PVP/CS/Phyllanthus emblica Nanofiber Membranes for Dry Facial Masks: Manufacturing Process and Evaluations

In the wake of increasing demands on skin health, we propose simple, natural, and safe dry facial masks that restrict melanin synthesis. Phyllanthus emblica (P. emblica) is made into powders via a low-temperature extraction and freeze-drying process to serve as a natural agent. Next, it is added to mixtures containing Polyvinylpyrrolidone (PVP) and Chitosan (CS), after which the blends are electrospun into PVP/CS/P. emblica nanofiber membrane dry facial masks using the electrospinning technique. The dry facial masks are evaluated using the calibration analysis method, extraction rate test, scanning electron microscopy (SEM), release rate test, tyrosinase inhibition assay, biocompatibility test, and anti-inflammatory capacity test. Test results indicate that when the electrospinning mixture contains 29.0% P. emblica, the nanofibers have a diameter of ≤214.27 ± 74.51 nm and a water contact angle of 77.25 ± 2.21. P. emblica is completely released in twenty minutes, and the tyrosinase inhibition rate reaches 99.53 ± 0.45% and the cell activity ≥82.60 ± 1.30%. Moreover, the anti-inflammatory capacity test results suggest that dry facial masks confine inflammatory factors. PVP/CS/P. emblica nanofiber dry facial masks demonstrate excellent tyrosinase inhibition and are hydrophilic, biocompatible, and inflammation-free. The dry facial masks are a suitable material that is worthwhile exploring and applying to the cosmetic field.


Introduction
Nowadays, the majority of facial masks are commonly made of cellulous or polymer matrices (i.e., carriers) with a liquid blend that consists of an active substance. To achieve the purposes of forming, sale, and preservation, the effective substance is usually composed of chemical or synthetic elements. Meanwhile, the liquid in facial masks is also incorporated with essence, moisturizer, thickener, and antiseptic agents, and these additives may harm the health and living environment of people [1][2][3][4][5]. However, the manufacturing process can be adjusted to improve this disadvantage. For example, the electrospinning technique is introduced to the cosmetic field in order to produce dry fabrics that contain no extra additives while retaining biocompatible and eco-friendly attributes. In addition, nanofiber membranes are used as the carrier in which effective substances can be directly

Extraction of P. emblica
The alcohol and deionized water (7:3 v/v%) are formulated into a solvent. Ten grams of P. emblica is weighed and then completely mixed with 100 mL of solvent, forming the P. emblica mixture. A magnet mixer is used to extract the P. emblica mixture at a temperature of 40 • C and an extraction rate of 100 rpm for six, twelve, twenty-four, and forty-eight hours. The impurities are removed from the extract with a piece of 0.22 µm filter paper and the filtrate is placed in a refrigerator at −20 • C overnight until it is frozen. Finally, the 24 h freeze-drying is employed to create P. emblica powders.

Preparation of Nanofiber Membranes
To begin, PVP (20 wt%) and CS (2 wt%) are separately processed at 60 • C for twelve hours using a magnet mixer. When both of them are dissolved completely, the PVP solution and CS solutions are blended for thirty minutes. Next, 0, 0.005, 0.01, and 0.02 wt% of the P. emblica powders are separately mixed with PVP/CS blends for thirty minutes using a magnet mixer, after which the ultrasonic vibration machine is used to remove the bubbles. Finally, the electrospinning process is conducted as follows. The PVP/CS/P. emblica mixtures are infused into a 15-mL syringe with needle #22. An electrospinning syringe pump (KD Scientific, KDS220, Sanit Louis, MO, USA) is equipped with a static generator (COSMI, SC-80H, Taichung, Taiwan). The mixture is pushed through the needle to form a Taylor cone and then drawn from the charged end by the electric field and drafted to the collect plate in another non-charged end. The repetitively collected electrospinning nanofibers are accumulated into nanofiber membranes. During the electrospinning process, the needle is designed to sway back and forth in order to broaden the collection range of the membranes. PVP/CS/P. emblica nanofiber membranes are thus formed and then stored in a moisture-proof case immediately. The spinning rate is 0.025 mL/min; the electrospinning distance is 15 cm; the electrospinning voltage is 15 kV; the temperature is 25-30 • C; and the humidity is 35%. Figure 1 shows the diagram of the process. Samples are measured with a water contact angle meter (OCA-15 PLUS, SCA20, Munich, Germany). Next, the test solution (i.e., deionized water) is used to fill a syringe, and the photograph device is adjusted to a suitable position. A sample is fixed on a plate be-  The immersion method is used. P. emblica is weighed (W0) with a specified weight and then placed in a beaker for extraction. The extraction is conducted at a temperature of 40 • C with an extraction time of 6, 12, 24, and 48 h. The individual extraction solutions are freeze-dried into powders (Wt) and the extraction rate is computed with the equation as follows.

Scanning Electron Microscopy
Samples are affixed to the foundation of the scanning electron microscope (HITACHI S-4800, HORIBA EMAX400, Kyoto, Japan) with carbon tape, and observed for the surface morphology at an operation voltage of 15 kV.

Water Contact Angle Test
Samples are measured with a water contact angle meter (OCA-15 PLUS, SCA20, Munich, Germany). Next, the test solution (i.e., deionized water) is used to fill a syringe, and the photograph device is adjusted to a suitable position. A sample is fixed on a plate beneath the syringe, after which 20 µL of deionized water is dripped over the sample surface. The photograph device is activated as soon as the drop contacts the nanofiber membrane. The highest point along with two points on both sides of the drop are measured and recorded for contact angle analysis. Six samples for each specification are used for the average.

Release Rate Measurement
Samples are trimmed into cubes of 1 cm × 1 cm, and then placed in a spectrometer tube filled with deionized water for specified lengths of time (e.g., 5, 10, 15, and 20 min). The samples are removed and measured for the optical density using a UV-Vis spectrophotometer (Thermo Fisher, Genesys 10S UV-Vis, Pleasanton, CA, USA). The calibration analysis linear regression is used to compute the release rate.  Table 1. The tyrosinase inhibition rate is computed with the equation as follows and vitamin C serves as the control group. Tyrosinase inhibition rate (%): where A, B, C, and D separately means the optical density of corresponding solutions.

Biocompatibility Measurement
The NIH/3T3 cells are cultured in 96-well plates (5 × 103 cells/mL) for a 24 h culture in a carbon dioxide incubator. Next, the culture solution is removed and then replaced with the culture solutions in which samples were once immersed. The cells in the 96-well plates are once again cultured in a carbon dioxide incubator for twenty-four and seventy-two hours. Afterwards, the plates are removed and a PrestoBlue agent is added for a reaction in 15-20 min. With a specified optical density of 570, the Micro plate ELISA Reader (Tecan, Sunrise, Switzerland) is used for the measurement. The data are yielded and computed for cell viability as follows.
Cell Viability (%) = (Wt/A) × 100% Wt refers to the optical density for different culture solutions while A refers to the optical density for the pure culture solution.

Anti-Inflammatory Capacity Measurement
The IL-1α and TNF-α are separately used for the anti-inflammatory capacity. The NIH/3T3 cells are cultured in 96-well plates (5 × 103 cells/mL) for twenty-four hours in a carbon dioxide incubator. The culture solution is removed and then replaced with another culture solution in which the samples were once immersed, after which the cells are once again cultured in the carbon dioxide incubator for twenty-four and seventy-two hours. The cells in the 96-well plates are removed in order to remove 50 µL of supernatant fluid. The fluid is added to other 96-well plates, followed by 50 µL of antibody cocktail. Afterwards, the mixtures are oscillated at 400 rpm for one hour, and the solution is removed. After the plates are rinsed with wash buffer and 100 µL of TMB development solution is added, the plates are oscillated at 400 rpm in darkness for ten minutes. Finally, 100 µL of stop solution is added for interaction, and then a micro plate ELISA Reader (Tecan, Sunrise, Zurich, Switzerland) is used for the measurement with a specified OD of 450.  Figure 3 shows the solvent for the extraction of P. emblica is composed of water and alcohol. The weight of P. emblica is specified as ten grams and the extraction time is 6, 12  Figure 3 shows the solvent for the extraction of P. emblica is composed of water and alcohol. The weight of P. emblica is specified as ten grams and the extraction time is 6, 12, 24, or 48 h as in Table 2. The extract weight of powders is 0.58 g, 2.9 g, 2.53 g, and 2.5 g, respectively. The computation of extraction rate indicates that the corresponding extraction rates are 5.80 ± 0.30%, 29.00 ± 1.00%, 25.33 ± 1.15%, and 25.00 ± 1.00%.  Figure 3 shows the solvent for the extraction of P. emblica is composed of water and alcohol. The weight of P. emblica is specified as ten grams and the extraction time is 6, 12 24, or 48 h as in Table 2. The extract weight of powders is 0.58 g, 2.9 g, 2.53 g, and 2.5 g respectively. The computation of extraction rate indicates that the corresponding extrac tion rates are 5.80 ± 0.30%, 29.00 ± 1.00%, 25.33 ± 1.15%, and 25.00 ± 1.00%.

Tyrosinase Inhibition Assay for P. emblica Powders
As the presence of melanin is attributed with an interaction with tyrosinase, the restriction on tyrosinase is evaluated to examine whether the melanin synthesis is successfully blocked. P. emblica powders are evaluated for a tyrosinase inhibition rate. Tyrosinase is allowed to thaw in advance. Tyrosine, the extract, and tyrosinase are added to the 96-well plates in order, after which the tyrosinase inhibition rate is computed with the equation where the tyrosinase inhibition rate (%) is The tyrosinase inhibition rates are 99.83 ± 0.15%, 97.03 ± 0.97%, 92.20 ± 1.58%, and 90.46 ± 0.97% for the extraction times of six, twelve, twenty-four, and forty-eight hours, respectively. Vitamin C is used as the control group. Figure 4 shows that the Tyrosinase inhibition rate is dependent on the extraction time. There are more different extracts or impurities with an increase in the extraction time. With a specified concentration but a longer extraction time, the content of the tyrosinase inhibitor may be decreased, which in turn causes a decrease in the tyrosinase inhibition rate.
Vitamin C is used as the control group. Figure 4 shows that the Tyrosinase inhib is dependent on the extraction time. There are more different extracts or impur an increase in the extraction time. With a specified concentration but a longer e time, the content of the tyrosinase inhibitor may be decreased, which in turn cau crease in the tyrosinase inhibition rate.

SEM Observation of Nanofiber Membranes
The morphology, nanofiber diameter, and porosity of nanofiber membran observed via the SEM images. Finer nanofibers possess good specific surface ar enabling membranes to be well attached to skins as well facilitating the release blica. Meanwhile, a porous structure has a positive influence over the air ventilati on Table 3 and

SEM Observation of Nanofiber Membranes
The morphology, nanofiber diameter, and porosity of nanofiber membranes can be observed via the SEM images. Finer nanofibers possess good specific surface areas, thus enabling membranes to be well attached to skins as well facilitating the release of P. emblica. Meanwhile, a porous structure has a positive influence over the air ventilation. Based on Table 3 and The presence of P. emblica contributes toward a significant improvement in conductivity, which facilitates drafting during the electrospinning process, namely a significantly lower fiber diameter. In particular, the group of PVP/WCS/P. emblica 0.01 wt% exhibits the lowest nanofiber diameter. As for the group of PVP/WCS/P. emblica 0.02 wt%, the viscosity is increased, which subsequently changes the nanofiber diameter. Meanwhile, a rise in the nanofibers also means that there are P. emblica embedded. The variation in the nanofiber diameter is superior to that of PVP/WCS/P. emblica 0 wt% nanofiber membranes. Figure 5E presents a greater distinguish rate.

Hydrophilicity of Nanofiber Membranes
The hydrophilicity of nanofiber membranes can be well perceived according to the water contact angle. Namely, a water contact angle lower than 90 degrees means the materials are hydrophilic and the lower the angle, the higher the hydrophilicity. In this study, the dry facial masks are different from the wet types that contain liquor components. To provide the dry facial masks with convenient and efficient uses, the masks require a certain hydrophilicity for purposes including moisture absorption, adhesion to skins, decomposition of masks, and the release of effective substances. Hence, the hydrophilicity for dry facial masks also affects the P. emblica release. Figure 6 shows that the water contact angles of PVP/WCS/P. emblica 0, PVP/WCS/P. emblica 0.005, PVP/WCS/P. emblica 0.01, and

Hydrophilicity of Nanofiber Membranes
The hydrophilicity of nanofiber membranes can be well perceived according to the water contact angle. Namely, a water contact angle lower than 90 degrees means the materials are hydrophilic and the lower the angle, the higher the hydrophilicity. In this study, the dry facial masks are different from the wet types that contain liquor components.
To provide the dry facial masks with convenient and efficient uses, the masks require a certain hydrophilicity for purposes including moisture absorption, adhesion to skins, decomposition of masks, and the release of effective substances. Hence, the hydrophilicity for dry facial masks also affects the P. emblica release. Figure 6 shows that the water contact angles of PVP/WCS/P. emblica 0, PVP/WCS/P. emblica 0.005, PVP/WCS/P. emblica 0.01, and PVP/WCS/P. emblica 0.02 wt% are 65.00 ± 1.82 • , 69.00 ± 1.82 • , 76.00 ± 2.16 • , and 77.25 ± 2.21 • , respectively. PVP/WCS is a hydrophilic material and with a rise in the P. emblica content, the water contact angle of the membranes is also increased. When the water contact angle is lower than 90 degrees, the PVP/WCS/P. emblica 0 wt% nanofiber membranes still exhibit excellent hydrophilic properties.
The hydrophilicity of nanofiber membranes can be well perceived accordin water contact angle. Namely, a water contact angle lower than 90 degrees means terials are hydrophilic and the lower the angle, the higher the hydrophilicity. In th the dry facial masks are different from the wet types that contain liquor compon provide the dry facial masks with convenient and efficient uses, the masks requi tain hydrophilicity for purposes including moisture absorption, adhesion to skins position of masks, and the release of effective substances. Hence, the hydrophi dry facial masks also affects the P. emblica release. Figure 6 shows that the water angles of PVP/WCS/P. emblica 0, PVP/WCS/P. emblica 0.005, PVP/WCS/P. emblica 0 PVP/WCS/P. emblica 0.02 wt% are 65.00 ± 1.82°, 69.00 ± 1.82°, 76.00 ± 2.16°, and 2.21°, respectively. PVP/WCS is a hydrophilic material and with a rise in the P content, the water contact angle of the membranes is also increased. When the wa tact angle is lower than 90 degrees, the PVP/WCS/P. emblica 0 wt% nanofiber mem still exhibit excellent hydrophilic properties.

Release Rate of Nanofiber Membranes
The PVP/WCS/P. emblica nanofiber membranes are tested for their release rates, which are 0 for PVP/WCS/P. emblica 0 wt%, 0.057 for PVP/WCS/P. emblica 0.005 wt%, 0.13 for PVP/WCS/P. emblica 0.01 wt%, and 0.27 for PVP/WCS/P. emblica 0.02 wt%. To sum up, the release rate is in direction proportion to the P. emblica content. Figure 7B demonstrates the release rate of PVP/WCS/P. emblica 0.02 wt% as related to different lengths of time. The release rates with corresponding times are 0% (0 min), 56.63 ± 1.49% (5 min), 75.10 ± 0.91% (10 min), 96.73 ± 0.35% (15 min), and 100.0 ± 0% (20 min). P. emblica is efficiently released in the first five minutes, then isometrically released in the subsequent 5-15 min, and eventually completely released in the final 15-20 min, thus demonstrating an excellent release rate curve as is required for facial masks. Figure 7B shows the diagram of releasing process of P. emblica from the nanofiber membranes. PVP/WCS/P. emblica nanofibers are decomposed in water, during which P. emblica is released from the nanofibers. The SEM image indicates the morphology when the nanofibers are decomposed. Nanofibers are gradually melted into membrane and then completely dissolved in water, due to which P. emblica is totally released in water. reaches the optimal rate when the P. emblica content is 0.02 wt%. The strengthened activity of tyrosinase facilitates the transformation of tyrosine into levodopa (i.e., hydroxylation) and the series of reactions trigger the melanin synthesis. Consisting of tannins and vitamin C, P. emblica inhibits enzyme activity and then confines the melanin synthesis, which substantiates the incorporation of P. emblica with PVP/WCS nanofiber membranes that have a positive influence on confining the melanin synthesis [45].

Tyrosinase Inhibition Assay of Nanofiber Membranes
PVP/WCS/P. emblica nanofiber membranes are tested for their tyrosinase inhibition rate. Figure 8 shows that the control group (vitamin C) has an inhibition rate of 100% while the inhibition rates are 62.86 ± 1.68% for PVP/WCS/P. emblica 0.005 wt%, 97.03 ± 0.97% for PVP/WCS/P. emblica 0.01 wt%, and 99.53 ± 0.45% for PVP/WCS/P. emblica 0.02 wt%. To sum up, the tyrosinase inhibition rate is in direct proportion to the P. emblica content and reaches the optimal rate when the P. emblica content is 0.02 wt%. The strengthened activity of tyrosinase facilitates the transformation of tyrosine into levodopa (i.e., hydroxylation) and the series of reactions trigger the melanin synthesis. Consisting of tannins and vitamin C, P. emblica inhibits enzyme activity and then confines the melanin synthesis, which substantiates the incorporation of P. emblica with PVP/WCS nanofiber membranes that have a positive influence on confining the melanin synthesis [45].

Biocompatibility of Nanofiber Membranes
PVP/WCS/P. emblica nanofiber membranes are tested for cell viability. Figure 9 shows that the cell viability for the control group is 100%. On day 1, the PVP/WCS/P. emblica 0 wt%, PVP/WCS/P. emblica 0.005 wt%, PVP/WCS/P. emblica 0.01 wt%, and PVP/WCS/P. emblica 0.02 wt% showed cell viabilities of 98.92 ± 2.04%, 92.54 ± 1.64%, 89.53 ± 1.68, and 87.01 ± 2.56%, respectively. On day 3, the control group showed a cell viability of 100%, and the four groups show cell viabilities of 98.13 ± 1.54%, 93.06 ± 2.83%, 85.80 ± 2.65%, and 82.60 ± 1.30%. The higher the P. emblica content, the lower the cell viability. In sum, regardless of whether it is day 1 or day 3, the PVP/CS/P. emblica nanofiber membranes exhibit an average cell viability of over 80%, suggesting that the nanofiber membranes are biocompatible for cell growth [45]. The cytocompatiblity results in Figure 9B-F show the status of NIH/3T3 cells on day 3, the findings of which correspond to the cell activity. The PVP/WCS/P. emblica 0 wt% group shows that the dense cells are at a form of fine fusiform and firmly attached to the well. A small proportion of fibroblast cells have a round shape mainly because NIH/3T3 presents contact inhibition and an excessive number of cells trigger the contact inhibition. The cell density is inversely proportional to the content of P. emblica but still preserves the complete cell morphology and status, which confirms the cytocompatibility. age cell viability of over 80%, suggesting that the nanofiber membranes are biocompatible for cell growth [45]. The cytocompatiblity results in Figure 9B-F show the status of NIH/3T3 cells on day 3, the findings of which correspond to the cell activity. The PVP/WCS/P. emblica 0 wt% group shows that the dense cells are at a form of fine fusiform and firmly attached to the well. A small proportion of fibroblast cells have a round shape mainly because NIH/3T3 presents contact inhibition and an excessive number of cells trigger the contact inhibition. The cell density is inversely proportional to the content of P. emblica but still preserves the complete cell morphology and status, which confirms the cytocompatibility.

Anti-Inflammatory Capacity of Nanofiber Membranes
PVP/CS/P. emblica nanofiber membranes are tested for their anti-inflammatory capacity. Two inflammatory factors (IL-1α and TNF-α) are used for the test. IL-1α and TNFα are involved with inflammation and also trigger acute phase reaction proteins. A rise in the inflammatory factors inflicts cells with death, allergy, septicemia, or inflammatory reactions. As a result, the anti-inflammatory capacity tests with IL-1α and TNF-α as inflammatory factors can determine whether nanofiber membranes trigger the inflammation of

Anti-Inflammatory Capacity of Nanofiber Membranes
PVP/CS/P. emblica nanofiber membranes are tested for their anti-inflammatory capacity. Two inflammatory factors (IL-1α and TNF-α) are used for the test. IL-1α and TNF-α are involved with inflammation and also trigger acute phase reaction proteins. A rise in the inflammatory factors inflicts cells with death, allergy, septicemia, or inflammatory reactions. As a result, the anti-inflammatory capacity tests with IL-1α and TNF-α as inflammatory factors can determine whether nanofiber membranes trigger the inflammation of cells and skins. Figure 10 shows that in the IL-1α test, the OD for the control group is 0.31 ± 0.01, while the ODs for PVP/CS, PVP/CS/P. emblica 0.005, PVP/CS/P. emblica 0.01, and PVP/CS/P. emblica 0.02 are 0.32 ± 0.02, 0.27 ± 0.01, 0.21 ± 0.01, and 0.18 ± 0.01. Figure 11 shows that in the TNF-1α test, the OD for the control group is 2.18 ± 0.17 while the ODs for PVP/CS, PVP/CS/P. emblica 0.005, PVP/CS/P. emblica 0.01, and PVP/CS/P. emblica 0.02 are separately 2.54 ± 0.08, 2.00 ± 0.04, 1.81 ± 0.04, and 1.61 ± 0.05. Based on the two anti-inflammatory capacity tests, the incorporation of PVP/CS induces the occurring inflammatory factors. In contrast, with an increase in the P. emblica content, PVP/CS/P. emblica groups show a declining trend in the inflammatory factor regardless of whether it is IL-1α or TNF-α. Specifically, the PVP/CS/P. emblica 0.02 demonstrates the optimal inhibition efficacy. It is proved that the nanofiber membranes do not cause an inflammatory reaction and that the incorporation of P. emblica can further reduce the presence of inflammatory factors [46].

Conclusions
In this study, electrospinning is employed and successfully creates PVP/CS/P. emblica nanofiber membranes to serve as dry facial masks with excellent melanin-confining effects and biocompatibility. The test results show that the maximal P. emblica extraction rate is 29.0% with an optimal extraction time of 12 h. However, the tyrosinase inhibition rate is adversely affected by a longer extraction time, which suggests that there are more differ-

Conclusions
In this study, electrospinning is employed and successfully creates PVP/CS/P. emblica nanofiber membranes to serve as dry facial masks with excellent melanin-confining effects and biocompatibility. The test results show that the maximal P. emblica extraction rate is 29.0% with an optimal extraction time of 12 h. However, the tyrosinase inhibition rate is Figure 11. TNF-α inflammatory factor of nanofiber membranes as related to the P. emblica content.

Conclusions
In this study, electrospinning is employed and successfully creates PVP/CS/P. emblica nanofiber membranes to serve as dry facial masks with excellent melanin-confining effects and biocompatibility. The test results show that the maximal P. emblica extraction rate is 29.0% with an optimal extraction time of 12 h. However, the tyrosinase inhibition rate is adversely affected by a longer extraction time, which suggests that there are more different extracts or impurities. In addition, the resulting nanofiber membranes exhibit evenly distributed nanofibers and the PVP/CS/P. emblica 0.02 wt% group exhibits a diameter of 165.33 ± 51.66 nm. The nanofiber membranes also demonstrate good hydrophilicity with a water contact angle of 77.25 ± 2.21 • , which facilitates the interaction between the membrane and water while improving the degradation level. Moreover, the release rate test indicates that the nanofiber membranes can reach a P. emblica release rate of 96.73 ± 0.35% in fifteen minutes and P. emblica can be totally released in twenty minutes. Furthermore, PVP/CS/P. emblica nanofiber membranes have tyrosinase inhibition rates as high as 99.53 ± 0.45%, thus suggesting an excellent restriction on melanin synthesis. PVP/CS/P. emblica nanofiber membranes demonstrate biocompatibility with a lowest cell activity of ≥82.60 ± 1.30%. Regarding the cytocompatibility of PVP/CS/P. emblica nanofiber membranes, it is proved that the cells demonstrate a good morphology and adhesion degree. As for the anti-inflammatory capacity, PVP/CS/P. emblica nanofiber membranes show inhibition capacity against IL-1α and TNF-α inflammatory factors that are separately reduced to 0.18 ± 0.01 and 1.61 ± 0.05. This result substantiates the view that ill inflammation is absent. Serving as dry facial masks, PVP/CS/P. emblica nanofiber membranes demonstrate excellent melanin-confining, hydrophilic, biocompatible, and inflammation-free features, and can be developed for and applied to the cosmetics field.