A natural and sustainable UV blocker and tyrosinase enzyme inhibitor cosmetic raw material and a preparation method thereof

Abstract

The invention relates to a natural, sustainable, eco-friendly, cost-effective ultraviolet (UV) blocker and tyrosinase enzyme inhibitor cosmetic raw material, obtained from silkworm excrement, which is improved in quality, stabilized to prevent activity loss, free of side effects, stable and long-lasting, and to a preparation method thereof, for use in the cosmetics sector in particular, as well as in various other industrial fields. The inventive ultraviolet (UV) blocker and tyrosinase enzyme inhibitor cosmetic raw material is obtained by using sericin protein, fulvic acid, and optimized silkworm excrement extract.

Description

[0001] A NATURAL AND SUSTAINABLE UV BLOCKER AND TYROSINASE ENZYME

[0002] INHIBITOR COSMETIC RAW MATERIAL AND A PREPARATION METHOD

[0003] THEREOF

[0004] Technical Field of the Invention

[0005] The invention relates to a natural, sustainable, eco-friendly, cost-effective ultraviolet (UV) blocker and tyrosinase enzyme inhibitor cosmetic raw material, obtained from silkworm excrement, which is improved in quality, stabilized to prevent activity loss, free of side effects, stable and long-lasting, and to a preparation method thereof, for use in the cosmetics sector in particular, as well as in various other industrial fields.

[0006] State of the Art

[0007] Tyrosinase inhibitors are raw materials found in skin whitening products, which are especially preferred in cosmetics today. Inhibition of cellular tyrosinase enzyme reduces melanin production and eliminates aggressive pigmentation problems[1].

[0008] In the state of the art, many different products and methods are used to prevent the loss of activity of UV blocker and tyrosinase enzyme inhibitor materials. These include chemical stabilization, physical stabilization, antioxidants, encapsulation technologies, and other protective methods.

[0009] In the present art, chemical stabilization, which is one of the methods used to prevent the loss of activity of UV blocker and tyrosinase enzyme inhibitor materials, provides protection against the effects of rays by changing the structure of UV blocker and tyrosinase enzyme inhibitor materials. However, this method can negatively affect the quality of the products and cause side effects in the long term. The chemical stabilization method can lead to changes in product quality and potential side effects, while maintaining the effectiveness of UV blocker and tyrosinase inhibitor materials. This method can lead to allergic skin reactions, irritation, and long-term health risks due to changes in the structure of the products. In the state of the art, physical stabilization, which is another method used to prevent the loss of activity of UV blocker and tyrosinase enzyme inhibitor materials, provides protection by methods such as microemulsions or nanoemulsions without changing the structure of the materials; however, these methods cannot maintain the stability of the products for a long time and offer short-lived products.

[0010] In the present technique, the antioxidants used to prevent the loss of activity of UV blocker and tyrosinase enzyme inhibitor materials, on the other hand, aim to provide protection by destroying free radicals present in the structures of UV blocker and tyrosinase enzyme inhibitor materials; however, this method has limitations in terms of the stability of antioxidants and may have negative effects on the quality of products. The limitations mentioned here relate to the stability of antioxidants used for the protection of UV blocker and tyrosinase enzyme inhibitor materials and their potential negative impact on the quality of the products. Antioxidants degrade over time and lose their effectiveness, leading to stability problems. This can negatively affect the efficacy and shelf life of products. Furthermore, the chemical structure of antioxidants can affect the quality of the product and cause changes in color, odor, and texture. Antioxidants can react with other components of the product to form unwanted by-products, which can reduce the efficacy of the product or lead to side effects. For all these reasons, it is clear that antioxidants in the current method may have negative effects on product stability and product quality.

[0011] Antioxidants can degrade over time and lose their effectiveness, which negatively affects the efficacy and shelf life of the product. Furthermore, antioxidants can alter the chemical structure of the product, causing differences in color, odor, and texture, and can react with other ingredients to form unwanted by-products. These limitations complicate the effective protection of UV blocker and tyrosinase inhibitors.

[0012] In the state of the art, on the other hand, encapsulation technologies, which is another method used to prevent the loss of activity of UV blocker and tyrosinase enzyme inhibitor materials, UV blocker and tyrosinase enzyme inhibitor materials are protected in microcapsules or nanocapsules, ensuring stability without any change in the structure of the substances; however, these methods can be expensive due to the encapsulation agents and negative environmental impacts are inevitable if the encapsulation materials are produced from non-natural materials. The non-natural materials referred to here are usually man-made components such as synthetic polymers, petroleum derivatives, and chemical additives. Non-natural materials often used in encapsulation technologies include synthetic polymers such as polyethylene glycol (PEG), polyvinyl alcohol (PVA), and polycaprolactone (PCL). These materials can cause negative environmental impacts as they are not biodegradable and tend to accumulate in the environment. Some synthetic polymers used in the encapsulation of bioactives have environmental impacts and these materials have biodegradability challenges. In particular, degrading polymers such as acrylic acid and polylactic acid in the biological environment also has challenges and potential toxic effects[2J. In addition, synthetic polymers have negative effects on environmental and biological systems[3].

[0013] Due to reasons such as the limitations and shortcomings of the products and methods used to prevent the loss of activity of UV blocker and tyrosinase enzyme inhibitor materials in the present art, the fact that the chemical stabilization method, which is one of the methods used to prevent the loss of activity of UV blocker and tyrosinase enzyme inhibitor materials and which provides protection against the effects of rays by changing the structure of UV blocker and tyrosinase enzyme inhibitor materials, negatively affects the quality of the products and causes side effects in the long term, and that the physical stabilization method, which is another of the said methods, is unable maintain the stability of the products for a long time, the fact that, in the present art, the antioxidants used to prevent the loss of activity of UV blocker and tyrosinase enzyme inhibitor materials have limitations in terms of the stability of antioxidants and these antioxidants have negative effects on product quality, and the fact that, in the present art, the encapsulation technologies, which are another method used to prevent the loss of activity of UV blocker and tyrosinase enzyme inhibitor materials, are costly due to the encapsulation agents used and these encapsulation materials are harmful to the environment due to being produced from non-natural materials, it has become necessary to present an ultraviolet (UV) blocker and tyrosinase enzyme inhibitor cosmetic raw material in which all these problems are eliminated, and a preparation method thereof. Summary and Objects of the Invention

[0014] The invention describes a natural, sustainable, eco-friendly, cost-effective ultraviolet (UV) blocker and tyrosinase enzyme inhibitor cosmetic raw material, obtained from silkworm excrement, which is enhanced in quality, stabilized to prevent activity loss, free of side effects, stable and long-lasting, and to a preparation method thereof, for use in the cosmetics sector in particular, as well as in various other industrial fields. The inventive ultraviolet (UV) blocker and tyrosinase enzyme inhibitor cosmetic raw material was obtained using sericin protein, fulvic acid, and optimized silkworm excrement extract and contains raw silkworm excrement extract encapsulated with fulvic acid and sericin protein.

[0015] The object of the invention is to provide an ultraviolet (UV) blocker and tyrosinase enzyme inhibitor cosmetic raw material with no loss of activity and stability. An ultraviolet (UV) blocker and tyrosinase enzyme inhibitor cosmetic raw material with no loss of activity and stability is provided by encapsulation performed with sericin protein, which is found in the invention as a waste in the silk industry. Encapsulation performed with said sericin protein prevents the loss of activity and stability of the ultraviolet (UV) blocker and tyrosinase enzyme inhibitor cosmetic raw material against the harmful effects of heat and UV. The invention provides a long-lasting ultraviolet (UV) blocker and tyrosinase enzyme inhibitor cosmetic raw material that can remain stable for long periods of time.

[0016] Another object of the invention is to provide a quality-improved ultraviolet (UV) blocker and tyrosinase enzyme inhibitor cosmetic raw material. A quality-improved ultraviolet (UV) blocker and tyrosinase enzyme inhibitor cosmetic raw material is provided in the invention by using silkworm excrement extract encapsulated with sericin protein and fulvic acid, a natural resource characterized as a waste in the silk industry. In this way, the stability of the raw material is increased, loss of activity is prevented, and the longterm efficacy of the product is maintained. Sericin protein is particularly effective in providing protection against UV rays, while fulvic acid is known for its antioxidant properties and acts as an enzyme inhibitor. In this way, the quality of this raw material used in cosmetic products is improved and an environmentally friendly and sustainable option is offered. The invention provides an ultraviolet (UV) blocker and tyrosinase enzyme inhibitor cosmetic raw material without any side effects. An ultraviolet (UV) blocker and tyrosinase enzyme inhibitor cosmetic raw material without any side effects is provided with natural ingredients derived from silkworm excrement. The main components of the raw material used in the invention are silkworm excrement extract enriched with sericin protein and fulvic acid. Sericin protein shows protective properties against UV rays, while fulvic acid is known for its antioxidant properties and also reduces the formation of skin blemishes by inhibiting the enzyme tyrosinase. The encapsulation of these ingredients increases the stability of the raw material and ensures that the active ingredients are safe and effective when applied to the skin. The absence of any chemical additives in the formulation of the inventive cosmetic raw material and the selection of natural ingredients minimizes the risk of side effects on the skin.

[0017] The invention provides a low-cost ultraviolet (UV) blocker and tyrosinase enzyme inhibitor cosmetic raw material. The cost is reduced by using silkworm excrement in the production of the inventive ultraviolet (UV) blocker and tyrosinase enzyme inhibitor cosmetic raw material.

[0018] The invention provides a natural, sustainable, and environmentally friendly ultraviolet (UV) blocker and tyrosinase enzyme inhibitor cosmetic raw material. A sustainable and environmentally friendly ultraviolet (UV) blocker and tyrosinase enzyme inhibitor cosmetic raw material is provided by using silkworm excrement in the production of the inventive ultraviolet (UV) blocker and tyrosinase enzyme inhibitor cosmetic raw material. In addition, in the invention, an encapsulation is performed with protein, which is a waste in the silk industry, and by using the said sericin protein, waste is recycled and an environmentalist policy is followed.

[0019] Description of the Drawings

[0020] Fig. 1. Three-dimensional (3D) plot of ethanol (%) and extraction time interaction for total antioxidant (A), 3D plot of ethanol (%) and solid / liquid ratio interaction for total antioxidant (B).

[0021] Fig. 2. 3D plot of ethanol (%) and extraction time interaction for sun protection factor (SPF) (A), 3D plot of ethanol (%) and solid / liquid ratio interaction for SPF (B). Fig. 3. 3D plot of ethanol (%) and solid / liquid ratio interaction for tyrosinase inhibitory effect (A), 3D plot of ethanol (%) and extraction time interaction for tyrosinase inhibitory effect (B), 3D plot of extraction time and solid / liquid ratio interaction for tyrosinase inhibitory effect (C).

[0022] Fig. 4. 3D and contour plot of the effect of 3 compounds on SPF (A), 3D and contour plot of the effect of 3 compounds on tyrosinase enzyme inhibitory effect (B), 3D and contour plot of the effect of 3 compounds on antioxidant (IC50 pg / ml) response variable (C).

[0023] Fig. 5. Ramp plots generated for raw excrement extraction optimization.

[0024] Fig. 6. Ramp graphs obtained for the 11th solution proposal in formulation optimizations (Formulation 1 (F1) - inventive raw material).

[0025] Fig. 7. Ramp graphs obtained for the 4th solution proposal in formulation optimizations (Formulation 2 (F2)).

[0026] Fig. 8. UV sensitive paper images for seven different applications under direct sunlight at UV index 3. For each trial, from left to right, positive control, negative control, sample.

[0027] Fig. 9. UV sensitive paper images under UVB lamp. For each trial, from left to right, positive control, negative control, sample.

[0028] Detailed Description of the Invention

[0029] The invention relates to a natural, sustainable, eco-friendly, cost-effective ultraviolet (UV) blocker and tyrosinase enzyme inhibitor cosmetic raw material, obtained from silkworm excrement, which is improved in quality, stabilized to prevent activity loss, free of side effects, stable and long-lasting, and to a preparation method thereof, for use in the cosmetics sector in particular, as well as in various other industrial fields. The inventive ultraviolet (UV) blocker and tyrosinase enzyme inhibitor cosmetic raw material was obtained using sericin protein, fulvic acid, and optimized silkworm excrement extract and contains raw silkworm excrement extract encapsulated with fulvic acid and sericin protein. The inventive ultraviolet (UV) blocker and tyrosinase enzyme inhibitor cosmetic raw material has thermal stability, as well as showing antioxidant properties and showing UV blocking (sun protection factor) and depigmentation (tyrosinase inhibitor) effects.

[0030] Obtaining Extracts from Silkworm Excrement in Powder Form

[0031] Extraction Optimization

[0032] A total of 17 different extraction procedures were performed for ethanol-water optimization of raw silkworm excrement. Ground raw excrement were subjected to convectional solvent extraction without any pretreatment. Convectional extractions were carried out using different ratios of water and ethanol. Design Expert Version 12.0.0 (Stat-Ease Inc., Minneapolis, MN, USA) was used to create the experimental design for the extraction. The Box Behnken Design (BBD) method, which is a response surface methodology, was applied for optimization. The Box-Behnken method was chosen to examine three factors and three levels of these factors. In the experimental design, a design matrix giving 17 trials for three factors and five center points was obtained. Replication and blocking were not performed. Factors examined during the extraction phase were determined as; A: Ethanol (%), B: Extraction time (hours), C: solid / liquid ratio (Table 1). The extraction process was applied at the values given in Table 1. At the end of the extraction applied, yield (R1), total protein (R2), total antioxidant (R3), total phenol (R4), sun protection factor (SPF; R5), tyrosinase enzyme inhibitory effect (R6) values were evaluated (Table 4). Surface response plots for optimization are given in Figure 1-3.

[0033] Table 1. Experimental ranges and levels of independent extraction variables Extraction Efficiency

[0034] Ethanol-water extraction yields were obtained by dividing the amount of extract obtained by the amount of solids entering the extraction. The highest yield was 16.426% (24 hours, 100% water, solid / liquid ratio 1:30) and the lowest yield was 1.415% (24 hours, 100% ethanol, solid / liquid ratio 1 :30) (Table 4).

[0035] Protein Quantification

[0036] Bradford method was used for protein analysis. For the method, the color reagent was first prepared, 100 mg of Coomassie Blue G-250 dye was weighed and dissolved in 50 mb of 95% ethanol, 100 mb of 85% phosphoric acid was added and the volume was completed to 1 b with distilled water. The prepared dye was filtered through a 0.45 pm pore diameter filter and stored at +4°C for experimentation and diluted 1:4 with distilled water before use. Then 200 pl of dye solution was added to 4 pl of sample and read at 595 nm. To express the amount of protein in the sample in mg BSA / g dry extract, a BSA standard curve was constructed using 7 different concentrations. Each sample was then plated at four different concentrations of 5-2-1-0.5 mg / ml. The amount of protein in the sample at four different concentrations was consistent with each other. The amount of protein obtained was very similar for each condition and ranged from 8.32-9.9 mg BSA / g dry extract (Table 4).

[0037] Determination of Antioxidant Capacity

[0038] ABTS method was used for total antioxidant determination. 7 mM ABTS+and 2.45 mM potassium persulfate (K2S2O8) stock solution in a 1 :1 ratio was incubated in a dark environment for 12-16 hours at room temperature. The prepared ABTS solution was diluted 1 :10 with methanol. The dilute ABTS solution was adjusted to 0.7 A at a wavelength of 734 nm in a microplate reader. Adjustment process was performed with methanol and ABTS. If the absorbance value was above 0.7, dilution was made with methanol, if it was lower than 0.7, the absorbance was adjusted with ABTS and the measurement was performed. Each of the 17 extracts obtained by ethanol-water extraction was studied at different concentrations, at three concentrations. After this experiment, the best antioxidant capacity with an IC50 value of 13.28 pg / ml was obtained under 50% Ethanol / 50% water - 4 hours - 1 / 20 solid liquid ratio conditions (Table 4).

[0039] Total Phenol Amount

[0040] Folin-Ciocalteu method was used for total phenol determination. Three parallel analyses were performed for samples prepared at different concentrations. The extract samples were plated in 20 pl volume in three parallel 96-well plates. Folin-Ciocalteu reagent diluted at a ratio of 1 :10 (Folin: Distilled water) with distilled water was added to the samples in a volume of 100 pl and incubated in the dark for 5 minutes. After 5 minutes of incubation, 80 pl of 7% sodium carbonate (Na2CO3) was added and left to incubate for 1 hour in the dark at room temperature. At the end of incubation, absorbance values were measured in a microplate reader at 750 nm wavelength. Total phenol was entered into the program as mg GAE / g dry extract. The extract with the highest phenol content was obtained under 100% water - 14 hours - 1 / 20 solid-liquid ratio conditions with 204.76 mg GAE / g dry extract value (Table 4).

[0041] Determination of Sun Protection Factor (SPF)

[0042] Sunscreens act as a barrier against UVA and UVB from the sun. UVA-UVB wavelength is in the range 290-320 nm. Spectrophotometric measurements made in this range provide information about the SPF values of the samples. Method of implementation: diluting 2 mg / ml sample with ethanol at a ratio of 1:9, diluting the mixture again with ethanol at a ratio of 1 :4 and reading at 290-320 nm in a quartz cuvette, calculating SPF using Equation-1

[0043] Here the values of EE and I are constants and can be found in the literature. The "CF" value is calculated based on a product taken from the market with a known SPF value. A calculation is then made for the samples. In order to find the CF value, this method was applied to some sunscreens available in the market and the CF value was calculated for each of them. These values were averaged to find a constant CF value. As a result of this experiment, the highest SPF of 17.7 was obtained with 100% Ethanol - 14 hours - 1 / 20 solid-liquid ratio (Table 4).

[0044] Tyrosinase Enzyme Inhibition Activity

[0045] Method for determination of tyrosinase enzyme inhibition activity comprises the process steps of: preparing tyrosinase enzyme with 0.1 M pH 6.8 phosphate buffer to a concentration of 250 U / rnl, using L-tyrosine as substrate and preparing L-tyrosine substrate at 3 mM with 0.1 M pH 6.8 buffer, preparing samples with 2% DMSO solution at different concentrations, using kojic acid as a positive control, performing the analyses in a 96-well plate and firstly adding 100 pl of 0.1 M pH 6.8 phosphate buffer to the wells, followed by adding 20 pl of 250 U / rnl enzyme solution,

[0046] - adding 20 pl of sample solution of different concentrations to the enzyme wells, pre-incubating at 25°C for 10 minutes,

[0047] - after incubation, adding 20 μl of 3 Mm L-tyrosine substrate to the wells and incubating at 25°C for 30 min and recording absorbances at half-hourly intervals for 2 h at 492 nm on a microplate reader.

[0048] The most effective extract with an IC50 value of 0.17 mg / ml was obtained at 50% ethanol - 50% water - 24 hours - 1 / 20 solid liquid ratio (Table 4).

[0049] Optimization Phase

[0050] In the optimization, 6 different responses were examined. From these responses, the design was aimed to be optimized for conditions where the sun protection factor (SPF) and tyrosinase enzyme inhibitory effect were maximized. In the optimization phase, yield, total protein, total phenol content were left between the obtained response values and the minimum condition was selected for total antioxidant. The maximum condition for SPF and the minimum condition for tyrosinase enzyme inhibitory effect given in IC50 were chosen.

[0051] Ramp plots generated for raw excrement extraction optimization are shown in Fig. 5. For the specified conditions, the program suggested 56 different solutions. From these results, solution 4 was selected with tyrosinase inhibitory effect 0.179 (mg / ml); SPF 16.199; total antioxidants 34.169 (pg / ml); total protein 9.322 (mg BSA / g dry extract); total phenol 125.564 (mg GAE / g dry extract), and yield 7.385%. The desirability of the selected solution is 0.919. Desirability is an objective function ranging from zero to a target value of 1. This is the best achievable level of desirability for the optimization conditions.

[0052] Standardized Production of Multi-functional Bioactive Mixtures with Powder Form

[0053] Extracts

[0054] Design Expert Version 13.05.0 (Stat-Ease Inc., Minneapolis, MN, USA) program was used to create and interpret the experimental matrix in order to obtain standardized multi-functional bioactive mixtures in powder form. Within the scope of the conditions to be studied, the Optimal (Custom) Design option was selected in the program. In the formulation study, fulvic acid and sericin protein were used with the raw extract to improve bioactive properties. The minimum and maximum values used in the design are given as 0 and 1.

[0055] The aim of the ternary mixtures prepared to obtain powder formulations is to encapsulate the raw extract with fulvic acid and sericin protein. For this purpose, mixtures were prepared with the coefficients given in Table 2 with a total weight of 100 mg and dissolved with 10 ml distilled water. In the dissolving stage, the samples were first kept in an ultrasonic bath for half an hour. The mixture was then stirred in a cooled shaking incubator at 25°C, 150 rpm for 6 hours to ensure complete homogeneity of the mixing and dissolving process. Samples were frozen at 20°C for 24 hours and then lyophilized for 48 hours to complete the encapsulation process.

[0056] In the designed experimental matrix, the aim was to observe the effect of single, binary, and ternary mixtures of raw extract, sericin protein, and fulvic acid used in the formulation. For this purpose, SPF, tyrosinase enzyme inhibitory effect, and antioxidant tests were applied to lyophilized samples. Table 2. Multi-functional Bioactive Mixtures with Powder Form Extracts and Results.

[0057] Optimization Phase for Standardized Production of Multi-functional Bioactive Mixtures with Powder Form Extracts

[0058] Optimization phase has started. Sericin protein (A), fulvic acid (B), raw silkworm excrement extract (%) (C) values were kept within the ranges studied during optimization. While the sun protection factor and tyrosinase enzyme inhibitory effect response, which are considered to be the most important within the scope of the project, were desired to be maximum, it was decided to be within the range since the antioxidant capacity was very high among the 3 components. The program proposed 13 solutions.

[0059] Table 3. Ingredients and quantities contained in the formulations.

[0060] The inventive ultraviolet (UV) blocker and tyrosinase enzyme inhibitor cosmetic raw material comprises raw silkworm excrement extract encapsulated with fulvic acid and sericin protein.

[0061] In an embodiment of the invention, the inventive ultraviolet (UV) blocker and tyrosinase enzyme inhibitor cosmetic raw material comprises 539 mg of raw silkworm excrement extract encapsulated with 266 mg of fulvic acid and 141 mg of sericin protein in a 1 gram mixture.

[0062] In an embodiment of the invention, the inventive ultraviolet (UV) blocker and tyrosinase enzyme inhibitor cosmetic raw material comprises 539 mg of raw silkworm excrement extract encapsulated with 266 mg of fulvic acid and 141 mg of sericin protein in a 1 gram mixture.

[0063] A preparation method of ultraviolet (UV) blocker and tyrosinase enzyme inhibitor cosmetic raw material comprises the process steps of: i. preparing a mixture from raw silkworm excrement extract, fulvic acid, and sericin protein, and adding distilled water to the obtained mixture, ii. keeping the mixture in an ultrasonic bath followed by a mixing process in a cooling-shaking incubator, iii. freezing the resulting mixture and then lyophilizing it to complete the encapsulation process.

[0064] In an embodiment of the invention, a preparation method of ultraviolet (UV) blocker and tyrosinase enzyme inhibitor cosmetic raw material comprises the process steps of: i. preparing a mixture of totaling 1 gram (g) from 593 milligrams (mg) of raw silkworm excrement extract, 266 mg of fulvic acid, and 141 mg of sericin protein, and adding distilled water to the obtained mixture, ii. keeping the mixture in an ultrasonic bath for 15-60 minutes followed by a mixing process in a cooling-shaking incubator at 20-35°C, 100-200 rpm for 3-12 hours, iii. freezing the resulting mixture at -20°C for 12-48 hours and then lyophilizing it for 36-72 hours to complete the encapsulation process.

[0065] In an embodiment of the invention, a preparation method of ultraviolet (UV) blocker and tyrosinase enzyme inhibitor cosmetic raw material comprises the process steps of: i. preparing a mixture of totaling 1 gram (g) from 593 milligrams (mg) of raw silkworm excrement extract, 266 mg of fulvic acid, and 141 mg of sericin protein, and adding distilled water to the obtained mixture, ii. keeping the mixture in an ultrasonic bath for 30 minutes followed by a mixing process in a cooling-shaking incubator at 25°C, 150 rpm for 6 hours, iii. freezing the resulting mixture at -20°C for 24 hours and then lyophilizing it for 48 hours to complete the encapsulation process.

[0066] SPF analysis, tyrosinase enzyme inhibitory effect and antioxidant capacity analysis were performed on the samples obtained in powder form.

[0067] 3D and contour plots of the effect of raw silkworm excrement extract, fulvic acid, and sericin protein on SPF, tyrosinase enzyme inhibitor and antioxidant activity are shown in Fig. 4.

[0068] The effect of antioxidant capacity of raw silkworm excrement extract, sericin protein, and fulvic acid was evaluated as IC50 (pg / ml). Plots showing the effects of the three components are shown in Fig. 4. At the maximum values of the raw silkworm excrement extract, the concentration at which the IC50 value is reached decreases. It is seen that increasing the raw silkworm excrement extract has a positive effect on the response value. However, in ternary compositions where the amount of sericin increases, the concentration at which the IC50 value is reached also increases and has a negative effect on the response value. Looking at the points where sericin proteinraw silkworm excrement extract and fulvic acid-raw silkworm excrement extract were combined in the same ratio, it was observed that the combination with fulvic acid had a positive effect on the antioxidant effect response.

[0069] After the selected conditions, the program proposed 14 solutions. The solution proposal numbered 11, which is the ternary mixture expected to show the best effect here, has been selected and named formulation 1 (F1). In binary mixtures, the solution proposal number 4, which shows the best effect and has a desirability function of 1 , was selected and named as formulation 2 (F2). It was evaluated whether sericin protein, which has the lowest sun protection factor and tyrosinase inhibitory effect in the mixtures, would increase its effect when combined with raw silkworm excrement extract and whether it would show protective properties. In Formulation 2, which has a better sun protection factor and tyrosinase inhibitory response, fulvic acid and raw silkworm excrement extract were used together.

[0070] The ramp plots obtained for the 11th and 4th solution proposals in the formulation optimizations are given in Figs. 6 and 7.

[0071] Stability Tests of Formulations

[0072] Both formulations (Formulation 1-(F1) and Formulation 2-(F2)) and the raw extract were subjected to a thermal stability test. The samples were exposed to a temperature of 70°C for 5 and 21 hours. Sun protection factor, antioxidant capacity determination, and tyrosinase enzyme inhibition properties of the heat-treated samples were investigated (Table 5). When the results of the analysis after the heat treatments were examined, it was observed that there was a gradual decrease in all properties of the raw silkworm excrement extract. With the formulations developed, the aim was to obtain a more stable raw material by preventing this loss of activity. Formulation 1 (F1) represents raw silkworm excrement extract and fulvic acid encapsulated with sericin protin. Table 5 clearly shows that encapsulation with sericin protein provides thermal stability for each bioactivity. Formulation 2 (F2) containing fulvic and sericin protein provided good protection in terms of antioxidant capacity. As seen in Table 5, the antioxidant capacity of raw silkworm excrement extract decreases when it is heat treated alone. Here, it is seen that this feature is preserved with the effect of fulvic acid.

[0073] As a result, two new thermally stable raw materials for use in cosmetic formulations were identified and these materials were mixed with cream base to obtain dual-action products.

[0074] Application of Powder Form Extracts to Prototype Cosmetic Products

[0075] The UV-blocking effect of the products obtained by adding at a ratio of 1 / 100, 1 / 1000 and 1 / 5000 directly into the cream base was tested using UV-sensitive papers. UV sensitive paper images for seven different applications under direct sunlight at UV index 3 and under UVB lamps are given in Figs. 8 and 9. When the results were examined, no color change was observed on the applied part due to the fact that the base cream used creates a physical barrier between UV rays and paper. Products with higher extract concentrations (Fig. 8.1 and Fig. 8.4) were more protective than the base cream.

[0076] Due to the fact that the base cream used creates a physical barrier between the UVB rays and the paper, there was no partial discoloration of the applied part. It was found that the products with higher extract concentration (Fig. 9.1 and Fig. 9.4) provided more protection than the base cream and also showed similar results to sunscreen. Formulation numbered 1 (Formulation 1-(F1)) encapsulated with sericin protein was more effective at high concentration and also remained more stable than the cream containing only fulvic acid-extract. Under UVB lamp, formulated creams containing different ratios of Formulation 1 and Formulation 2 showed more protective effect than the base cream. The creams containing Formulation 1 and Formulation 2 were found to be as protective as the 30 factor sunscreen used as a positive control. Table 4. Optimization results of raw silkworm excrement extraction. Table 5. Thermal stability analysis results of heat treated samples

[0077] References

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[0079] [2] Parhi, R. Drug delivery applications of chitin and chitosan: a review. Environ Chem

[0080] Lett 18, 577-594 (2020). https: / / doi.Org / 10.1007 / s10311 -020-00963-5

[0081] [3] Das, A., Ringu, T., Ghosh, S. et al. A comprehensive review on recent advances in preparation, physicochemical characterization, and bioengineering applications of biopolymers. Polym. Bull. 80, 7247-7312 (2023). https: / / doi.Org / 10.1007 / s00289-022- 04443-4

Claims

CLAIMS1. An ultraviolet (UV) blocker and tyrosinase enzyme inhibitor cosmetic raw material, characterized in that it comprises raw silkworm excrement extract encapsulated with fulvic acid and sericin protein.

2. A raw material according to claim 1, characterized in that it comprises 539 mg of raw silkworm excrement extract encapsulated with 266 mg of fulvic acid and 141 mg of sericin protein in a 1 gram mixture.

3. A preparation method of ultraviolet (UV) blocker and tyrosinase enzyme inhibitor cosmetic raw material, characterized in that it comprises the process steps of: i. preparing a mixture from raw silkworm excrement extract, fulvic acid, and sericin protein, and adding distilled water to the obtained mixture, ii. keeping the mixture in an ultrasonic bath followed by a mixing process in a cooling-shaking incubator, iii. freezing the resulting mixture and then lyophilizing it to complete the encapsulation process.

4. A preparation method according to claim 3, characterized in that it comprises the process steps of: i. preparing a mixture of totaling 1 gram (g) from 593 milligrams (mg) of raw silkworm excrement extract, 266 mg of fulvic acid, and 141 mg of sericin protein, and adding distilled water to the obtained mixture, ii. keeping the mixture in an ultrasonic bath for 15-60 minutes followed by a mixing process in a cooling-shaking incubator at 20-35°C, 100-200 rpm for 3-12 hours, iii. freezing the resulting mixture at -20°C for 12-48 hours and then lyophilizing it for 36-72 hours to complete the encapsulation process.

5. A preparation method according to claim 4, characterized in that it comprises the process steps of: i. preparing a mixture of totaling 1 gram (g) from 593 milligrams (mg) of raw silkworm excrement extract, 266 mg of fulvic acid, and 141 mg of sericin protein, and adding distilled water to the obtained mixture,ii. keeping the mixture in an ultrasonic bath for 30 minutes followed by a mixing process in a cooling-shaking incubator at 25°C, 150 rpm for 6 hours, iii. freezing the resulting mixture at -20°C for 24 hours and then lyophilizing it for 48 hours to complete the encapsulation process.

6. An ultraviolet (UV) blocker and tyrosinase enzyme inhibitor cosmetic raw material prepared by a method according to any one of claims 3-5.

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