Technical Field

The invention relates to cosmetic products.

The invention particularly relates to formulations containing elderberry seed oil obtained by encapsulation method as a phyto-phospholipid complex suitable for use in the field of cosmetics as personal care products without synthetic components.

Known State of the Art

Nipple pain and tenderness are a common condition for breastfeeding women in the early postpartum period and are usually associated with the cessation of breastfeeding. This condition, which is caused by many reasons, manifests itself in some people by the fact that the nipples are only painful and sensitive, while itching can also be added to pain and tenderness. Menstruation, pregnancy, breastfeeding, infection, cancer, exercise-related friction, allergies are the most commonly observed causes.

Pregnancy causes several breast and nipple-related changes, for example, nipple sensitivity and hypersensitivity may occur, nipples and areolas may (the skin around the nipple) darken, areola and nipples may grow and the nipples may tend to protrude more, small glands on the surface of the areolas (Montgomery tubercles) may become fluffy bumps, breast enlargement may occur, veins may darken due to increased blood flow to the breasts, a yellowish, thick discharge (colostrum) may come from the breasts.

A fungus infection (also called thrush) is the most common type of infection that can occur on the nipples. The pores and hair follicles located around the nipples can become blocked and infected. Fungal infections can also develop in the nipples of women who wear bras made of materials that unsuitable and do not allow the skin to breathe. The nipples of breastfeeding women are especially prone to fungal infections and breast infections such as mastitis. Infections can occur in men or women with nipple piercings, especially when the piercings are not performed with a good technique or are not properly cared for afterwards. Another disease that can cause nipple pain and soreness is a type of cancer called Paget's disease. It usually affects only one nipple and two breasts and it is rare. Symptoms of skin changes such as an inverted or flattened nipple, yellowish or bloody discharge from the nipple, itching or tingling at the nipple, reddish, crusty or scaly skin around the nipple and areola are observed.

The friction of inappropriate fabrics worn during exercise that come into contact with the breast also usually causes nipple pain. For this reason, the skin may dry out or crack.

It is not uncommon to have pain and tenderness in the nipples during or after breastfeeding, especially in the first few minutes when the baby holds the breast for the first time. However, the nipples should not remain painful or bleed for a long time. If the nipple pain is severe or constant, or repeats, it may be because of a problem that needs to be corrected. The problem includes: the baby's inability to get the nipple and areola, problems with the baby's inability to latch on the breast, the baby's incorrect positioning, inverted nipples, moving the baby's mouth away from the breast without stopping sucking, babies with short frenulum (frenulum is the piece of tissue that connects the tongue to the lower part of the mouth), also known as "tongue tied".

Although there are many factors that contribute to a woman's decision to start and continue breastfeeding, evidence shows that most women stop breastfeeding early due to perceived difficulties. Unfortunately, the treatment of a painful/ sore nipple is complicated, due to the baby's repetitive sucking, infections caused by the entry of microorganisms through the nipple fissure, and constant exposure of the nipple skin with the baby's oral flora.

Modified lanolin, cold compresses, hydrogel pads and breast protectors can help reduce the pain.

Lanolin is a pale yellow, sticky substance with a slight characteristic odor, which is derived from sheep wool and chemically classified as a wax. Lanolin is considered a pure and safe intervention (free of preservatives, additives, water, chemicals or perfumes) aimed at creating a humid healing environment for nipple trauma and providing a semi-occlusive barrier that supports the retention of internal moisture and prevents dryness. Healthcare professionals often recommend the administration of lanolin to treat painful/damaged nipples, but a randomized controlled trial evaluated the effectiveness of lanolin on nipple pain and breastfeeding outcomes. It has been shown that applying lanolin to painful nipples immediately after childbirth does not significantly reduce nipple pain, nor does it improve the duration of breastfeeding. Therefore, the provision of lanolin in the hospital and/or the recommendation of lanolin by health professionals to treat nipple pain related to breast feeding is not as effective as desired.

For this reason, new formulations with clean ingredients and rich fatty acids are needed that will relieve nipple pain and sensitivity. However, the direct inclusion of plant extracts and oils in various products poses some difficulties. In particular, many of the plant bioactive components are prone to degradation. Factors such as light, temperature, oxygen, pH, as well as processing can affect their storage stability. Also, the poor solubility of hydrophobic compounds in water prevents them from dissolving and being absorbed. In the case of essential oils, it further limits their application due to their high volatility.

Therefore, due to the inadequacy of existing solutions on the subject, it became necessary to make a development in the relevant field in order to prevent and eliminate problems related with the subject.

Object of the Invention

The present invention relates to formulations that meet the requirements mentioned above, eliminate all the disadvantages and provide some additional advantages.

The primary object of the present invention is to develop a clean formulation that reduces nipple pain or tenderness or soreness by means of its content of vegetable oils, encapsulated elderberry seed oil and amino acid mixture and does not contain harmful substances.

Another object of the invention is to develop a product suitable for use in different cosmetic applications, thanks to its encapsulated content.

In order to achieve the objects described above, the invention is a cosmetic formulation comprising water and elderberry seed oil encapsulated with phosphatidylcholine as a phyto-phospholipid complex, which does not contain synthetic components and which nourishes, protects, moisturizes and balances the skin.

The structural and characteristic features of the invention and all its advantages will be understood more clearly by means of the detailed description written below and the related figures, and therefore evaluation should be made by taking these detailed descriptions and figures into consideration.

Figures to Help Explain the Invention

Figure 1 a : First in vitro cytotoxicity experiment

Figure 1 b : Second in vitro cytotoxicity experiment

Figure 1 c : Cell photographs from in vitro cytotoxicity experiment of nipple formula

Figure 2 : The effect of nipple formula on the expression of COL1A1 , HAS3, TGFB1 , FBLN 5, ELN and LOXL1 genes.

Figure 3 : Microscope image of the Nipple formula of the Scracth experiment

Detailed Description of the Invention

In this detailed explanation, the invention is described only for a better understanding of the subject in such a way that it does not create any limiting effects.

A cosmetic formulation that contains no synthetic ingredients and that nourishes, protects, moisturizes and balances the skin, in its basic form comprises elderberry seed oil encapsulated with phosphatidylcholine as a phyto-phospholipid complex and water (preferably deionized water). In a preferred embodiment of the invention, the formulation comprise 83-91% (preferably 88%) water, 8-12% (preferably 10%) elderberry seed oil and 1 -5% (preferably 2%) phosphatidylcholine by total weight. The molecular size of the encapsulated elderberry seed is preferably 208 nm.

The formulation that is the subject of the invention is suitable for use to reduce nipple pain or tenderness or soreness. In addition, the invention is also effective for reducing/eliminating the problem of dry skin (xerosis), which is a common symptom of a number of chronic skin diseases, such as atopic dermatitis.

In a preferred embodiment of the invention, the formulation developed comprises shea butter, avocado oil, squalene obtained from olives and amino acid mixture in the amounts given in Table 1 in the preferred ratios. In addition, it also preferably comprise sodium benzoate as a preservative in the amount given in Table 1. The developed formulation can be used for nipple pain and soreness, which is a common problem in breastfeeding women, and is preferably in the form of balm. The formula is suitable for use on sensitive skin and it has properties that nourish, protect, moisturize, balance the skin and also it will not harm the baby, even if it leaves residue. The mentioned amino acid mixture is preferably selected from a group that includes serine, alanine, glycine, glutamic acid, lysine, threonine, arginine, proline or a combination thereof.

Table 1

In an exemplary embodiment of the formulation that is the subject of the invention, a liposome comprising elderberry seed oil is obtained first. To prepare a 100 g liposome solution, 2 grams of 0.06% L-alpha phosphatidylcholine is weighed and dissolved in 88 grams of water by mixing at 20,000 rpm using a homogenizer. It is mixed at 20,000 rpm using a homogenizer for another 5-10 minutes by adding 10 grams of elderberry seed oil to the resulting aqueous liposome solution. The resulting mixture is made homogeneous by passing it 3 times (passes) using a micro fluidizer device at 20,000 psi. The pH of the formula is set at a value between 5.5-6. Then, after the shea butter is thoroughly melted at 40 degrees, avocado oil is added. Squalene obtained from olives is mixed into the mixture with the help of a mechanical mixer and taken into a container. This container is placed in a second container with water at 35-40 degrees in such a way that water does not enter the firsts container. It is mixed for 3 minutes so that the maximum temperature is 35-40 degrees. Encapsulated elderberry seed oil, amino acid mixture and Na benzoate as preservative are added to this mixture and left to cool by stirring at 30 RPM. The amounts to be added are given in Table 1 .

Liposomes are mainly used for transporting substances very selectively, in a way that increases the effectiveness of active substances and reduces their undesirable toxic properties. In other cases, liposomes are used to prolong the effect, improve adsorption, or simply stabilize active substances. They are also useful in gene transfer and various diagnostic and industrial applications. Their size and physicochemical properties lead to the penetration and diffusion of these structures into the tissue in various ways by releasing the encapsulated product.

Physicochemical studies focused on the properties of liposomes such as fluidity, permeability and molecular organization reveal the importance of double-layered lipid layers as a structural element of natural membranes. In cell biology, liposome-cell interactions constitute a model element for physiological processes such as fusion and adhesion. In addition, they can be used to improve cell-cell fusion, change the phospholipid and cholesterol content of membranes, and transfer molecules that are normally soluble in water, but difficult to penetrate into the cell.

Fixed and essential oils (EO) obtained from plants are natural, complex compounds. They are found in the structure of aromatic plants as secondary metabolites. They can be synthesized by all plant organs, such as buds, flowers and are found in leaves, stems, branches, seeds, fruits, roots, trees or tree barks and secretory cells, cavities, ducts, epidermic cells or glandular trichomes. Mono-, sesqui- and di-terpenes, including terpenes, are the main molecules of essential oils. These oils have a wide range due to their biological effects such as antifungal, antioxidant and bactericidal etc. properties. For this reason, the use of essential and fixed oils in the food industry and pharmacy has been increasing steadily in recent years. However, it is well known that most vegetable oils are not biologically stable. They are sensitive to oxygen, light and temperature. Some essential oils contain unstable functional groups that are affected by oxygen, oxygen can be oxidizing for alcohols and aldehydes, radiation can change the structure of unsaturated monoterpenic and sesquiterpenic hydrocarbons. In addition, the water solubility of oils is poor. For this reason, encapsulation method is used to increase the solubility and bioavailability of vegetable oils, as well as to increase their effectiveness. The composition of the lipid vesicle membrane, especially the type of phospholipids, cholesterol content, molar ratio of essential oils to lipids, preparation method and varieties can affect liposome size and encapsulation efficiency. Essential oils can reduce the size of liposomes, homogenize liposomal dispersions, increase fluidity and reduce the oxidation of the lipid layer.

As a rich source of fatty acids, vegetable oils have taken their place in the market of medicinal and cosmetic products. The fatty acids present in these oils form a barrier film on the skin that reduces trans epidermal water loss (TEWL), thereby contributing to the preserving proper hydration of the epidermis. In addition, fatty acids protect, renew and soften the stratum corneum, thereby contributing to the continuity of the intracellular structure of the skin. Depending on the percentage of the content of individual components, especially fatty acids, the effect of oils on the skin and human health may vary. For example, lack of lipids causes the skin to be deprived of an adequate protective layer and to dry out. The reason why vegetable oils are one of the main ingredients in cosmetic products is that they provide protection against excessive water loss through the skin by forming a barrier film that mainly covers the epidermis, that is, they provide skin moisture.

Encapsulation method is used for this purpose in the invention. In this method, a formulation has been developed to increase the solubility and bioavailability of elderberry seed oil, as well as to control its release and to increase its effectiveness, by bringing elderberry seed oil encapsulated with phosphatidylcholine into balm form with avocado and shea oils and enriching it with squalene and amino acids mixture, to nourish, calm and moisturize the skin.

The physiochemical parameters of the formulation, which is the subject of the invention, was evaluated in terms of, pH, particle size and consistency. The results of the stability study show that there is no change in the visual appearance, homogeneity and particle size. Since the pH value is extremely important for sensitive nipple tissue, the pH adjustment of the formulation was set to 6.8 by measuring the pH value of water first, then homogenized shea butter.

Studies have been conducted on the zeta potential and the molecular size with percutaneous absorption efficiency for the encapsulated elderberry seed structure, which is one of the main components of the developed invention. The zeta potential allows many important properties of colloidal systems to be understood, controlled, and the electrical charge or potential on the particles to be determined. The zeta potential is a measurement of the repulsion or attraction value between molecules. This measurement gives detailed information about the dispersion mechanisms and is the key to electrostatic dispersion control. A particle attracts ions of the opposite charge in suspension at a certain charge, as a result, a strong bond surface forms on the surface of the charged particle, and then a surface is formed that spreads outwards from the surface of the charged particle. There is a boundary called the "sliding surface" within this spread surface. The potential on this sliding surface is called the zeta potential and is affected both by the surface structure of the grain and the content of the liquid in which it is located. The behavior of molecules in liquids is determined by the zeta potential values, not by the electrical charge on their surfaces. For this purpose, zeta potential measurements of the encapsulated molecules were made at concentrations of ¹ /2, 1/3, 1/5, and 1/8 diluted with deionized water. The related results are given in Table 2.

Table 2

The zeta potential is related to the surface charge density and double layer thickness. The surface charge density depends on the concentration of potential determining ions. Since the H+ ion is the potential determining ion in many systems, the zeta potential depends on the pH. The zeta potential is positive for low pH values and negative for high pH values. Since the pH of the developed formulation is set to 6.8, a formulation with a negative value, 1/3 concentration and a nanometer size of 203 nm is preferred. Furthermore, the PI of this concentration is 0.11 , in other words, it shows a homogeneous distribution. Physicochemical analyses, microbiological activity tests and stability studies were performed by encapsulating elderberry seed oil and preserving the contents of phenolic substances consisting of acetic acid, tartaric acid and citric acid (Table 3). The invention was formulated by determining the non-cytotoxic dose of the obtained content.

Table 3

The antimicrobial results of the developed product are given in Table 4, Table 5 and Table 6 below. Table 4 refers to the amount of inoculated microorganisms, Table 5 refers to the neutralizing activity value, and Table 6 refers to the analysis results performed on the 7th, 14th and 28th days. Table 4 Table 5

Table 6 The analysis of the expression of COL1 A1 , HAS3, TGFB1 , FBLN 5, ELN, LOXL1 genes of the final formula and its effect on tissue regeneration in dermal fibroblast cells were investigated at the molecular level, and the Scratch assay test was also studied to visually show cell proliferation. Physical and chemical analyses, microbiological activity test and stability studies were performed. For this purpose, a formulation was made in this study to take advantage of the conditioning and soothing effect of elderberry seed oil, which contains rich fatty acids. Elderberry seed oil fatty acids are as follows:

C16:0 Palmitic acid 5.0 - 7.2 %

C18:0 Stearic acid 1 .0 - 3.0 % C18:1 (n-9) Oleic acid 12.4 - 19.0 % C18:2 (n-6) Linoleic acid 37.0 - 46.2 %

C18:3 (n-3) Linolenic acid 30.0 - 40.0 %

C20:0 Icosanoic acid 0.1 - 0.4 %

C22:0 Docosanoic 0.0 - 0.1 %

The medicinal potential of elderberry comes from its antioxidant potential, a property shared by numerous phytochemicals. The human body uses antioxidants from plant sources to neutralize harmful free radicals, and the total antioxidant capacity of elderberry is one of the highest of all small fruits.

Sambucus L. consists of 5-30 species and 6-11 subspecies depending on the taxonomic system. The most common species is Sambucus nigra L. Its common names are elderberry, black elderberry, European elderberry, European elderberry and European black elderberry. The chemical composition of Sambucus nigra is rich and depends on different factors such as variety, location, ripening stage and climatic conditions. In terms of carbohydrates, elderberry fruits contain 7.86-11 .50% total sugar. Elderberry fruit and flowers also contain essential oils (about 0.01% in the fruit).

Elderberry contains large amounts of bioactive compounds, especially polyphenols with high biological activity, such as ascorbic acid, flavonols, phenolic acids and anthocyanins; anthocyanins are also a source of the characteristic black-purple color of fruits. Elderberry is an extremely useful resource due to its high anthocyanin content, which is widely used as a coloring agent in various beverages and can also provide nutritional benefits. Anthocyanins and other flavonoids exhibit antioxidant, anticarcinogenic, immunostimulating, antibacterial, antiallergic and antiviral properties. It also has skin conditioning, soothing and revitalizing properties. In the invention, elderberry seed oil, which has a high antioxidant property as well as many beneficial effects, is used in formulas by encapsulating it with phosphatidylcholine.

Phosphatidylcholine (PC) is an industrially produced phospholipid-type phytosomes in the glycerophospholipid class. Phospholipid-type phytosomes are abundant in egg yolks and plant seeds. Currently, industrially produced phospholipids are available. Phospholipids can be divided into glycerophospholipids and sphingomyelins depending on their structure. In addition to phosphatidylcholine (PC), glycerophospholipids include phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylinositol (PI), and phosphatidylglycerol (PG). Phytosomes, on the other hand, are produced by the interaction of metabolites found in plant extracts with phosphatidylcholine. The formulations exhibit good solubility and bioavailability properties. Isolated compounds (e.g. silymarin, curcumin) and extracts (e.g., thistle, green tea, grape seed, ginkgo biloba) have been made complex in phytosomes, which exhibit improved properties compared to free forms.

PC, PE and PS are the main phospholipids used to prepare complexes consisting of a hydrophilic head group and two hydrophobic carbon chains. The benefits of PC include its amphipathic properties, which give it a moderate solubility in water and lipid media. In addition, PC is an important component of cell membranes and, accordingly, exhibits robust biocompatibility and low toxicity.

1. Determination of in vitro Cytotoxicity of Formulations by MTT Method

The in vitro cytotoxicity of the formulations of the subject of the invention was determined by the MTT method. This test is a sensitive method in which 3, [4,5- dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) tetrazolium salt is used to measure cell proliferation.

CRL-2076 human normal fibroblast cells were planted in 24-well plates with 1x10 ⁵ cells/well, and in 96-well plates with 1x10 ⁴ cells/well amounts. The cells were incubated for 24 hours. The samples were prepared in IMDM nutrient medium containing 10% fetal bovine serum (FBS) in a ratio of 1/16 (extract/medium, w/v). The samples were studied in 6 repetitions. As a result of the first in vitro cytotoxicity experiment, the appropriate dose range was determined. In the 2 ^nd experiment, the dose range determined on 24 well plates was studied in 4 repetitions. Only the nutrient medium was added to the control group cells. At the end of 24 hours, the nutrient media in the wells was removed and the nutrient medium containing the test sample was applied to each well. After incubation for 24 hours with the nutrient medium containing the test sample, the nutrient media was removed and the nutrient medium prepared with MTT (5mg/ml) was added to all wells and the plates were left to incubate for 3 hours. At the end of the period, the MTT solution in the wells was poured and DMSO was added to each well and the plates were incubated on the mixer for 15 minutes. The absorbance values of the plates were measured at 570 nm on the microplate reader and the % viability was calculated according to the absorbance values. 1.1. In vitro Cytotoxicity of Nipple Formulation

The results regarding the in vitro cytotoxicity of the developed formulation on the nipple are given in Figures 1 a and 1 b. Figure 1 c shows cell photos of the in vitro cytotoxicity experiment of the nipple formula (application of Nipple Elderberry formulation 0.8 and 3.12% dose). In the first experiment (shown in Figure 1 a), the viability was determined to be 66% in cells where a 3.12% dose of Nipple Elderberry formulation was applied, 73% at a 1 .56% dose, and 77% at a 0.8% dose. Concentrations of Elderberry below 0.8 and 0.8% were used here.

As a result of the second MTT experiment (shown in Figure 1 b) and microscopic observations, no cytotoxic effect was observed at all doses of nipple formula (0.2-0.8%).

2. Determination of Gene Expressions by RT-qPCR Analyses

The effect of Nipple formula on the expression of COL1A1 , HAS3, TGFB1 , FBLN 5, ELN and LOXL1 genes in human normal fibroblast cells was determined by RT-qPCR analysis. The studies performed for these analyses are described below.

2.1. Application of the Formulations

Human normal fibroblast cells were planted into T25 flasks in 5x10 ⁵ cells/flask amounts and incubated at 3713 for 24 hours in an environmen t containing 5% CO2. After 24 hours of incubation, the doses of each test sample determined according to the MTT results were administered in 5 ml nutrient medium (IMDM+10% FBS) and the cells were incubated at 3713 for 24 hours in an environment co ntaining 5% CO2.

The doses of the nipple formula were applied as 0.2 and 0.4%.

2.2. RNA Isolation

RNA isolation was performed according to the protocol recommended by the company. The amount and purity of the obtained RNA’s were determined by nanospectrophotometer. 2.3. cDNA synthesis

RNA samples were diluted to 100 ng'ml with RNase/DNase-free water. From the diluted RNA’s, cDNA synthesis was performed according to the procedure recommended by the cDNA producer company.

2.4. RT-qPCR Analysis

The reaction was carried out by providing the thermal cycle recommended by the SYBR green manufacturer in the RT-qPCR device. Fold change was determined by proportioning the expression levels of the target genes to the expression level of the reference gene (ACTB) and normalizing them according to the 2 ^-AACT method. [2]

2.4.1. Effect of Nipple Formulation on Gene Expression:

The effect of Nipple formula on the expression of COL1A1 , HAS3, TGFB1 , FBLN 5, ELN and LOXL1 genes is shown in Figure 2. Accordingly, the following increases were determined in the cells that the developed formula was applied compared to the control group cells;

• 2.5-fold increase in the expression of the COL1A1 gene at a dose of 0.2% and 2.8- fold increase at a dose of 0.4%,

• 2.6-fold increase in the expression of the TGFB1 gene at a dose of 0.2% and 3-fold increase at a dose of 0.4%,

• 1 .05-fold increase in the expression of the HAS3 gene at a dose of 0.2% and 1 .2- fold increase at a dose of 0.4%,

• 1 .9-fold increase in the expression of the ELN gene at a dose of 0.2% and 2-fold increase at a dose of 0.4%,

• 1 .4-fold increase in the expression of the FBN5 gene at a dose of 0.2% and 1 .5-fold increase at a dose of 0.4%,

• In the expression of the LOXL1 gene, 1.2-fold expression at a dose of 0.2% and 1 .5-fold expression at a dose of 0.4%. The evaluation of the effect of Nipple formula on the expression of COL1A1 , HAS3, TGFB1 , FBLN 5, ELN and LOXL1 genes (compared to control group cells) is given in Table 3.

Table 3

5

3. Determination of the Effects of the Nipple Formulation on Cell Migration by Scratch Method

The Scratch method is an in vitro test that allows studying of the migration of cells in the two-dimensional cell layer created by the wound. Human normal fibroblast cells (CRL- 10 2076) added to the plate wells with nutrient medium were incubated for 24 hours. In the cell layer that became confluent after incubation, plus-shaped wound areas were formed with a 200 microliter pipette tip. Only nutrient media or nutrient media containing different concentrations of substances were added to the cells that created a wound area, and the cells were left to incubate. The cells were imaged and photographed with 15 an inverted microscope in 8-18 hour intervals. The Scracth experiment microscope image of the Nipple formula is given in Figure 3. The migration of cells to the wound area created in the culture area was imaged and photographed through a microscope. When comparing the cells in the control group with the cells applied with the Nipple formula, it has been observed that cell migration increases occurred in all applications at the 14 ^th hour, depending on the dose.

With the emulsion formula containing the developed encapsulated phyto-phospholipids, a solution has been obtained that reduces nipple pain/soreness in particular, and this encapsulated content is suitable for use in many cosmetic formulas. With the product that is the subject of the invention, the difficulties experienced regarding the direct inclusion of plant extracts or oils in various products have been eliminated. In particular, plant bioactive components, which are known to be prone to degradation, can be used without deterioration in the presence of factors such as light, temperature, oxygen, pH, and also without degradation in their processing and storage stability. In addition, the problems related to the poor water solubility of hydrophobic compounds, their dissolution and absorption have also been overcome.