Formulation and Evaluation of a Sunscreen Ingredient

The skin, referred to as the cutaneous membrane, is the body's outer layer.¹ While barrier integrity matures by six years of age, complete physiological and microbiological maturation extends into later childhood.^{2, 3, 4, 5} Of the UV radiation reaching Earth, about 95% is UVA and 5% is UVB. UVA causes skin aging and tanning, while UVB leads to sunburn by penetrating deeper layers of skin. Both types generate ROS, initiating photochemical reactions and photosensitization. Prolonged UV exposure is linked to eye disorders, immune suppression, and skin cancers. UV radiation induces DNA mutations essential for carcinogenesis. Chronic exposure causes hyperpigmentation, collagen degradation, and mutation accumulation, with 86% of melanomas and 90% of non-melanoma skin cancers attributed to UV exposure. UVB (280–320?nm) causes DNA lesions like Cyclobutane pyrimidine dimers (CPDs) and Pyrimidine-pyrimidone (6-4) photoproducts, disrupting replication and transcription. UVA (320–400?nm) is needed in small amounts for circadian rhythm regulation but at high doses suppresses immunity and promotes ROS formation. ROS damages DNA, lipids, and proteins, contributing to photoaging and cancer. Melanin protects against UV but can act as a pro-oxidant under exposure, leading to CPD formation in melanocytes and facilitating melanoma.⁶ Melanocytes and Interfollicular epidermal stem cells are present in the basal layer of skin. When these cells get high UV exposure in childhood, risk of developing skin cancer later in life increases. This is because epidermal stem cells have lifelong persistence, thus being an ideal site for carcinogenic effect. Also such UV exposure serves to be a starting point for melanocytic nevi development.⁷ Skin sensitivity to UV varies by skin tone. Darker skin has greater resistance to sunburn and better UV tolerance but remains susceptible to damage. The World Health Organization (WHO) recommends using sunscreen along with protective clothing, hats, sunglasses, and shade. Daily sunscreen use helps prevent premature aging and skin cancer. Hence, it is recommended to use sunscreens daily, irrespective of skin type.⁸

All organic sunscreen agents can cause adverse effects like irritation, allergic contact reactions, photoallergy, and phototoxic effects.⁹ Zinc oxide (ZnO) and Titanium dioxide (TiO₂) are widely used inorganic UV filters. However, ZnO’s thicker consistency may cause clogged pores in certain people, particularly when used with other comedogenic substances. For those with acne-prone skin, this may result in outbreaks or skin irritation.¹⁰ For TiO₂ the two main issues raised were the possibility that TiO₂ nanoparticles could produce ROS in response to UV light, which could harm skin cells' DNA, and the nanoparticles' ability to penetrate the skin.¹¹

Vitamin C is a powerful antioxidant found in the aqueous compartment of the cells. It counteracts oxidative damage caused by a variety of causes, most prevalent of which is UV damage. Deep stratum corneum layers contain a large amount of Vitamin E, which serves as the skin’s main defence against oxidative stress brought about by UV radiations. Topically Vitamin E shields the skin from UV- induced cutaneous damage as well as chemical agent’s carcinogenic and mutagenic properties.¹² According to one study, tocotrienols have 1600 times the antioxidant capacity of alpha tocopherol. According to some reports, the unsaturation in the aliphatic tail of tocotrienol may be responsible for its superior antioxidant activity by facilitating easier tissue penetration.¹³

In the current research work attempt is being made to develop a sunscreen ingredient that can be incorporated in any base to develop Sunscreen formulation making use of antioxidants and vegetable oils. A blend of vegetable oils is expected to reflect the UV rays, thus contributing to the Sun Protection Factor (SPF). L- Ascorbic acid and Tocotrienol will contribute to antioxidant activity of “Sunscreen ingredient”, this will take care of oxidative stress generated due to UV radiations exposure. Kaolin being an inorganic sunscreening agent, will help to improve the SPF. Also, it will aid in the stability of “Sunscreen ingredient” which is Oil- in- Water (O/W) emulsion by acting as a suspending agent.

MATERIALS AND METHODS:

Materials:

Gift sample of Tocotrienol with brand name Orah Vit E (OE) was obtained from Orah NutriChem Pvt. Ltd., Pune. L- ascorbic acid (AA) was procured from Molychem, Mumbai. Olive oil, Almond oil, Sesame oil were purchased from Vishal Chem, Mumbai. Glycerin, Propylene glycol, Kaolin, Tween 80, Span 80, Stearic acid and Potassium hydroxide were procured from renounced sources.

Methods:

1. Formulation of Sunscreen Ingredient (SI)

Step 1: Determination of ratio of surfactant mixture (S_mix)

Hydrophilic Lipophilic balance (HLB) value was determined assuming ratio of oils as 5: 3: 2 for Olive oil: Almond oil: Sesame oil and considering required HLB (RHLB) values of individual oils.

HLB of Oil blend = (Parts of Olive oil x RHLB of Olive oil) + (Parts of Almond oil x RHLB of Almond oil) + (Parts of Sesame oil x RHLB of Sesame oil)        

HLB of Oil blend = 6.7

Thus, tentatively Oil blend was assumed to have RHLB of 6.7.

Now, by doing theoretical calculations the ratio of surfactant quantities was determined for getting HLB of 6.7.

Step 2: Procedure followed for developing emulsion

Aqueous phase consisted of AA, Kaolin, Tween, Glycerin, Propylene glycol and Water. Aqueous phase was stirred for 10 min. for uniformly dispersing Kaolin. Kaolin was weighed separately, then sieved from sieve no. 100 to break lumps, make it free flowing and then transferred to aqueous phase. The oil phase consisted of a blend of oils, OE and Span. Oil phase was stirred manually and added drop wise to aqueous phase. This was followed by stirring for 30 min. on a magnetic stirrer. Resultant course emulsion was subjected to probe sonication for 15 min., with 5 sec. pulse cycle.

Step 3: Determination of Concentration of the internal phase that can be loaded in emulsion

Various batches of emulsion were prepared having different concentrations of oil with 5% (w/w) S_mix. Emulsions were then exposed to stress conditions. Those stress conditions were Centrifugation (1000 RPM, 20 min.), Accelerated conditions (40?, 75 % RH; 1 week) and Freeze Thaw cycles (48 hrs each, 3 cycles; freezing at 0? and thawing at room temperature). The objective was to maximize the quantity of oils in emulsion.

Step 4: Minimizing the S_mix concentration

Emulsions were prepared of the selected S_mix by gradually reducing concentration of S_mix in them. Developed emulsions were exposed to above mentioned stress conditions to evaluate their stability. The objective was to minimize the quantity of S_mix in emulsion.

Step 5:  Optimization of oil ratio

Using 2³ Factorial design ratio of olive oil: almond oil: sesame oil was optimized, so that maximum SPF can be achieved. Output factor/ Response was in- vitro SPF. Input factors with their levels are summarized below,

Table no. 1: Coded and Uncoded levels of Factors for Factorial design

Step 6: Development of Optimized emulsion, Sunscreen Ingredient

By executing experimental design and analyzing data using StatEase software, software recommended a 7: 1: 1 ratio of Olive oil: Almond oil: Sesame oil for achieving maximum SPF. Using oils in said ratio and executing procedure given in Step: 2 optimized emulsion was developed in triplicates.

2. Formulation of Prototype formulation (PF)

Step 1: Formulation of Vanishing cream base

Vanishing cream base was used to develop PF. Formula of Vanishing cream given in literature (14) was modified to achieve desired organoleptic characteristics in final formulation. Stearic acid and Potassium hydroxide in formula will generate in- situ emulsifier, glycerin will act as humectant in the formulation. Modified formula for vanishing cream base is as follows:

Table no. 2: Formula of Vanishing Cream base

Procedure: Aqueous phase and Oil phase were prepared separately. Aqueous phase consists of Potassium hydroxide, glycerin and water. The oil phase has Stearic acid. Required quantities of ingredients were weighed and transferred into respective beakers. Both beakers were heated in a water bath until stearic acid melts completely. Once both phases were liquid, the aqueous phase was transferred to the Oil phase. This was followed by vigorous stirring until formulation cooled. Thus, forming a vanishing cream base.

Step 2: Blending of Vanishing cream base and SI

It was predecided that base and ingredient will be mixed in 1: 1 ratio. For that purpose in one beaker vanishing cream base was weighed. Pre- formulated SI was transferred into the same beaker in small portions followed by mixing (Geometric dilution). This ensured uniform and thorough mixing. Once all the quantity of SI was added, the resultant mixture was stirred for 10 to 15 min. Thus, developing the Prototype formulation.

3. Evaluation of SI and PF

1. Organoleptic properties (Color, Odor): SI and PF were subjected to evaluation of color and odor. Visual observation was performed to record color. Identification of the odor was performed by manual assessment.
2. Texture: PF was evaluated for its texture by rubbing a small quantity of formulation over skin.
3. pH: 1 % w/v solution of SI and PF were made in distilled water. pH was determined using Digital pH meter. To prepare 1 % w/v solution 0.5 gm of each was weighed in a beaker and diluted with 50 ml of distilled water.
4. Dilution test¹⁵: A dilution test was performed to determine the type of emulsion. A small amount of SI was mixed with water in one beaker and another small portion with oil in another beaker.
5. Homogeneity¹⁵: The PF was tested for Homogeneity by visual appearance and by touch.
6. Spreadability: To determine spreadability of PF two clean glass slides were used. On one of the glass slides 50 mg of formulation was placed. Over this slide another clean glass slide was placed. Now over these two slides weight of 100 gms was placed. Sample was allowed to spread for 5 min. Then by removing weight, spread of sample was recorded from three different sites and averaged to get spreadability of sample.
7. Washability: Glass slides used for spreadability testing of PF were used for accessing washability of formulation. A glass slide was held under running tap water to evaluate washability.
8. Viscosity¹⁵: PF’s viscosity was measured using Brookfields viscometer spindle 7. Three replications of the measurements were made at 100 rpm. Viscosity was calculated in Centipoise (cP). Formula to calculate viscosity is given below,

Viscosity (cP) = Dial reading x Factor

1. In- vitro SPF determination^{16, 17}:  In- vitro SPF values were recorded for both PF and SI. 0.1 gm of sample was diluted to 10 ml with Ethanol: Water (40: 60) to get 1 % w/v solution. 1 ml of this 1 % w/v solution was diluted to 10 ml with the same solvent to get 0.1 % w/v solution. Absorbance of this 0.1 % w/v solution was recorded from 290 nm to 320 nm, at 5 nm intervals. Then using the Mansur mathematical equation and reported constants, SPF was calculated. Values of EE (????) x I (????) are constant and reported in literature.

• 

  Where, 

• CF = Correction factor (10)
• EE (????) = Erythmogenic effect of radiation with wavelength ????
• Abs (????) = Spectrophotometric absorbance values at wavelength ????
• I (????) = Intensity of solar light of wavelength ????

Table no. 3: Values of EE (????) x I (????) ¹⁶

10. Antioxidant study, DPPH (2, 2- Diphenyl-1- picrylhydrazyl) method¹²: Antioxidant study was performed for both PF and SI. 1% w/v solution of sample was prepared by dissolving 0.1 gm in 10 ml Ethanol. 100 ppm concentration solution of DPPH was prepared in ethanol. In a test tube, 1 ml sample solution was mixed with 2 ml DPPH solution. The test tube was covered and subjected to sonication for 10 min. in a Bath sonicator. This was followed by incubation of samples at room temperature for 30 min. A Blank/ Control solution was also prepared. Post incubation absorbance of blank and sample were recorded at 517 nm, and anti- oxidant activity calculated using given formula,

Antioxidant activity (%) =

( Absorbance of Blank - Absorbance of sampleAbsorbance of Blank ) x 100

11. Particle Size and Polydispersity Index (PDI): SI was subjected to evaluation of particle size and PDI. 0.1 gm of emulsion was diluted with double distilled water to 10 ml, to get 1 % w/v solution. 1 ml of 1 % w/v solution was diluted to 10 ml to get 0.1 % w/v solution. This 0.1 % w/v solution was used for analysis. Before analyzing, samples were filtered from a 0.45 µm pore filter.

50. Zeta potential determination: SI was subjected to evaluation of zeta potential. Sample prepared for Particle Size and PDI determination was used for determination of Zeta potential.

1000. Assay (for AA and OE content): 10 mg of PF was weighed and diluted to 10 ml with methanol to get stock solution. 4 ml of stock solution was diluted to 10 ml, this solution was used to record absorbance at 293 nm to quantitate OE in sample solution. 0.1 ml of stock solution was diluted to 10 ml, this solution was used to record absorbance at 247 nm in-order to quantitate AA. OE has maximum absorbance at 293 nm and AA at 247 nm in methanol. UV Visible spectrum revealed wavelength of maximum absorption of AA and OE.

{a}                                                                               {b}

Fig. no. 1: UV Visible spectrum {a}: OE, {b}: AA

14. In- vitro SPF determination according to COLIPA- 2011 guidelines: A sample of 5 gms of PF was given for in- vitro SPF determination at KET’s Scientific research center, Mulund.

4. Accelerated Stability Studies of SI and PF:

SI and PF were kept for Stability studies under Accelerated conditions, 40? and 75 % RH for a duration of 45 days. Samples of both were collected periodically and analyzed. The FTIR spectrum of samples taken on day 1 and day 45 were compared, to comment on content of AA and OE in formulation. For that purpose, a small quantity of sample was solubilized in Chloroform and spread over a blank KBr pellet, then FTIR spectrum was recorded.

RESULTS:

1. Formulation of SI

Determination of ratio of surfactant mixture (S_mix)

Following were the three ratios that were screened for emulsion development,

Determination of Concentration of the internal phase that can be loaded in emulsion.

Results of stability on exposure to stress conditions of various batches of emulsion are summarized below. Based on the results it was observed that, S_mix of Tween 80 and Span 80 gave stable emulsion with 40 % w/ w oil. However this emulsion showed separation of one oil droplet over the surface. Thus, this surfactant combination was chosen for development of SI and the quantity of oil in emulsion was reduced to 36 % w/ w.

Table no. 4: Stability data of various batches of emulsion prepared with different concentrations of oil

Minimizing the S_mix concentration

Attempt was made to reduce surfactant concentration ensuring that maximum quantity of oil is being loaded in emulsion with minimum S_mix concentration, such that resulting emulsions are stable enough. For that purpose emulsions having 40 % oil phase were prepared by gradually reducing the concentration of S_mix. Stability data for this study is summarized in table no. 5.

Table no. 5: Stability data of various batches of emulsion prepared with different concentrations of Smix

From this study it was observed that 5.0 % w/ w and 4.5 % w/ w S_mix gave emulsions which were stable under multiple stress conditions. Emulsion with 4.0 % w/ w S_mix showed phase separation on centrifugation. Thus, the S_mix concentration was decided to be kept 4.5 % w/ w.

Optimization of oil ratio

StatEase software was used for optimization. Experimental design with response values was as follows,

Table no. 6: Experimental design with response values

Software provided a Pareto chart. In that Pareto chart bars of A, B, C and AC were of blue color suggesting that the effect of these factors is Negative on the response. Bars of BC and ABC were of orange color suggesting that these factors have Positive effect on response.

Fig. no. 2: Pareto chart

Software generated ANOVA table for the data which is given below (Table no. 7). Model F- value of 343.27 implies that the model is significant. Model terms A, AC and BC have their P- value less than 0.0500, indicating that those model terms are significantly affecting the response i.e. in- vitro SPF.

Table no. 7: ANOVA table

Software also provides equation in terms of Coded factors which helps to predict the relative effect of each factor, interaction factor and higher order interaction factor on the response by comparing the factor coefficients. Coded equation is,

Y = 19.83 - 0.4041A - 0.1494B - 0.2391C - 0.3300 AC + 0.5951BC + 0.1863ABC

2D Contour plots and 3D Surface response plots were generated by the software giving graphical representation of the relationship between factors and response variable.

   Fig. no. 7: Overlay plot for factor B and factor C      Fig. no. 8: Overlay plot for factor A and factor C

Table no. 8: Fit Statistics

Table no. 9: Optimized oil ratio

The results depict that the response values predicted by the software have been reproduced and hence the oil ratio 7: 1: 1 was concluded to be the Optimized oil ratio.

Through laboratory work and by analysing statistical data given by the StatEase software, a formula of the SI was developed which is given in table no. 10. Orah Vit E and L- ascorbic acid work as antioxidant, oil blend as Sunscreen agent, Glycerin and Propylene glycol as humectant, Span and Tween 80 as emulsifier, Kaolin plays dual role, it acts as Sunscreen agent and emulsion stabilizer. Optimized SI was formulated in triplicates and evaluated.

Table no. 10: Formula of SI

Fig. no. 9: Optimized SI formulated in triplicates

2. Evaluation of SI

Table no. 11: Results of evaluation of SI

3. Formulation and evaluation of PF

Formulation:

Vanishing cream was decided to be used as a base for developing sunscreen cream. For that purpose a vanishing cream base was prepared.

                                Fig. no. 10: Vanishing cream base       Fig. no. 11: Prototype formulation

Evaluation:

Table no. 12: Results of evaluation of PF

4. Accelerated stability studies of SI and PF

SI was subjected to stability studies for a duration of 45 days. Periodically samples were withdrawn and analyzed to characterize stability of the SI over a period. To the results obtained, a statistical test Single sample t- test was applied to evaluate if results vary significantly or not.

Table no. 13: Accelerated stability study data for SI

FTIR spectra of SI samples kept for accelerated stability studies were recorded on Day 1 and Day 45. FTIR spectra of SI were studied to ensure that AA and OE are present in the sample, sufficient to develop characteristic peaks. Given below are the FTIR spectra of samples,

Table no. 14: FTIR peaks specifically shown by AA and OE in sample of SI

PF was subjected to stability studies for a duration of 45 days. Periodically samples were withdrawn and analyzed to characterize stability of the PF over a period. To the results obtained, a statistical test Single sample t- test was applied to evaluate if results vary significantly or not.

Table no. 15: Accelerated stability study data for PF

FTIR spectra of PF samples kept for accelerated stability studies were recorded on Day 1 and Day 45. FTIR spectra of PF were studied to ensure that AA and OE are present in the sample, sufficient to develop characteristic peaks. Given below are the FTIR spectra of samples,

Table no. 16: FTIR peaks specifically shown by AA and OE in sample of PF

DISCUSSION:

1. Formulation of SI

Determination of Concentration of the internal phase that can be loaded in emulsion: Initially emulsions with 20 % w/ w oil were developed and exposed to stress conditions. However, emulsion having Tween 60 and Span 80 showed phase separation. Thus this combination was ruled out. With the remaining two S_mix using 40 % oil phase, emulsions were developed. From those emulsions, Tween 20 and Span 80 containing emulsion showed phase separation on exposure to stress conditions. The only left combination was of Tween 80 and Span 80, forming a stable emulsion containing 40 % oil. However post freeze thaw cycles, this emulsion showed separation of tiny droplet of oil. Further Tween 80 and Span 80 with 45 % oil showed phase separation on centrifugation. Thus, the selected surfactant combination was Span 80 and Tween 80 and Oil phase concentration was decided to be kept 40 % w/w.  

Minimizing the S_mix concentration: Exposure to excessive surfactants can lead to drying, irritation, itching of skin due to dehydration. So surfactants should be used in minimum concentration. Emulsion with 4.0 % w/ w S_mix showed phase separation on centrifugation. However, emulsion with 4.5 % w/ w S_mix was stable under all three stress conditions. Thus, S_mix quantity was reduced to 4.5 % w/ w.

Optimization of oil ratio: Considering the data given by Pareto chart, ANOVA table, Coded equation, 2D Contour plots and 3D Surface response plots we observe that factor A, interaction factor AC and interaction factor BC significantly affect the In- vitro SPF value. Effect of factor A i.e. Olive oil and interaction factor AC (Olive oil and Sesame oil) is Negative. This means that with an increase in the amount of Olive oil in the formulation, in- vitro SPF will reduce. However, the coefficient of factor A in the coded equation is small. This signifies that though an increase in the quantity of Olive oil will reduce SPF, this reduction will not be drastic. Interaction factor AC will also reduce SPF but as indicated by its small coefficient, reduction will not be drastic. When we observe the 3D Surface Response plot for AC factors, we can clearly observe that, as the quantity of Olive oil increases (with constant quantity of sesame oil), significant reduction in SPF is not observed. However, with increasing quantities of Sesame oil, SPF is drastically reducing. Thus, highlighting the need to minimize the quantity of Sesame oil to achieve maximum SPF. This also becomes evident through the negative coefficient associated with factor C, Sesame oil. The effect of interaction factor BC is Positive and largest amongst other factors. This means that interaction between Almond oil and Sesame oil is improving SPF. However, this positive trend is not observed throughout the concentration range of Almond oil and Sesame oil, after a certain concentration trend is getting reversed. Further increase in quantity of both oils is reducing SPF. The optimized oil ratio recommended by StatEase software is 7: 1: 1 for olive oil: almond oil: sesame oil to achieve maximum in- vitro SPF. High amount of Olive oil is acceptable due to minimum SPF reduction. Low amount Sesame oil is essential to minimize its strong negative effect on SPF. A low amount of Almond oil will avoid mild negative effects and will also preserve the beneficial effect of interaction factor BC.

2. Evaluation of SI

Table no. 17: Significance of evaluation of SI

3. Formulation and evaluation of PF

Formulation: Vanishing cream is designed to spread easily on skin and then rapidly vanish leaving behind a thin, invisible layer. (Rieger, 2009) They are light in feel being oil- in- water emulsion, formula is well established and formulation is simple. Thus, vanishing cream was decided to be used for developing PF.

Evaluation:

Table no. 18: Significance of evaluation of PF