Jose, CA, USA).

Characterization and Stability Study of Shape Memory Gel
The pH and rheology of the shape memory gel containing VFL and VFL phytosomes were measured using a pH meter and rheometer (AMETEK Brookfield, Middleboro, MA, USA) equipped with a plate and plate geometry, respectively [62]. The physical stability of the products was investigated by measuring the pH and rheology of the gel after six heating/cooling cycles and after storage at 4 • C, 30 • C, and 45 • C for 1 month.

Release Study of VDL and VFL Extracts, Phytosomes, and Shape Memory Gel
The release profile of the VDL and VFL extracts, VDL and VFL phytosomes, and shape memory gels containing VDL and VFL phytosomes were evaluated by the method described by Liu et al. [63]. Phytosomes containing the same concentration of VDL and VFL extracts (13.33 mg/mL, 200 µL) and gels containing 0.1% VDL and VFL phytosomes were put in the upper chamber of Transwell ® (Corning, Glendale, AZ, USA), where the donor chamber contained the PBS buffer (1000 µL). The total phenolic compounds released from extracts, phytosomes, and gels were collected at predetermined time intervals, i.e., 15 min, 30 min, 1, 2, 4, 6, 8, 12, and 24 h. The total phenolic contents were measured by the Folin-Ciocalteau method.

Statistical Analysis
The data were statistically analyzed using a one-way ANOVA, followed by the Newman-Keuls method as a post hoc test to determine the significance of differences (GraphPad Prism 7.02, La Jolla, CA, USA). In all cases, p < 0.05, p < 0.01, p < 0.001, and p < 0.0001 were deemed statistically significant. Data were presented as the mean ± SD of the % cell viability (n = 3).