Production of extract from amphimallon solstitiale larvae, pupae and / or adult insect for the treatment of dermatitis disease and the extract obtained as a result of the production method thereof

Abstract

The present invention relates to an extract obtained from Amphimallon solstitiale larvae, pupae and / or adult insects for the treatment of dermatitis and to the therapeutic use of this extract. Our invention is the production method of Amphimallon solstitiale larval, pupal and / or adult insect extract, the extract produced as a result of this method and the use of this extract in the treatment of dermatitis, which is used in the elimination of itching, blistering, scaling, rash, redness and burning on the skin, which are common symptoms of various skin diseases such as dermatitis, preventing its recurrence and treating it without side effects.

Description

[0001]DESCRIPTION PRODUCTION OF EXTRACT FROM AMPHIMALLON SOLSTITIALE LARVAE, PUPAE AND / OR ADULT INSECT FOR THE TREATMENT OF DERMATITIS DISEASE AND THE EXTRACT OBTAINED AS A RESULT OF THE PRODUCTION METHOD THEREOF Field of the Invention The present invention relates to an extract obtained from Amphimallon solstitiale larvae, pupae and / or adult insects for the treatment of dermatitis and to the therapeutic use of this extract. State of the Art: Various skin diseases such as eczema, vitilligo, psoriasis, rose urticaria, lichen, dermatitis are widely observed in humans and significantly affect the daily life of individuals. Today, drugs with a high side effect profile including various chemicals and hormone derivatives developed for the treatment of these diseases are used orally, by injection or topically as ointments and creams. Skin diseases tend to recur over time. Therefore, natural, economical methods are being investigated to reduce the patient's exposure to hormones and drugs, to reduce the use of treatment tools with a high side effect profile, and to reduce the financial burden. The high rate of side effects and the formation of undesirable effects on the patient's biological systems have led to the development of more natural, alternative, low side effect profile products in this field. Hormone therapies, especially those containing cortisone, cause a decrease in the immune system and increase the risk of microbial diseases. Over time, resistance to the drugs used develops in the biological system and the disease symptoms return in a more aggressive form. Undesirable effects such as thinning of the skin, changes in skin color and enlargement of capillaries can also be observed. This also requires treatment. Topical cream, spray, ointment formulations supporting the use of herbal extracts for the treatment of each disease for various skin diseases such as eczema, psoriasis, urticaria, lichen, vitilligo, rosacea are available in the literature and in the market. However, in the known state of the art, there is a need for a formulation with herbal extracts to relieve the common symptoms of all skin diseases. Since current treatment approaches are generally symptomatic, no permanent cure is realised. This may cause recurrence of the disease. In addition, while existing treatments mostly use compounds of chemical origin (such as corticosteroids and phosphodiesterase inhibitors), our invention uses larval, pupal and / or adult insect extracts of biological origin, which are completely side-effect-free. With our invention, dermatitis can be cured completely, irreversibly and without side effects. It has necessitated a development in the relevant technical field. Object of our invention and its advantages over the prior art The present invention relates to an insect extract of Amphimallon solstitiale larvae, pupae and / or adults for relieving the symptoms of skin diseases, which eliminates the above-mentioned disadvantages and brings new advantages to the relevant technical field. The main object of the present invention relates to an insect extract of Amphimallon solstitiale larvae, pupae and / or adults, which provides complete and irreversible, side-effect-free relief of itching, blistering, scaling, rash, redness and burning of the skin, which are symptoms of dermatitis. The object of the present invention is to provide a larval, pupal and / or adult insect extract having anti-inflammatory and anti-irritant activity on the skin. A large number of anticancer, antioxidant, antimicrobial, antibacterial, anti-inflammatory, anti- degenerative active substances have been identified in the content of insects in the family Scarabaeidae of the Coleoptera order. Amphimallon solstitiale extract has been used in dermatitis due to its high content of anti-inflammatory substances. In order to obtain the extract in the method of obtaining Amphimallon solstitiale larvae, pupae and / or adult insects to be used in the treatment of dermatitis disease, Amphimallon solstitiale larvae, pupae and / or adult insects stored in -800C deep freezer were subjected to the following processes to obtain the extract: 1-Application of phosphate buffer to prevent deterioration of the content of larvae, pupae and / or adult insects during disintegration 2- Larvae, pupae and / or adult insects are subjected to beating in a mortar and pestle 3-Completion of disintegration in order to discovery of larvae, pupae and / or adult insect content 4-Passing the product obtained in the sonicator through filter paper for use in cell culture stages 5-Cultivating HaCaT cells and changing the medium every two days to preserve the viability of the cell 6-Passaging of the obtained HaCaT cells 7- Washing in phosphate buffered saline (PBS, phosphate buffered saline) to prevent cells from sticking together and to maintain cell viability 8-Subjecting the cultivated cells to cytotoxicity test after reaching a sufficient number 9- Determination of the IC50value occurs 10-Establishment of in vitro dermatitis-induced cell model by administering Interleukins to HaCaT cells 11-Application of the dose and duration determined in the cytotoxicity stage to the in vitro dermatitis-induced cell model 12-Obtaining cell lysate from HaCAT cells and subsequent RNA isolation from these samples in order to detect gene expression changes in the in vitro dermatitis-induced cell model before and after the application of larval, pupal and / or adult insect extract in order to perform quantitative PCR 13- Performing quantitative PCR on RNAs obtained from cell lysates in order to detect gene expression changes in the in vitro dermatitis-induced cell model in larvae, pupae and / or adult insects before and after extract application. The results obtained by applying the extract obtained in our invention to the in vitro dermatitis model; Flagrin expression was found to be higher in eczema cells than in HaCaT cells at 0 and 24 hours. However, Flagrin expression was found to be significantly lower in in vitro dermatitis-induced cells at 48 h. The in vitro dermatitis-induced cell model occurred at 48 h after IL-4 and IL-13 administration. This Flagrin value shows that our dermatitis model is working. Loricrin expression was found to be higher in dermatitis cells than HaCaT cells at 0 and 24 hours. However, Loricrin expression at 48 hours was found to be significantly higher in in vitro dermatitis-induced cells. This result is due to the therapeutic effect of Amphimallon solstitiale larval, pupal and / or adult insect extract. In our study, Keratin-1 (Krt-1) expression was found to be lower in Dermatitis cells than in HaCaT cells at 0 and 24 hours. However, at the 48th hour, Krt-1 expression was found to be significantly higher in in vitro dermatitis-induced cells. This result is due to the therapeutic effect of Amphimallon solstitiale larval, pupal and / or adult insect extract. In our finding, it was determined that Krt-10 expression was lower than HaCaT cells at 0 and 24 hours in the in vitro HaCaT cells Dermatitis model. However, at 48 hours, Krt-10 expression was found to be significantly higher in the in vitro HaCaT cells dermatitis model. This result is due to the therapeutic effect of Amphimallon solstitiale larva, pupa and / or adult insect extract. In our study, it was determined that Krt-14 expression was lower than HaCaT cells at 0 and 24 hours in the in vitro HaCaT cells dermatitis model. However, at 48 hours, Krt-14 expression was found to be significantly higher in the in vitro HaCaT cells dermatitis model. This result is due to the therapeutic effect of Amphimallon solstitiale larva, pupa and / or adult insect extract. Description of Figures: Figure 1. Outline of the molecular pathways involved in the mechanism of dermatitis Figure 2. Diagram of the 3-year life cycle of the family Scarabaeidae Figure 3 Microscope image of HaCaT cells Figure 4. Appearance of study groups where larva, pupa and / or adult insect extract was used Figure 5. Invert microscope images of cells. (A) HaCaT cells (H0) without treatment with larval, pupal, and / or adult extracts. (B) HaCaT cells (H24) treated with larval, pupal, and / or adult insect extract for 24 h. (B) HaCaT cells (H48) treated with larval, pupal, and / or adult insect extract for 48 h. (D) In vitro dermatitis-induced cells (E0) without larval, pupal and / or adult insect extract treatment. (E) In vitro dermatitis-induced cells (H24) treated with larval, pupal and / or adult insect extract for 24 hours. (E) In vitro dermatitis-induced cells (H48) treated with larval, pupal and / or adult insect extract for 48 hours. Figure 6. Comparison view of Flagrin expression in cells. Figure 7. Comparison view of Loricrin expression in cells. Figure 8. Comparison view of Krt-1 expression in cells. Figure-9: Comparison view of Krt-10 expression in cells. Figure-10: Comparison view of Krt-14 expression in cells. Table-1. Table of cDNA Synthesis Kit contents Table-2: Table showing Reverse Transcriptase Reaction Conditions Table 3. Table showing the primers and their sequences used in our study Table-2: Table showing Quantitative PCR Kit components Table-5. Table showing Quantitative PCR Cycle conditions Part numbers in Figure-1: 1- Reduced barrier structure proteins 2- Increased hyperplasia barrier inhibition Detailed Description of the Invention The present invention relates to an extract obtained from Amphimallon solstitiale larva, pupa and / or adult insects for the treatment of dermatitis and to the therapeutic use of this extract. The Amphimallon solstitiale larva, pupa and / or adult insect extract, which was supplied and stored at -80°C, was subjected to the following processes: 1- Phosphate buffer was used to prevent the insect content from being spoiled while dissecting the larva, pupa and / or adult insect. Phosphate buffer used here is obtained by dissolving 0,78005 g of Sodium dihydrogen phosphate (NaH2PO4) in 80 ml of water. The buffer pH is adjusted by adding HCL to be between 6,8-7,40 and the final volume is completed to 100 ml. 2- Larva, pupa and / or adult insects are subjected to a pounding process in a ceramic mortar that has been sterilized by autoclaving. The beating process was carried out in a cold environment on ice. The disintegration was not carried out continuously but intermittently to prevent heating. 3- In order to completely reveal the contents of the larva, pupa and / or adult insect, it was crushed in a sonicator at intervals working on the principle of sonic disintegration. This process was carried out on ice to preserve the integrity of the resulting content. 4- The larva, pupa and / or adult insect content obtained in the sonicator was passed through filter paper to be used in the cell culture stages. The aim here is to ensure that larva, pupa and / or adult insect contents do not contaminate cell culture studies and do not affect viability. 5- HaCaT cells were cultivated in DMEM High Glucose medium. The aim here is to ensure that cell viability and proliferation continue. In order to maintain cell viability, DMEM High Glucose medium was changed every two days. As a result, healthy and sufficient cells will be obtained for subsequent studies. 6- Following this, passaging HaCaT cells. Passaging is performed in order to change the medium of the cells and prepare a stock culture from there. In the flask removed from the oven, the cells were allowed to flow by pull-and-release action with the help of a pipette towards the wall where the cells were located. Afterwards, a slow pull-and-release was performed towards the bottom of the flask with the help of a pipette. Then, 2 mL was added to the flask and suspended. It was centrifuged at 2000 rpm for 10 minutes and the supernatant was discarded. Medium was added to the pellet and divided into flasks. Medium was added to make the final volume 5 mL. The cells were incubated under 37°C and 5% CO2 conditions. 7- In order to prevent the passaged cells from sticking to each other and to preserve cell viability, these cells were washed with phosphate buffered saline (PBS) with a pH between 6.8-7.4. 8- Cultured and washed cells were subjected to cytotoxicity test after reaching sufficient number. Cytotoxicity study is performed to show the biological effect of any chemical or biological molecule or a product such as a medical device. Here, at which dose (amount) and time interval, the dose and time value at which the cell viability decreases to IC50, i.e.50%, is determined. The larva, pupa and / or adult insect extract prepared in volumes of 2.5 µL, 5 µL, 10 µL, 25 µL, 50 µL, 100 µL was applied to HaCaT cells in 105numbers for 24 and 48 hours. The cells were incubated under 37°C and 5% CO2conditions. After the incubation periods of 24 and 48 hours, 10 µl of WST-1 reagent was added to all wells and incubated at 37°C and 5% CO2. After 4 hours of incubation, measurements were made in a spectrophotometer at 450 nm wavelength. The IC50 values of larva, pupa and / or adult insects were determined by creating graphs. 9-The dose and time determined at the cytotoxicity stage were applied in the in vitro dermatitis model. According to the results obtained from Graphpad Prism programme, the approximate amount to be applied in 24 hours was found to be 12-16μl and the amount to be applied in 48 hours was found to be approximately 80-90μl. 10- Creation of an in vitro dermatitis model by giving interleukins to HaCaT cells. In order to create a dermatitis model, HaCaT cells were grown in medium containing IL-4 and IL-13 at 10 ng / mL for 24 hours. Larva, pupa and / or adult insect extracts were added to the dermatitis-induced cells at specified amounts and times (for 24 and 48 hours). Cell groups are determined as Dermatitis 0th Hour, 24th Hour and 48th Hour. HaCaT 0th Hour, 24th Hour and 48th Hour samples were used as controls. 11- RNA isolation kit was used in the RNA isolation process. First of all, the cells were homogenized at room temperature for 5 min with 1 ml of lysis buffer solution per 1 x 107cells. Then, 200 µl chloroform was added and incubated for 2 minutes. The aqueous phase was transferred to a new tube by centrifugation at 12.000 x g for 15 min at 4°C.1 volume of buffer solution was added and mixed. The mixture was transferred to a 700 column and centrifuged at 10.000 x g for 30 seconds. Then 500 µl of buffer solution 2 and 500 µl of buffer solution 3 were added to the column and centrifuged at 10.000 g for 30 s each. In the last step, 50µl of nuclease-free, i.e. ultrapure water free from RNA and DNA degrading enzymes, was placed in the centre of the column and centrifuged at 10.000 x g for 1 minute. The quality and quantity of RNA obtained were determined by spectrophotometer OD260 / 280. Samples with OD260 / 280 values in the range of 1.8-2 were used in the next step of quantitative PCR. 12- Quantitative PCR was performed to detect gene expression changes in the in vitro dermatitis model before and after application of larval, pupal and / or adult insect extract. In order to determine the expression of target genes, RNAs isolated in the first step were translated into cDNA. The cDNA Synthesis kit was used for this process. The kit components are mixed in a tube. Table 1. cDNA Synthesis Kit Components Table 2. Quantitative PCR Reaction Conditions After the reaction conditions were adjusted, the study was carried out on the PCR device. A quantitative PCR kit was used for the quantitative PCR process. DNA sequences for the primers used in the reaction are presented below. Table 3. DNA sequence of the primers used in the study Table 4. Quantitative PCR Kit Components After the reaction conditions were adjusted, the study and result reading were carried out on the quantitative PCR device. The results obtained in our invention are presented below: Flagrin expression was found to be higher in in vitro dermatitis-induced cells than in HaCaT cells at 0 and 24 hours. However, Flagrin expression at 48 hours was found to be significantly lower in in vitro dermatitis-induce cells. The dermatitis pattern occurred at 48 hours after administration of IL-4 and IL-13. This Flagrin value shows that our dermatitis model is working. Loricrin expression was found to be higher in in vitro dermatitis-induced cells than in HaCaT cells at 0 and 24 hours. However, Loricrin expression at 48 hours was found to be significantly higher in in vitro dermatitis-induced cells. This result is due to the therapeutic effect of Amphimallon solstitiale larva, pupa and / or adult insect extract. Keratin-1 (Krt-1) expression was found to be lower in in vitro dermatitis-induced cells than in HaCaT cells at 0 and 24 hours. However, at the 48th hour, Krt-1 expression was found to be significantly higher in in vitro dermatitis-induced cells. This result is due to the therapeutic effect of Amphimallon solstitiale larva, pupa and / or adult insect extract. It was determined that Krt-10 expression was lower than HaCaT cells at 0 and 24 hours in the in vitro HaCaT cells dermatitis model. However, at the 48th hour, Krt-10 expression was observed to be significantly higher in in vitro HaCaT cells of in vitro dermatitis-induced cells. This result is due to the therapeutic effect of Amphimallon solstitiale larva, pupa and / or adult insect extract. It was determined that Krt-14 expression was lower than HaCaT cells at 0 and 24 hours in the in vitro HaCaT cells dermatitis model. However, at 48 hours, Krt-14 expression was significantly higher in in vitro HaCaT cells of in vitro dermatitis-induced cells. This result is due to the therapeutic effect of Amphimallon solstitiale larva, pupa and / or adult insect extract. Detailed study details of the in vitro dermatitis model using Amphimallon solstitiale are presented below: Larva, Pupa and / or Adult Insect Homogenization After obtaining the larval, pupal and / or adult insect, the provided larva, pupa and / or adult insect must be dissected to reveal its contents. For this purpose, the dissection process was carried out using a laboratory mortar. Phosphate buffer content was used to preserve the content structure intact during dissection. Phosphate buffer content: Phosphate buffer / sodium dihydrogen phosphate is obtained by dissolving 0,78005 g NaH2PO4 in 80 ml of water, then HCl is added to make the pH between 6,8-7,40 and completed to 100 ml. The phosphate buffer at different pHs obtained was used in homogenisation processes. For the larval, pupal and / or adult insect extract to be used in our invention, the pH at which it will be most effective in the dermatitis cell model created in our invention has been determined as pH 7.00. Cultivation of Cells Cells were removed from the nitrogen tank and heated in a water bath at 37°C before cultivation. The thawed cells were collected in a 50 mL sterile flask, 10 mL medium was added and centrifuged at 800 rpm for 5 minutes. The supernatant was removed after centrifugation. The pellet was homogenised with 2 mL medium and cultivated into 25 cm2(T25) flasks depending on the number of cells and the final volume was completed to 5 mL with medium. They were left to incubation at 37°C and 5% CO2conditions. Medium Change The medium was changed every two days to maintain the viability of the cells. When the cells spread over the entire surface of the flask (when they reached 80-90% density), passaging was performed because there was no space for them to grow. Passaging of Cells In the passaging process, the medium in the flask was first removed. Then, washing was performed with 1 mL PBS (Phosphate Buffered Saline) (Figure 10). After washing, PBS was removed, 1 mL Trypsin-EDTA was added and the cells were incubated in an oven for 3 min. With trypsin-EDTA, cells adherent to the bottom of the flask were detached from the surface. After the cells were removed from the oven, they were examined under an inverted microscope to check whether they were separated from the base. In the flask removed from the oven, the cells were allowed to flow by pull-and-release action with the help of a pipette towards the wall where the cells were located. Then, with the help of a pipette, a slow pull-and-release action was made 5 times towards the bottom of the flask. Then, 2 mL was added to the flask and suspended. It was centrifuged at 2000 rpm for 10 minutes and the supernatant was discarded. Medium was added to the pellet and divided into flasks. Medium was added to make the final volume 5 mL. The cells were incubated under 37°C and 5% CO2 conditions. WST-1 Test In the study, the effect of Amphimallon solstitiale larvae, pupae and / or adult insects was determined in HaCat cells and in vitro atopic dermatitis model cells. For this purpose, firstly, the larvae, pupae and / or adult insects were crushed in a mortar and pestle, since this part is rich in chitin, then a sonicator (90% amplify) was used to increase the homogenisation efficiency and the content of larvae, pupae and / or adult insects in 5 ml phosphate buffer. Larvae, pupae and / or adult insects were homogenised and tested at 2.5 µL, 5 µL, 10 µL, 25 µL, 50 µL, 100 µL for 24 and 48 hours respectively. Then, 10 µl of WST-1 reagent was added to all wells and after 4 hours of incubation, measurements were made at 450 nm wavelength in a microplate reader spectrophotometer. The IC50 value of the larva was calculated by creating graphs. Creating a Dermatitis Model In order to create an atopic dermatitis model, HaCaT cells were grown in medium containing IL-4 and IL-13 at 10 ng / mL for 24 hours. Then, larval, pupal and / or adult insect extracts were added to the dermatitis-induced cells at specified amounts and times (for 24 and 48 hours). Cell groups are determined as Dermatitis 0th Hour, 24th Hour and 48th Hour. HaCaT 0th Hour, 24th Hour and 48th Hour samples were used as controls (Figure 4). Preparation of Cell Lysate In order to prepare cell lysate, cells were first removed with Trypsin-EDTA. It was then lysed with lysis buffer containing 50mM Tris, 150 µM NaCl, 1% NP-40 and 1% Proteinase inhibitor factor. After treating the cells with lysis buffer, the cells were centrifuged (+4⁰C, 13000rpm, 13-15 min.). Then, the supernatant was removed and stored in a -20°C deep freezer. Quantitative PCR Quantitative PCR method was applied to determine the effectiveness of larva, pupa and / or adult insect against HaCaT and Atopic dermatitis model cells. The steps and details are given below. RNA Isolation RNA isolation kit was used in the RNA isolation process. First of all, the cells were homogenized at room temperature for 5 min with 1 ml of RNA lysis solution per 1 x 107cells. Then, 200 µl chloroform was added and incubated for 2 minutes. The aqueous phase was transferred into a new tube by centrifugation at 12.000 x g for 15 min at 4°C. 1 volume of buffer solution 1 was added and mixed. The mixture was transferred to a 700 column and centrifuged at 10.000 x g for 30 seconds. Then 500 µl of buffer solution 2 and 500 µl of buffer solution 2 were added to the column and centrifuged at 10.000 g for 30 s each. In the last step, ultrapure water devoid of 50 DNA and RNA degrading enzymes was placed in the middle of the column and centrifuged at 10.000 x g for 1 minute. The quality and quantity of RNA obtained were determined by spectrophotometer OD260 / 280. Samples with OD260 / 280 values in the range of 1.8-2 were used in the next step of quantitative PCR. Complementary DNA (cDNA) Synthesis In order to determine the expression of target genes, RNAs isolated in the first step were translated into cDNA. The cDNA Synthesis kit was used for this process. The kit components are mixed in a tube. After the reaction conditions were adjusted, the reaction and evaluation were performed on the PCR device. Determination of Gene Expression Level by Quantitative PCR For the quantitative PCR process, the PCR Master Mix kit, which contains a fluorescent dye that binds to double-stranded DNA, was used. Primer and nucleotide sequences used in this study are presented in Table 3. After the reaction conditions were adjusted, the reaction took place in the real-time PCR device. Statistical Analysis After the quantitative PCR analyses, statistical analyzes were performed in the GraphPad Prism 8.0 program. One-way ANOVA test was applied for comparisons. Statistical significance level was accepted as p<0.05. Findings WST-1 Dosage Determination According to the results obtained from Graphpad Prism programme, the approximate amount to be applied in 24 hours was found to be 12-16μl and the amount to be applied in 48 hours was found to be approximately 80-90μl. Microscopic Examination of Cells The study in our invention consists of 6 groups. Cells were viewed with an inverted microscope. They are named as follows; HaCaT cells not treated with larval, pupal and / or adult insect extract (H0), HaCaT cells treated with larval, pupal and / or adult insect extract for 24h (H24), HaCaT cells treated with larval, pupal and / or adult insect extract for 48 h (H48), in vitro Atopic dermatitis model applied cells not treated with larval, pupal and / or adult insect extract (E0), in vitro Atopic dermatitis model cells treated with larval, pupal and / or adult insect extract for 24 hours (H24), in vitro Atopic dermatitis model cells treated with larval, pupal and / or adult insect extract for 48 hours (H48). The change in the cell number of larval, pupal and / or adult insect extract is shown in Figure 5. Invert microscope images of cells. (A) HaCaT cells (H0) not treated with larval, pupal, and / or adult insect extracts. (B) HaCaT cells (H24) treated with larval, pupal, and / or adult insect extract for 24 h. (B) HaCaT cells (H48) treated with larval, pupal, and / or adult insect extract for 48 h. (D) In vitro atopic dermatitis-model applied cells (E0) not treated with larval, pupal and / or adult insect extract. (E) In vitro atopic dermatitis model applied cells (H24) treated with larval, pupal and / or adult insect extract for 24 hours. (E) In vitro atopic dermatitis model applied cells (H48) treated with larval, pupal and / or adult insect extract for 48 hours. It was observed that the number of HaCaT and in vitro dermatitis model applied cells without larval, pupal and / or adult insect extract treatment was higher than the cells with 24 and 48 hours of extract treatment. Evaluation of Quantitative PCR Results Delta-Delta Ct method was used to analyze Flagrin, Loricrin, Krt1, Krt 10 and Krt14 genes. According to this method, average ΔCt values were determined by subtracting the average Ct value of the Beta-Actin gene from the average Ct values of the genes in question. ΔΔCt values were calculated by subtracting the average ΔCt values from the ΔCt values of the relevant genes. For the analysis of fold changes, 2^-ΔΔCt values were calculated. The formulas for the calculations are presented below. dCt= Applied genCt–housekeepingCt, ddCt= Application Group dCt – Control group dCt Fold Change = 2^-ddCt qPCR Results The graphics obtained from the Graphpad prism 8.0 program show the changes in Flagrin, Loricrin, Krt-1, Krt-10 and Krt-14 gene expressions in the cells (Figure-6, Figure-7, Figure-8, Figure-9 and Figure-10). Flagrin expressions in cells are shown in the graph obtained from the Graphpad Prism 8.0 program (Figure 6). Flagrin expression was found to be higher in in vitro dermatitis model applied cells than in HaCaT cells at 0 and 24 hours. However, Flagrin expression at 48 hours was found to be significantly lower in in vitro atopic dermatitis model applied cells. In line with these results, it was determined that the in vitro atopic dermatitis applied model occurred at the 48th hour after the application of IL-4 and IL-13. Loricrin expressions in cells are shown in the graph obtained from Graphpad prism 8.0 program. Loricrin expression was found to be higher in in vitro dermatitis model applied cells than in HaCaT cells at 0 and 24 hours. However, at the 48th hour, Loricrin expression was found to be significantly higher in in vitro Atopic dermatitis model applied cells (p<0.0001). Krt-1 expressions in cells are shown in the graph obtained from the Graphpad Prism 8.0 program (Figure 8). Krt-1 expression was found to be lower in in vitro dermatitis model applied cells than in HaCaT cells at 0 and 24 hours. However, at the 48th hour, Krt-1 expression was found to be significantly higher in in vitro Atopic dermatitis model applied cells (p<0.0001). It was determined that Krt-10 expression was lower than HaCaT cells at 0 and 24 hours in the in vitro HaCaT cells atopic dermatitis model. However, at 48 hours, Krt-10 expression was found to be significantly higher in the in vitro HaCaT cells atopic dermatitis model (p<0,0001). It was determined that Krt-14 expression was lower than HaCaT cells at 0 and 24 hours in the in vitro HaCaT cells atopic dermatitis model. However, at 48 hours, Krt-14 expression was found to be significantly higher in the in vitro HaCaT cells atopic dermatitis model (p<0,0001). As a result: In our study, Flagrin expression was found to be higher than HaCaT cells in the in vitro HaCaT cells Atopic dermatitis model at 0 and 24 hours. However, at 48 hours, Flagrin expression was found to be significantly lower in the in vitro HaCaT cells Atopic dermatitis model. In our study, Loricrin expression was found to be higher than HaCaT cells in the in vitro HaCaT cells Atopic dermatitis model at 0 and 24 hours. However, at 48 hours, Loricrin expression was found to be significantly higher in the in vitro HaCaT cells Atopic dermatitis model. In our study, Krt-1 expression was found to be lower than HaCaT cells in the in vitro HaCaT cells Atopic dermatitis model at 0 and 24 hours. However, at 48 hours, Krt-1 expression was found to be significantly higher in the in vitro HaCaT cells Atopic dermatitis model. In our finding, the reason why Loricrin and Krt1 gene expressions are found at higher levels at 48 hours in the in vitro HaCaT cells Atopic dermatitis model is due to the therapeutic properties of the larval, pupal and / or adult insect extract. The expression of these genes was found to be significantly higher in dermatitis cells that entered the healing pathway.

Claims

CLAIMS 1- Production method of Amphimallon solstitiale larval, pupal and / or adult insect extract, which is used in the elimination of itching, blistering, scaling, rash, redness and burning on the skin, which are common symptoms of various skin diseases such as dermatitis, preventing its recurrence and treating it without side effects, characterized in that, it comprises the following process steps; - Applying phosphate buffer to prevent the contents of the supplied Amphimallon solstitiale larval, pupal and / or adult insect from being destroyed while being dissected - Subjecting larval, pupal and / or adult insect extracts to beating in a mortar and pestle - Completion of sonicator disruption and complete extraction of larval, pupal and / or adult insect extract contents - Passing the product obtained in the sonicator through filter paper for use in cell culture stages. 2-Amphimallon solstitiale larval, pupal and / or adult insect extract production method according to claim 1, characterized in that, phosphate buffer obtained by dissolving 0.78005 g of dihydrogen phosphate (NaH2PO4) in 80 ml of water is used. 3-Amphimallon solstitiale larval, pupal and / or adult insect extract production method according to claim 1, characterized in that, the pH of the phosphate buffer used is adjusted by adding HCl to be between 6,8-7,40 and the final volume is completed to 100 ml.

4. 2-Amphimallon solstitiale larval, pupal and / or adult insect extract production method according to claim 1, characterized in that, larvae (L1, L2, L3), pupae and / or adult (female and male) insect forms of the Amphimallon solstitiale species are used.

5. Amphimallon solstitiale larval, pupal and / or adult insect extract obtained as a result of the method according to any one of the preceding claims, characterized in that, it is used to relieve itching, blistering, scaling, rash, redness and burning of the skin, which are common symptoms of various skin diseases such as dermatitis, to prevent recurrence and to treat without side effects.

6. Amphimallon solstitiale larval, pupal and / or adult insect extract, which is used to relieve itching, blistering, scaling, rash, redness and burning of the skin, which are common symptoms of various skin diseases such as dermatitis, prevents recurrence and treats without side effects according to claim 5, characterised in that, the approximate amount to be applied in 24 hours is 12-16μl and the amount to be applied in 48 hours is approximately 80-90μl according to the results obtained from Graphpad Prism programme.

7. Amphimallon solstitiale larval, pupal and / or adult insect extract, which is used to relieve itching, blistering, scaling, rash, redness and burning of the skin, which are common symptoms of various skindiseases such as dermatitis, prevents recurrence and treats without side effects according to any one of the preceding claims, characterised in that, it is used in the treatment of dermatitis.

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