EFFECT OF TRANSFERSOME ON THE STABILITY AND ANTIOXIDANT ACTIVITY OF GLUTATHIONE IN ANTIAGING CREAMS

Objective: Glutathione is an important antioxidant compound that is added to various cosmetic preparations. This study compared the stability, antioxidant activity, and penetration of glutathione creams formulated with and without transfersome, a commonly used carrier system. Methods: The particle size of the transfersome was 55.65 nm, with a polydispersity index of 0.398 and an entrapment efficiency of 66.22%. During cycling and centrifugal testing, the creams (with and without transfersome) did not change color or demonstrate phase separation. Chemical stability analyses of the products were performed using high-performance liquid chromatography. Results: The remaining glutathione content in the transfersome cream was 83.44%, while that of the non-transfersome cream was 47.92%. In the penetration test using Franz diffusion cells, the transfersome cream demonstrated a cumulative penetration of 4474.44 µg/cm 2 , with a cumulative concentration percentage of 39.60% and a flux of 510.38 µg/cm −2 h −1 . In contrast, the non-transfersome cream demonstrated a cumulative penetration amount of 2793.80 µg/cm 2 , with a cumulative concentration percentage of 24.73% and a flux of 340.12 µg/cm −2 h −1 . In addition, the IC50 value of the transfersome cream preparation was 11.89 µg/mL, while that of the non-transfersome preparation was 15.57 µg/mL. Conclusion: Our findings indicate that the use of transfersome increases the stability and penetration of glutathione in cream preparations. Stability test, Antioxidant activity test, Penetration test.


INTRODUCTION
Glutathione is a potent antioxidant and is a thiol tripeptide compound found in every cell of the human body. Glutathione consists of three amino acids, namely, L-glutamine, L-cysteine, and L-glycine connected by gamma peptide bonds between the amine cysteine group (which is attached to the peptide bond by glycine) and the carboxyl group of the glutamate side chain [1]. In the body, two forms of glutathione exist reduced glutathione (GSH) and oxidized glutathione (GSSG) [2].
Glutathione in aqueous solutions is very unstable and easily decomposes, while GSSG is less susceptible to decomposition in aqueous solutions and is suitable for use in liquid form [3]. Glutathione can experience a decrease in quality due to the influence of heat, oxygen, and light, which can cause unpleasant odors such as sulfur odors and decreased preparation content [4]. Glutathione in topical preparations can be used to treat various skin issues, including dry and dull skin, the loss of elasticity, and the appearance of excessive lines and wrinkles [5].
Transfersome was first introduced in 1991 by Gregor Cevc. The term transfersome comes from the Latin transferre, which means to carry the whole, and from the Greek soma, which means the body [6]. Transfersome, in the broadest sense, is a compound or aggregate that very easily adapts and responds to its environment. In general, transfersome has a structure consisting of a hydrophilic part (aqueous layer) as its core surrounded by a hydrophobic part (double lipid layer) that can carry active substances with various solubilities [7].
Transfersome as a carrier in a given formulation has a number of advantages, including the ability to change the shape of the formulation so it can pass through narrow pores, improved penetration, the ability to carry active substances of a large solubility range efficiently (up to 90%), the ability to make compounds with a high molecular weight low, and the ability to release compounds slowly and gradually. In this study, we compared the stability, antioxidant activity, and penetration of glutathione creams formulated with and without transfersome, a commonly used carrier system.

Steps
Preparing the solvent phase Approximately 6.8 g of potassium dihydrogen phosphate was carefully weighed and then dissolved in 1000 mL of aquadest. The pH was then adjusted with dilute phosphoric acid (1:10) to pH 3.0.
Preparing the glutathione standard master solution 10 mg of glutathione standard was carefully weighted, then diluted with a buffered phosphate of pH 3.0 to concentrations of 1000 µg/mL, 100 µg/mL, and 10 µg/mL.

Determination of optimum analysis conditions
Before high-performance liquid chromatography (HPLC) analysis, the maximum wavelength was determined using UV-Vis spectrophotometry. The standard solution used for examining the maximum wavelength was 10 µg/mL. The determination of the optimum analysis conditions was performed through varying the flow rate at 0.6, 0.8, 1.0, and 1.2 mL/min.

System suitability test
The solution for the system suitability test was a standard glutathione solution at a concentration of 10 µg/mL, which was injected into the HPLC 6 times.

Selectivity test
The solution used for the selectivity test was a placebo or cream base that did not contain glutathione. The cream base used was 25 mg, which was sonicated for 15 min. After sonication, the cream was transferred into a centrifugation tube and vortexed for 5 min. After that, it was centrifuged at 4000 rpm for 15 min. The formed supernatant was then taken and filtered with a Whatman nylon filter syringe (0.45 µm).

Accuracy and precision test
Accuracy and precision tests were performed with glutathione concentrations of 80%, 100%, and 120%. For example, an 80% glutathione concentration was made by mixing 8 mg of standard glutathione with a cream base of up to 500 mg. The 100% concentration was made by mixing 10 mg of standard glutathione with a cream base of up to 500 mg. The 120% concentration was made by mixing 12 mg of standard glutathione with a cream base of up to 500 mg. For each concentration, we weighed the sample to ± 25 mg and then placed it into a 10-mL volumetric flask and sonicated it for 15 min, with final placement into a centrifuge tube. Before centrifugation, the solution was vortexed for 5 min then centrifuged at 4000 rpm for 15 min. The supernatant from each concentration was filtered using 0.45 µm nylon syringe filters. The 80% and 120% concentrations were injected into the HPLC 3 times, with the 100% concentration was injected 6 times.

Transfersome formulation and optimization of the sodium phosphate-buffered
The phosphate-buffered solution at pH 7.4 was made by mixing 50 mL of 0.2 M potassium dihydrogen phosphate with 39.1 mL 0.2 N sodium hydroxide in a 200-mL volumetric flask, then increasing the volume gradually with aquadest to 200 mL. Previously, 0.2 M potassium dihydrogen phosphate was made by weighing 1.36 g of potassium dihydrogen phosphate powder, dissolved in distilled water in a 50-mL volumetric flask, and a sufficiently small volume was added to reach the measuring flask limit. To prepare the 0.2 N sodium hydroxide solution, sodium hydroxide was weighed to 0.4 g, then dissolved in 50 mL of distilled water. The pH was checked using a pH meter. Data in Table 1.

Transfersome preparation
Transfersome was prepared by dissolving phosphatidylcholine and tween 80, then dissolving it into dichloromethane as much as 15 mL.
After complete dissolution, the solution was placed into a roundbottomed flask and applied with a rotary evaporator at 40°C with an initial speed of 50 rpm. This speed was increased by 25 rpm every 5 min to reach 150 rpm to remove any organic solvents for 60 min. The solution was then flowed with nitrogen gas for 2 min and left for 24 h in a closed state. Glutathione (0.6 gram) was then dissolved with phosphate buffer (pH 7.4) as much as 12 mL; then the solution was hydrated to a thin layer in a round-bottomed flask. The flask was then placed on a rotary evaporator without a vacuum at a temperature of 30 o C and rotated with an initial speed of 50 rpm; this speed was then increased by 25 rpm every 5 min to reach 150 rpm with the help of glass beads that easily peel off. The resulting suspension was removed from the rotary evaporator and then sonicated for 10 min.

Particle size test
This test used a particle size analyzer at 4°C. One drop of the sample from each transfersome formulation was dispersed in 10 mL aquadest, then placed in a cuvette to the ideal limit specified.

Entrapment efficiency test
Absorption efficiency was measured by calculating the total and free glutathione concentration. The total glutathione concentration was the concentration of glutathione that was absorbed and free in suspension. To separate the free glutathione, a centrifuge was used at a speed of 13,000 rpm for 2 h at 4°C. The supernatant formed then contained the free glutathione and was measured using HPLC at a wavelength of 200 nm. Absorption efficiency (EE) was measured using the following equation:

EE
Totalconcentration Freeconcentration Total concentrat Accelerated stability test Accelerated stability testing was performed by inserting a number of cream samples into plastic pots. Then they were stored in a climate chamber at storage conditions of 40±2°C, with humidity set at 75±5% RH. The creams were sampled at 0, 1, 2, and 3 months, followed by HPLC analysis. The samples were weighed as much as 25 mg for stability tests at 0, 1, 2, and 3 months, then placed in a 10-mL volumetric flask with solvents added to the limit. The solution was then sonicated for 15 min and placed into a centrifugation tube. Before centrifuging, the solution was vortexed for 5 min. The centrifuge speed was 4000 rpm for 15 min. The supernatant was taken and filtered using a Whatman nylon filter syringe (0.45 µm).

Stability cycling test
This stability test was performed by storing the cream at 4°C for 24 h, then removing it and placing it into an oven at a temperature of 40±2°C for 24 h; the treatment consisted of one cycle. This experiment was repeated for as many as six cycles over 12 days. The physical condition of the cream such as color, smell, and presence or absence of phase separation was then observed.

Centrifugal test
This stability test was intended to determine whether phase separation occurred in the cream samples. Cream samples were centrifuged for 3750 rpm for 5 h.

Antioxidant activity test and making the DPPH stock solution
Five milligrams DPPH was dissolved in 100 mL of methanol to obtain a concentration of 50 µg/mL. The solution was examined for its maximum wavelength using UV-Vis Spectrophotometry.

Making a blank solution
A total of 1 mL of the methanol was added to 3 mL DPPH, then vortexed for 20 s and incubated for 30 min. After 30 min, the absorbance was measured with the selected maximum wavelength.

Making sample solutions
The cream samples (500 mg containing 10 mg glutathione) were dissolved in 20 mL of methanol and then sonicated for 10 min. After sonification, the solution was centrifuged at 4000 rpm for 10 min. The centrifuged filtrate was then diluted to form concentrations of 3 µg/mL, 5 µg/mL, 8 µg/mL, 10 µg/mL, 15 µg/mL, and 20 µg/mL. As much as, 1 mL of each sample was added to 3 mL DPPH, then this solution was vortexed for 20 s and incubated for 30 min. After 30 min, the absorbance was measured with the selected maximum wavelength.

Determination of the % inhibition value
From the absorbance obtained from the blank solution and the sample, we calculated the % inhibition using the following formula:

Determination of IC50 values
After the % inhibition value was obtained, the data were plotted into a graph with concentration as the X-axis and % inhibition as the Y-axis. From the graph, we then obtained a linear regression equation in the form of y = a + bx, with the value of x representing the IC 50 value.

Extraction
The wavelength used for HPLC analysis was 200 nm, because the maximum wavelength can cause maximum absorbance. Based on these data, the selected flow rate was 0.8 mL/min. Even though the flow rate at 1.0 mL/min had a larger peak area, the tailing factor of the flow rate was larger than that at 0.8 mL/min. In addition, the retention time was similar to that shown on USP 41 NF 36, which is 5 min (Tables 2-3).
The system suitability test was needed because there were possible disturbing factors due to variations in the analysis tools and techniques used. The system suitability test was performed 6 times at a concentration of 10 µg/mL to produce a coefficient of variation. Based on the result of the analysis, the coefficient of variation obtained was 0.34% for the area, 0.34% for retention time, 1.18% for tailing factor, 0.90% for theoretical plate number (N), and 0.90% for the column efficiency value (height equivalent to a theoretical plate). The coefficient value of variation obtained meets the requirements, which is <2% ( Table 4).
The linear relationship is shown by the correlation coefficient parameters for the linear regression analysis of y = a + bx. From the calibration curve, the linear regression value obtained y = 14050x + 68846. With a correlation coefficient of 0.9992, the glutathione standard meets the linearity requirements because the correlation coefficient value is close to 1 or > 0.9990. Based on the results of the glutathione standard linearity test, the detection limit value was 6.79 µg/mL, and the quantitation limit value was 22.63 µg/mL. The smaller the limit of detection and limit of quantification values are, the more sensitive the analytical method. The data shown in Table 5 and Fig. 1.   The selectivity test aims to assess the ability of a method to measure a given substance specifically with the possibility of other components in the sample matrix. The results of the chromatogram obtained are then compared with those of the standard solution. Based on the results of this analysis, in the chromatogram of the placebo solution, there was no interference in the peak of other compounds around the glutathione retention time (Fig. 2).
Accuracy and precision tests were performed by mixing a number of standard glutathione solutions with the cream bases. This is called the simulation method. Calculation of the accuracy is performed by calculating the recovery value (% UPK). The recovery value is calculated by comparing the results of the analysis with the actual results. Based on the results of the study in Table 6, the %UPK at a concentration of 40 µg/mL was 101.32%, the concentration of 100% was 100.93%, and the concentration of 120% was 99.51%. From these data, the glutathione standard has a precision range between 99.51 and 101.32%, which is in accordance with the requirements of 98-102%. Precision testing showed that the coefficient variation at a concentration of 50 µg/mL was <2%.
Phosphatidylcholine acts as the main constituent in the manufacture of multiple lipid membranes. The phosphatidylcholine used in this study was Phospholipon 90G. Phosphatidylcholine has both hydrophilic and hydrophobic groups; thus it can form a double-layered structure. Phosphatidylcholine was chosen because it is relatively odorless. The surfactant used to make the glutathione transfersome was tween 80. Tween 80 is used as a surfactant because it also has a hydrophilic and lipophilic structure. Tween 80 has a long hydrocarbon chain that is flexible and not large enough to increase deformability. In addition, Tween 80 can also replace a portion of a double lipid membrane so that it can provide maximum deformability and make the transfersome more elastic. This can make vesicles easier to release the drug. In addition, Tween 80 is a non-ionic surfactant that is less irritant and is better tolerated by the body than other surfactants. Because it is a non-ionic surfactant, Tween 80 penetrates into the intracellular area of the stratum corneum, increasing fluidity and dissolving the lipid component [8][9][10].
Making transfersome using the layer hydration method consists of a thin-layer formation, with stages of hydration. The thin-layer formation uses a rotary evaporator with the help of a vacuum. Phospholipon and Tween 80 are dissolved first with dichloromethane. The temperature used to make transfersome is 40°C, at which the methanol as a solvent will evaporate. The use of a vacuum is intended to speed up the evaporation process of the solvents, but the vacuum is only used for a few moments so as not to cause a rapid evaporation process, which may cause uneven thin-layer formation. After the thin layer is formed, the thin layer is flowed with N 2 gas for 2 min then stored at 4°C for 24 h to prevent the oxidation of lipids and maximize solvent evaporation [9].
The next stage is the hydration of the thin layer that has been formed. The thin layer is hydrated using a phosphate-buffered at pH 7.4. The vesicle layer will form spontaneously and the hydrating fluid will enter into the dry thin-layer lipids. In the transfersome suspension that has been formed, particle size reduction is done. The reduction in particle size was done using ultrasonication at a 10% amplitude for 10 min. The time used to reduce the particle size will affect the size of the particles formed. The longer ultrasonication time, the smaller the particle size; however, if the ultrasonication time is too long, it can cause the oxidation and precipitation of phospholipids. Table 7 shown the Z-average particle size for formulation 1 was 55.65 nm; formulation 2 was 63.56 nm; and formulation 3 was 135.7 nm. Fig. 1: Calibration curve of the glutathione standard  The Z-average value was used to determine the particle size because the Z-average is the main and most stable parameter produced by the DLS method. Based on these data, F1 had the smallest particle size. This shows that the greater the concentration of phosphatidylcholine, the smaller the particle size obtained, and the greater the amount of surfactant, the greater the particle size obtained (Fig. 3).
The polydispersity index (PDI) value that can be obtained by the DLS method is in the range 0-0.6. If the PDI value obtained is small or close to 0, the spread of the particle size is evenly distributed or homogeneous. Whereas, if the IPD value obtained >0.6, samples have an increasingly broad distribution value [11]. PDI values can increase with increasing particle size because large particles tend to aggregate. From the results of the adsorption efficiency test, the greater the amount of phospholipids used, the greater the absorption efficiency. If the amount of phospholipids increases, the formation of the vesicles will also increase so that the absorption efficiency will be more optimal. In addition to the decreasing surfactant concentration, the absorption efficiency is even greater.
The purpose of this cycling test was to accelerate the changes in storage conditions that usually occur under normal conditions so that the preparation will experience varying levels of stress. The physical appearance of each glutathione preparation was observed before and after the test. The physical transfersome glutathione cream did not change color and still smelled of phosphatidylcholine. Whereas the non-transfersome cream did not change color, but there was a slight smell of sulfur in the cream preparation. However, from the two creams, no phase separation was observed and they remained homogeneous. Thus, it can be concluded that both creams are physically stable; however, glutathione transfersome creams appear to be more stable than non-transfersome creams.
This centrifugation test was likened to the gravitational force that will be received by a number of creams for 1 year of storage. Based on the results of the experiment, the transfersome glutathione cream and nontransfersome cream after centrifugation did not show any separation of the water phase and oil phase. The creams were likely protected from phase separation because of the use of emulsifying agents, which served to maintain the cream preparation. In addition, the homogenization process can also prevent separation. Our results indicated that both creams can withstand the force of gravity for 1 year.
In cosmetic preparations, the accelerated stability test can be performed in 30 days or 90 days, but for chemical stability and determining shelf life, it is recommended to carry out testing for a minimum of 3 months with the same conditions as in pharmaceutical products [12]  The amount of glutathione remaining from each cream decreased with increasing testing time. This is because glutathione is easily oxidized, with the thiol group easily oxidized to a disulfide group, which causes the glutathione to lose its antioxidant activity [13]. In addition, it can be caused by glutathione instability in oil cream formulations exposed to water and at high storage temperatures. Glutathione in the pH range of 5.0-8.0 can lose 10-15% of its level at room temperature storage, while storage at temperatures below 15°C, the glutathione lost is about 0-5% [14]. Reduced glutathione levels can also result because glutathione can be oxidized by oxygen molecules and the presence of metals (such as iron) in the preparation. From this accelerated stability test, we were able to determine the value of the shelf life for both creams using a zero-order reaction. The shelf life for the transfersome cream was determined to be 3.5 months, while that for the non-transfersome cream was 1.1 months. Result shown in Table 8 and Fig. 4.
Although the glutathione in this study was formulated in the form of transfersome, the storage period appears to be reduced. This is     The in vitro penetration test had two main parameters, namely, the cumulative amount of the penetrated substance (µg/cm 2 ) and the penetration rate (flux) [9]. Flux values are obtained from the slope of the line or the slope of the linear curve in steady-state and are obtained from a comparison between the cumulative amounts of penetration to time. Based on the data, it can be seen that the cumulative concentrated amount in the transfersome preparations is greater than that of the nontransfersome preparations. It can also be seen that the transfersome preparations have higher flux than the non-transfersome preparations. The cumulative amount of penetration can be influenced by factors such as the composition of the vesicles, vesicle size, membrane fluidity, elasticity, absorption efficiency, and method of manufacture [15].
The transfersome preparation contains phospholipids with a high affinity for biological membranes so that the phospholipid vesicles can join the intracellular lipid layer, which results in increased transfersome permeability. This can result in a greater transfer of the cream permeation compared with non-transfersome creams [9]. In addition, transfersome contains a surfactant that is an edge activator that functions to destabilize vesicles and increase deformability so that it allows increased glutathione penetration. Vesicles act as carriers of substances, where intact vesicles penetrate the stratum corneum under the influence of the transcutaneous hydration gradient. With the ability of vesicles to penetrate the stratum corneum spontaneously, the permeation ability of the transfersome is increased. Phospholipid vesicles serve to penetrate the stratum corneum and then modify the lipids between cells so as to increase overall fluidity, which causes an increased amount of glutathione to penetrate the skin.

CONCLUSION
The optimum analytical conditions for analyzing glutathione in antiaging cream preparations involved using an HPLC system with a C18 column and a UV-Vis detector, with an optimum wavelength of 200 nm and a flow rate of 0.8 mL/min. These conditions are in accordance with the requirements proposed, where linearity, selectivity, and accuracy and precision testing met the required criteria. Glutathione formulated as transfersome has a ratio of phosphatidylcholine to tween 80 of 90:10. This is because in comparison, these transfersome preparations have a small particle size and PDI, with a high absorption efficiency.
The results of the accelerated stability test showed that the amount of glutathione remaining in the cream transfersome preparations was 83.44%, while that of the non-transfersome cream was 47.92%. The results of the cycling test and the centrifugal test showed that the transfersome and non-transfersome preparations had no color change or phase separation and were resistant to gravity for 1 year of storage. However, for the non-transfersome cream, during the cycling test, a small amount of sulfur odor was detected from the preparation. In the transfersome and non-transfersome creams, the glutathione preparations were determined to have high antioxidant activity. In addition, penetration test results showed that the transfersome cream was able to increase the cumulative amount and flux of the penetrated glutathione better than the non-transfersome cream.
After this study, the cream preparations were stored at temperatures below 15°C so that the physical and chemical stability of the cream and the compounds contained could be maintained during the storage period, given the volatile nature of the glutathione compound. In addition, future studies are necessary to analyze other compounds that may be present in the formulations, including by-products of oxidized glutathione.