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FORMULATION AND EVALUATION OF ANTIAGING PHYTOSOMAL GEL

Objective: The aim of the present study was to prepare and evaluate antiaging phytosomal gel. Method: For this purpose, tender coconut water, Aloe vera extract, grape seed extract, vitamin E, and jojoba oil were taken. The cytokinins present in coconut water show astounding effects in preventing premature aging. A. vera contains Vitamin E and C and phenol compounds which contribute to aging. Grape seed extract contains polyphenols mainly in the form of flavonoids which are highly potent antioxidants. It also possessed an antiaging vitamin known as oligomeric proanthocyanidins which prevented premature aging. Vitamin E was chosen because of its antioxidant properties. Jojoba oil was chosen because it had good moisturizing properties and it was similar to human skin oil, sebum. Conventional dosage forms such as creams and gels were prepared using these ingredients, but they showed less antiaging effect. Therefore, phytosome was prepared by binding herbal extracts to phosphatidylcholine (acts as a carrier and nourishes the skin). They had a phospholipid molecular structure which included a watersoluble head and two fat-soluble tails, and due to this dual solubility, it was better absorbed and so it was used for the treatment of skin disorders, antiaging, and skin carcinomas. Result: Nine different formulations of the antiaging cream, gel, and phytosomal gel were prepared. Physicochemical parameters such as pH, viscosity, homogeneity, spreadability, and extrudability were determined. In vitro antioxidant studies were performed for the prepared antiaging cream, gel, and phytosomal gel. Among the three different formulations, phytosomal gel was chosen to be the best formulation to treat wrinkles and blemishes on the skin. When compared to the conventional dosage forms such as creams and gels, the phytosomal gel could release the herbal ingredients and showed better penetration into the skin. From the nine different formulations, F2 containing both tender coconut water and A. vera extract was chosen as the optimized formula. Optimization was done on the basis of in vitro antioxidant studies and physicochemical parameters. F2 acts as a potent free radical scavenger and inhibits oxidation by free radicals. 2,2-diphenylpicrylhydrazyl free radical scavenging assay was chosen as the best method in screening the antioxidant activity of the herbal extracts. The IC50 value of the prepared antiaging cream, gel, and phytosomal gel was found to be 70.5 μg/ml, 65.0 μg/ml, and 47.0 μg/ml, respectively. The lower the IC50 value, the highest the antioxidant activity. Thus, the antiaging phytosomal gel proved to show the highest antioxidant activity. The stability of the optimized formulation was carried out at two different temperatures, 30°C ± 2°C and 4°C ± 2°C, and the formulation was found to be stable at the end of 45 days. Conclusion: From the present study, it can be concluded that the prepared antiaging phytosomal gel was safe, convenient, and efficient carrier to deliver the herbal extracts. It also showed better penetration into the skin. Hence, the desired antiaging property was obtained. Hence, the desired antiaging property was obtained and is used in skin care cosmetics.


INTRODUCTION
Aging is a very common phenomenon to all living beings. It is signified by progressive deterioration and degeneration of cells and organs. As far as human beings are concerned, aging is a complex phenomenon of physical, psychological, and social changes. According to biologists, aging is a sum of all changes that occur in a living being with the passage of time and lead to a decreasing ability to survive stress, to functional impairment, and finally to death [1,2].
The effect of aging on the world population does have an indirect effect on the worldwide medical care and health scenarios [3]. The United Nations have stated that the number of people worldwide of the age group 60 or above will be rising one in 10 to the current one in five ratios by 2050 and the ratio in developing nations and developed nations are going to double and triple, respectively. These demographic statistics suggest healthcare scenario to be more careful with the changes associated with aging and to concentrate on more antiaging therapies [4,5].
The theories involved in the aging process are mutation accumulation and antagonistic pleiotropy theory. The symptoms include atrophy, laxity, wrinkling, sagging, dryness, blemishes, and sparse gray hair. Being the largest organ of the body, in case of body weight and surface area, skin has the most profound and observable changes of aging [6].
Cosmeceutical is a term used to refer to all the combinations of cosmeceutics and pharmaceuticals. These are mostly the hybrid products of pharmaceutics and cosmetics. These are meant for improving the beauty and skin tone through natural ingredients [7,8]. Cosmeceuticals are the most mushrooming arena of natural personal care industry. However, the sad part of story is that a large percentage of these products do have a lot of carcinogenic agents such as diethanolamine and triethanolamine (TEA). Due to these side effects, researchers have now turned to the natural side of the field. Herbal cosmetics are now a fine area of interest for researchers.
The antioxidants such as alpha-tocopherol and ascorbic acid are used widely these days as they are safer and have less adverse effects than the synthetic antioxidants. The mechanism of antioxidants is the scavenging of the free radicals produced in vivo results in cell death and tissue damage. Thus, antioxidants are vital substances which Antiaging cream reduces the wrinkles and blemishes on the skin. The advantage is that these have lesser penetrations and can be removed by sweating or a mild washing of the face. Gel is a semisolid preparation with two interpenetrating phases: A gelling phase and a liquid. The continuous liquid phase allows free diffusion of molecules through the skin. Gels are preferred to creams as they have less emollient effects and no pore clogging. There is also enhanced penetration of drugs [26,28].
Phytosomes are very recent introduction into herbal formulation as they are better absorbed and have higher bioavailability. The term phyto means "plant" while "some" means cell like. This are advanced forms of herbal formulation that contains bioactive phytoconstituents of herbal extract surrounded by a lipid. Phytosomes have a better pharmacokinetic and pharmacodynamic profile. In short, phytosomes are a bridge of connection between the conventional drug delivery system and the novel drug delivery system [30].
Advantages of phytosomes are increased bioavailability, efficient nutrient safety, and optimum entrapment efficiency. Phosphatidylchloride in phytosomes has a role both as a skin nourisher and also nourishes the skin. The phytosomes have better clinical benefits. The structural properties are also superior and are unique. Phytosomes also can cross cell membranes and enter cells. Being a complex between a natural products and natural phospholipid, there is the formation of hydrogen bond between the two compounds. With water, phytosomes assume a micelle shape. These are better absorbed, utilized, and have better results. It has higher bioavailability than conventional drug. There are increased pharmacokinetic and pharmacodynamic properties [32,33].
Phytosomes are used in liver diseases and heart diseases. It is also used as anti-inflammatory, lipolytic, vasokinetic, trophodermic, immunomodulatory, and antioxidant agents. Phytosomes are a patented technology developed by a leading manufacturer of drugs and nutraceuticals to incorporate standardized plant extracts or water-soluble phytoconstituents into phospholipids to produce lipid compatible molecular complexes called as phytosomes and so vastly improve their absorption and bioavailability [35].

Materials
Tender coconut water was obtained from CDB lab, South Ernakulum. Aloe vera extract was obtained from Elixir Extracts, Nellad Kochi.

Formulation of O/W cream
Required quantities of glyceryl monostearate (GMS), light liquid paraffin, isopropyl palmitate, emulsifying wax, stearic acid, and cetyl alcohol were weighed and transferred into a 200 ml beaker. To this, a required amount of jojoba oil and Vitamin E were added and heated in a water bath. Another 200 ml beaker was weighed, and required quantities of glycerin, TEA, methylparaben, and propylparaben were added followed by A. vera extract, grape seed extract, and tender coconut water. The beaker was heated to a temperature up to 60-70°C. After both the solutions had attained their respective temperature, beaker was removed from heat and poured the oily phase to water phase with continuous stirring. The stirring was continued until the mixture forms a creamy emulsion. A few drops of perfume with a small quantity of propylene glycol were mixed and were added to the creamy emulsion after cooling ( Table 1).

Formulation of gels
Antiaging gel was prepared using carbopol 940 as the gelling agent. Gelling agent was dispersed in a small quantity of distilled water and then stored overnight to ensure complete hydration. The active ingredients such as tender coconut water, A. vera extract, grape seed extract, Vitamin E, and jojoba oil in a suitable solvent such as propylene glycol were added to the dispersion. Other excipients such as methylparaben and propylparaben were also added slowly with continuous stirring. In carbopol gels, pH of the vehicle was brought to neutral by adding TEA. The final weight of the gel was adjusted to 50 g with distilled water. Entrapped air bubbles were removed by keeping the gels in vacuum desiccator ( Table 2).

Formulation of phytosomes
Phytosomes are prepared by reacting the herbal extract in an aprotic solvent such as methylene chloride, dioxane, and ethyl acetate with the phospholipid such as phosphatidylcholine, phosphatidyl ethanolamine, or phosphatidylserine dissolved in the same solvent. After solubilization has completed, the complex compounds are isolated by removing the solvent under vacuum, by freeze drying or by precipitation with nonsolvents such as n-hexane. Thus, the obtained complexes are lipophilic in character and soluble in a polar and aprotic solvent, in which the individual components of the complex are normally insoluble [62].

Formulation of phytosomal gel
Phytosomes are prepared by reacting the herbal extract and phospholipid such as soy lecithin in a ratio of 1:1 and dissolving them in an aprotic solvent such as ethyl acetate. After solubilization has completed, the complex compounds are removed by solvent evaporation technique. Thus, phytosomes are obtained. Gel was prepared using carbopol 940 as the gelling agent which was dispersed in a small quantity of distilled water and then stored overnight to ensure complete hydration. The active ingredients such as tender coconut water, A. vera extract, grape seed extract, Vitamin E, and jojoba oil in a suitable solvent such as propylene glycol were added to the dispersion. Then, preservatives such as methylparaben and propylparaben were also added slowly with continuous stirring. Then, the prepared phytosomes were incorporated into the gel, and thus, the phytosomal gel was obtained. This phytosomal gel showed better release of herbal extracts and better penetration to the skin, and as a result, desired antiaging property was obtained.

Evaluation studies
In vitro antioxidant studies [40] 2,2-diphenylpicrylhydrazyl (DPPH) free radical scavenging assay [41] Different concentrations of standard ascorbic acid and sample, namely, 20, 40, 60, 80, and 100 mcg/ml were prepared in methanol. 0.002% DPPH in methanol was used as free radical. Equal volume of different concentrations of standards and DPPH was mixed in a clean and labeled test tubes separately, and the tubes were incubated at room temperature in the dark for 30 min. The absorbance was measured at 517 nm using UV-Vis spectrophotometer. The degree of stable DPPH* decolorization to DPPHH (reduced form of DPPH) yellow indicated the scavenging efficiency of the sample. The scavenging activity of the sample against the stable DPPH* was calculated using the following equation:

Scavenging activity (%)=(A−B/A) 100
Where A is absorbance of control and B is absorbance of sample.
Reducing power assay Different concentrations of standard ascorbic acid and sample, namely, 20, 40, 60, 80, and 100 mcg/ml in 1ml of methanol were mixed with 2.5ml of phosphate buffer (200 mM, pH 6.6) and 2.5 ml of 1% potassium ferricyanide separately. The mixtures were placed in a water bath for 20 min at 50°C, cooled rapidly, mixed with 2.5 ml of 10% tri Joshua et al.
chloroacetic acid, and 0.5 ml of 0.1% ferric chloride. The intensity of iron (II)-ferricyanide complex was determined by measuring the formation of Perl's Prussian blue color at 700 nm after 10 min. The higher absorbance of the reaction mixture indicates increased reducing power. The scavenging activity of the sample was calculated using the following equation:

Scavenging activity (%)=(A−B/A) 100
Where A is absorbance of control and B is absorbance of sample.
Nitric oxide scavenging assay Sodium nitroprusside (5 µM) in standard phosphate-buffered solution (PBS) was incubated with different concentration of the samples dissolved in standard phosphate buffer (0.025M, pH 7.4) and the tubes were incubated for 25°C for 5 h. After 5 h, 0.5 ml of incubation solution was removed and diluted with 0.5 ml Griess reagent (prepared by mixing equal volume of 1% sulfanilamide in 2% phosphoric acid and 0.1% M naphthyl ethylenediamine dihydrochloride in water). The absorbance of chromophore formed was read at 546 nm. The control experiment was also carried out in similar manner, using distilled water in place of samples. The activity was compared with the standard ascorbic acid, which was used a standard antioxidant.
Hydrogen peroxide assay A solution of hydrogen peroxide (20 mM) was prepared in PBS (pH 7.4). Various concentrations of 1 ml of the samples or standards in methanol were added to 2 ml of hydrogen peroxide solutions in PBS. The absorbance was measured at 230 nm, after 10 min against blank solution that contained extracts in PBS without hydrogen peroxide. IC50 value is the concentration of the sample required to scavenge 50% free radical. The above experiments were performed (in triplicate) and the percentage inhibition was calculated using the following formula: Where A 0 was the absorbance of the standard (ascorbic acid) and A 1 was the absorbance of samples.

Physical evaluation of antiaging cream and gel
Preliminary evaluation was carried out as follows:

pH
The pH meter was calibrated using standard buffer solution such as pH 4 and 7. About 0.5 g of the cream was weighed and dissolved in 50.0 ml of distilled water and its pH was measured.

Viscosity
Viscosity of the formulation was determined by Brookfield Viscometer at 100 rpm, using spindle no 7.

Homogeneity
The formulations were tested for the homogeneity by visual appearance

Spreadability
Two glass slides of 20 cm × 20 cm were selected. A small amount of sample was sandwiched between the two glass slides. A 100 g weight  was placed on the upper slide so that the cream between the two slides was pressed uniformly to form a thin layer. The weight was removed and then fixed to a stand without slightest disturbance in such a way that the upper slide slides off freely, to the force of weight tied to it. The time taken for the upper slide to separate away from the lower one was noted using a stop clock. This parallel plate method is the most widely used method for determining and quantifying the spreadability of semisolid preparations. Simplicity and relative lack of expense are the advantages of this method.

Extrudability
It is an empirical test to measure the force required for the cream to extrude out from the tube. The prepared cream was filled into a collapsible tube and it was sealed and the weight of the tube was recorded. Placed a 500 g weight on the tube and the amount of cream that extruded out was collected and weighed. Then, the percentage of cream extruded was calculated. The packing of creams has gained a considerable importance in the delivery of desired quantity of cream; therefore, measurement of extrudability has become some important criteria for creams.

Characterization of phytosomal gel
The behavior of phytosomes in both physical and biological system is governed by the factors such as physical size, shape, stability, and its distribution. Therefore, the phytosomes are characterized for physical attributes, i.e. shape, size, and its distribution.

Visualization
Visualization of phytosome can be achieved using scanning electron microscopy (SEM).
Vesicle size and zeta potential The particle size and zeta potential can be determined by dynamic light scattering (DLS) using a computerized inspection system and photon correlation spectroscopy.

Vesicle stability
The stability of vesicles can be determined by assessing the size of the vesicles overtime. Mean size is measured by DLS.

Comparison studies of antiaging cream, gel, and phytosomal gel
The prepared formulations of cream, gel, and phytosomal gel were compared to find out which formulation had the highest antioxidant activity. The comparison study was done on the basis of in vitro antioxidant models.

Comparison studies based on the IC50 values of each formulation
A comparative study was also done on the basis of IC50 value of each formulation. The IC50 value is the concentration of the sample which scavenges 50% of the free radicals. The lower the IC50 value, the highest the antioxidant activity.

Stability studies
Stability studies were performed according to the ICH guidelines. The optimized formulation was kept at two different temperatures 30°C ± 2°C and 4°C ± 2°C for 45 days.

Comparison of the optimized phytosomal gel with marketed antiaging gel
The prepared antiaging phytosomal gel was compared with a marketed formulation, antiaging A. vera gel. This marketed formulation contained A. vera as antiaging ingredient because of the presence of phenol compounds and Vitamin E and C. The phytosomal gel containing tender coconut water and A. vera extract was compared with the antiaging A. vera gel.

RESULTS AND DISCUSSIONS
Physicochemical parametric evaluation studies Physicochemical parameters such as pH, viscosity, homogeneity, spreadability, and extrudability of antiaging creams and gels were found out, and it was found that F2 formulation showed optimum value.

Physicochemical parameter evaluation of antiaging cream pH measurement
It was previously reported that, for creams and gels to be non-irritant and safe for topical application, their pH has to be fall in the physiologic accepted range for topical preparations, i.e., pH 6-7 units. Table 3 shows that pH of various antiaging cream formulations ranged from 6.1 to 6.5 which lies in the normal physiologic range and thus produces no skin irritation.

Viscosity
The prepared antiaging creams and gels were formulated using Carbopol 940. Table 4 shows that the viscosity of various antiaging cream formulations ranged from 2455.577 to 5134.42.

Spreadability
The spreadability is an important criterion for uniform and ease of application of topical preparations. It also plays a major role from patient compliance point of view. Application of the formulation to the skin is more comfortable if the base spreads easily, exhibiting maximum "slip" and "drag." Spreadability of creams and gels are measured in terms of average diameter of the spread circle. Table 5 shows that

Extrudability
It is an empirical test to measure the force required for the cream to extrude out from the tube. For topical preparations, it is an important criterion to check the easiness of the cream to extrude out from the tube. Table 6 shows that the extrudability values of cream are ranged from 90.5 to 93.5.
Physicochemical parameter evaluation of antiaging gels pH measurement Table 7 shows that pH of various antiaging gel formulations ranged from 6.13 to 6.5 which lies in the normal physiologic range and thus produces no skin irritation.
Viscosity Table 8 shows that the viscosity of various antiaging gel formulations ranged from 2446.577 to 5334.423. Extrudability Table 10 shows that the extrudability values of all prepared gel formulations lie from 90.5 to 93.5.

In vitro antioxidant studies
In vitro antioxidant studies were performed for the prepared antiaging creams, gels, and phytosomal gels. The antioxidant activity of the prepared formulations in various concentrations was evaluated using in vitro models. It was observed that the test compounds scavenged free radicals in concentration-dependent manner in all models. The antioxidant activity was expressed as IC 50 (the amount of antioxidant needed to decrease the radical concentration by 50%), which is negatively related to antioxidant activity. The lower the IC 50 value, the higher is the antioxidant activity of the tested sample. The in vitro antioxidant studies included DPPH assay, reducing power assay, nitric oxide scavenging assay, and hydrogen peroxide assay. Among them, DPPH assay method was found to be the easiest, sensitive, and    The values are expressed as mean±SD. SD: Standard deviation  rapid method to assess the antioxidant activity of compounds. Nine different formulations of antiaging creams, gels, and phytosomal gels were prepared. Although the radical scavenging activity of the standard was found to be higher than that of the samples, the study showed that the samples have the proton-donating ability and could serve as free radical inhibitors or scavengers. Among them, F2 formulation showed the highest antioxidant activity. Physicochemical parameters such as pH, viscosity, homogeneity, spreadability, and extrudability were evaluated for both antiaging creams and gels. From the physicochemical parameter evaluation, it was found that F2 sample showed optimum values. Hence, the F2 formulation was taken as the optimized formula from all the nine different formulations. Characterization studies of antiaging phytosomal gel were also performed.

DPPH free radical scavenging activity
The result of antioxidant activity of different concentrations of samples and standard (ascorbic acid) is shown in Table 11. The samples exhibited marked antioxidant activity by scavenging DPPH* (free radical) and converting into DPPHH. A dose-dependent radical scavenging activity was observed. The scavenging activity of ascorbic acid was greater than that of all the nine different samples of cream.
Among them, F2 sample showed the highest antioxidant activity. In the presence of an antioxidant, DPPH radical obtains one more electron and the absorbance decreases.
In this study, the scavenging activity of F2 sample was found to be dose dependent, i.e., higher the concentration, more was the scavenging activity. Although the DPPH radical scavenging abilities of the samples were less than that of ascorbic acid, the study showed that the samples have the proton-donating ability and could serve as free radical inhibitors or scavengers, acting possibly as primary antioxidants.

Reducing power assay
The result of antioxidant activity of different concentrations of samples and standard (ascorbic acid) is shown in Table 12. In this study, the absorbance was found to increase with the dose of samples and standard which is suggestive of reducing power. In the Fe+3 reducing assay, the reducing power of antiaging cream and gel samples was found to increase with the dose. The presence of reductants such as antioxidant substances in the antioxidant samples causes the reduction of the Fe3+/ferricyanide complex to the ferrous form. Therefore, Fe2+ can be monitored by measuring the formation of Perl's Prussian blue at 700 nm. The reducing capacity of a compound may serve as a significant indicator of its potential antioxidant activity. However, the antioxidant activity of putative antioxidants has been attributed to various mechanisms, among which are prevention of chain initiation, binding of transition metal ion catalysts, decomposition of peroxides, prevention of continued hydrogen abstraction, and radical. Fig. 2 shows the reducing power assay of antiaging cream when compared with that of the standard ascorbic acid.

Nitric oxide scavenging assay
The result of antioxidant activity of different concentrations of samples and standard (ascorbic acid) is shown in Table 13. Nitric oxide free      Table 15 shows the percentage of DPPH free radicals scavenged by the sample and standard at increasing concentrations (Fig. 5). Table 16 shows the percentage of free radicals scavenged by the sample and standard at increasing concentrations (Fig. 6).

Reducing power assay of gels
Nitric oxide scavenging assay of gels Table 17 shows the percentage of free radicals scavenged by the sample and standard at increasing concentrations (Fig. 7).
Hydrogen peroxide assay of gels Table 18 shows the percentage of free radicals scavenged by the sample and standard at increasing concentrations (Fig. 8). Table 19 shows the percentage of free radicals scavenged by the sample and standard at increasing concentrations ( Fig. 9). Table 20 shows the percentage of free radicals scavenged by the sample and standard at increasing concentrations (Fig. 10).

Fig. 4: Percentage inhibition of the formulation by hydrogen peroxide assay when compared with the standard
radicals are released from the endothelial cells which are necessary for the inflammatory actions of skin. The results signify that the level of nitric oxide was reduced to a considerable amount by the samples. Thus, the prepared formulation can be used effectively for topical purposes. Table 13 suggests the potency of nitric oxide scavenging activity of the formulations with that of the standard ascorbic acid. Fig. 3 shows the nitric oxide scavenging assay of antiaging cream when compared with the standard.
Hydrogen peroxide assay H 2 O 2 is a weak oxidizing agent and can activate a few enzymes directly, usually by oxidation of essential thiol (-SH) groups. Hydrogen per oxide can cross cell membrane rapidly. Once inside the cell, H 2 O 2 can probably react with Fe2 + and possibly Cu2 + to form hydroxyl radical and this may be the origin of many of its toxic effects. It is therefore biologically advantageous for cells to control the amount of hydrogen peroxide that is allowed to accumulate. The decomposition of hydrogen peroxide by the formulations may result from its antioxidant and free radical scavenging activity.
The results given in Table 14 show the percentage of hydrogen peroxide radical scavenged by the samples and ascorbic acid at increasing concentrations. From them, it is evident that the formulations were capable of scavenging hydrogen peroxide in a concentration-dependent manner (Fig. 4).   Table 21 shows the percentage of free radicals scavenged by the sample and standard at increasing concentrations.

Nitric oxide assay of phytosomal gels
Hydrogen peroxide assay of phytosomal gel Table 22 shows the percentage of free radicals scavenged by the sample and standard at increasing concentrations (Fig. 11).
All the formulations showed potent antioxidant activity in all the assay methods. From the figures, the result represents that all the nine formulations had the capacity to scavenge the free radicals, but among them, the F2 formulation containing both tender coconut water and A. vera extract showed the highest antioxidant activity and also the radical scavenging activity by DPPH assay method showed highest antioxidant activity than the other methods. Hence, it was concluded that the prepared phytosomal gel could be used for the purpose of anti-wrinkle treatment as it could scavenge the harmful free radicals.

IC 50 values of optimized formulation, F2 of antiaging cream, gel, and phytosomal gel
IC 50 value of antiaging cream, F2 IC 50 value, i.e. the concentration of the sample which is required to scavenge 50% of the free radicals of the optimized formulation of the prepared antiaging cream, F2 is found to be 70.5 µg/ml (Fig. 12).        IC 50 value of antiaging Gel, F2 IC 50 value, i.e., concentration of the sample which is required to scavenge 50% of the free radicals of the optimized formulation of the prepared antiaging gel, F2 is found to be 65.05 µg/ml (Fig. 13).
IC 50 value of antiaging phytosomal gel, F2 IC 50 value, i.e., concentration of the sample which is required to scavenge 50% of the free radicals of the optimized formulation of the prepared antiaging phytosomal gel, F2 is found to be 47.0 µg/ml (Fig. 14).

Comparison studies of antiaging cream, gel, and phytosomal gel on the basis of in vitro antioxidant studies
The antioxidant property of the prepared formulations of antiaging cream, gel, and phytosomal gel was compared on the basis of in vitro antioxidant studies. From the comparison study, it was clear that antiaging. Phytosomal gel had the highest antioxidant activity, and thus, it could be concluded that the prepared phytosomal gel possessed the antiaging property. Comparison studies are shown in Table 23. Comparison studies of IC 50 values of antiaging cream, gel, and phytosomal gel [41] IC 50 values of the optimized formulations (F2) of antiaging cream, gel, and phytosomal gel were compared. The IC 50 value of antiaging cream was found to be 70.5 µg/ml, the IC 50 value of antiaging gel was found to be 65.0 µg/ml, and the IC 50 value of antiaging phytosomal gel was found to be 47.0 µg/ml. Therefore, it can be concluded that the antiaging phytosomal gel proved to show the highest antioxidant activity as it had an IC 50 value of 47.0 µg/ml. The lower the IC 50 value, the higher the antioxidant activity (Fig. 16).

Visual inspection
The prepared phytosomal gel was visually inspected and it showed that the formulation was homogeneous without any gritty particles and was of optimum consistency.

Globule size determination
Microscopic evaluation The prepared antiaging phytosomal gel was observed under optical microscope at 100× and observed that the formed vesicles were of uniform size.
Size and size distribution: The vesicular size and size distribution was evaluated using DLS, and the results showed that increase in extract phospholipid concentration increases the mean vesicular size. The vesicular size was between 52 nm and 115 nm. The polydispersity index was found to be low, shows that the particles were of low value and phytosomes formed by hydration was of uniform size (Figs. 17-21 and Table 24).

Zeta potential determination
The magnitude of zeta potential gives a potential stability of the colloidal dispersion. If the particles have large positive or negative charge reveals that they repel each other and there is dispersion stability. The zeta potential of the optimized formulation showed that the sample is sample which is highly stable. It was found as −22.21, and hence, this indicates that the prepared formulation is stable.

SEM
From Fig. 22, it is clear that the particle size of the optimized formulation was confirmed to be 52-115 nm. This was in accordance with the particle size of phytosomes in the literature.

Stability studies
The best formulation of the phytosomal gel was kept at varying conditions of temperature. The system was stable at 25°C. There were no significant changes in the formulation when kept at room temperature (30°C±2°C) and also at refrigerated temperature (4°C±2°C). No much change of pH, viscosity, homogeneity, spreadability, extrudability, and degradation of the samples were observed during 45 days period. Table 25 shows the data for stability studies for antiaging phytosomal gel F2. There was no much change in the zeta potential of the sample, and this proves that the phytosomal gel system remains stable.

Comparison of the phytosomal gel with marketed formulation
The prepared antiaging phytosomal gel was compared with that of an herbal marketed formulation, antiaging A. vera gel. It was found that the antioxidant activity was more for the phytosomal gel. Hence, it was proved that the prepared phytosomal gel containing the antiaging ingredients such as tender coconut water and A. vera had significant antiaging properties. The A. vera antiaging gel was taken for comparison as it had the antiaging ingredient, A. vera.
From Fig. 23, it was evident that the prepared antiaging phytosomal gel had the highest antioxidant property when compared with the conventional dosage forms such as antiaging cream and antiaging gel.

CONCLUSION
Nowadays, aging phenomenon has become a severe problem among people. Hence, they started to depend on antiaging cosmetics. However, they had severe adverse effects, and hence, they had to rely on cosmetics containing herbal ingredients as they had fewer side effects. Antioxidants are the major ingredients present in antiaging cosmetics. The antioxidants chosen for this study were tender coconut water, A. vera extract, grape seed extract, and Vitamin E. Nine different formulations of antiaging cream, gel, and phytosomal gel were prepared. Physicochemical parameters such as pH, viscosity, homogeneity, spreadability, and extrudability of prepared antiaging cream and gel were determined and the formula, F2 containing both tender coconut water and A. vera extract was chosen as the optimized formula. In vitro antioxidant studies were performed for antiaging cream, gel, and phytosomal gel, and the phytosomal gel was found to be the formulation having the highest antioxidant activity, and hence, it was the optimized formulation. Characterization studies of the optimized formulation such as vesicle size, size distribution, and stability were performed, and it was identified that the prepared antiaging phytosomal gel had all the parameters as optimum. Stability studies were done according to the ICH guidelines, and it was found that the optimized formulation was stable under both room temperature and refrigerated temperature. The optimized formulation was compared with a marketed antiaging gel and it was clear cut that the prepared antiaging phytosomal gel had highest antioxidant activity than the marketed formulation, and hence, it could be used for antiaging treatment. The result revealed that the natural ingredients such as tender coconut water and A. vera extract had significant antioxidant activity and they were found to be potent scavengers of free radicals which serve as a possible preventative intervention for the skin diseases. The activity may be due to the presence of cytokinins, phenol compounds, and Vitamin E and C.     Natural remedies are more acceptable in the belief that they are safer with fewer side effects than the synthetic ones. Hence, the prepared herbal antiaging phytosomal gel which is non-toxic, safe, and effective is found to be highly acceptable.

ACKNOWLEDGMENT
We would like to show our gratitude to the Verjinia CU and Dr. Deepa T Vasudevan for sharing their pearls of wisdom with us during the course of this research, and we thank the "anonymous" reviewers for their so-called insights and their comments on an earlier version of the manuscript.

AUTHOR'S CONTRIBUTIONS
Verjina CU, Deepa T Vasudevan developed and optimized the formulations. Julie Mariam Joshua and Athira Anilkumar wrote the manuscript with the support and supervision of Saritha A Surendran.