WO2018236304A2

WO2018236304A2 - A plant based raw material effective against skin blemishes and a method for its production

Info

Publication number: WO2018236304A2
Application number: PCT/TR2017/050520
Authority: WO
Inventors: Yildiz BODURLAR
Original assignee: Actv Biyoteknoloji Laboratuvar Sanayi Ve Ticaret Anonim Sirketi
Priority date: 2017-06-09
Filing date: 2017-10-24
Publication date: 2018-12-27
Also published as: WO2018236304A3

Abstract

The invention relates to a raw material for cosmetic products, with skin whitening and blemish removal effect, which contains a metabolite complex with antioxidant and anti- tyrosinase activity and obtained from plant cells using elicitors, and a method for its production

Description

A PLANT BASED RAW MATERIAL EFFECTIVE AGAINST SKIN BLEMISHES AND

A METHOD FOR ITS PRODUCTION

TECHNICAL FIELD

The invention relates to a raw material obtained from plant cells, with skin whitening and skin blemish removing effects, intended to be used in cosmetics industry, and to a method for production of the mentioned raw material.

The invention comprises a raw material for cosmetic products, which contains a metabolite complex with antioxidant and anti-tyrosinase activity and obtained from plant cells using elicitors, and a method for its production. THE PRIOR ART

Existence of skin color variations all over the earth depend upon genetic factors, skin pigments, exposure to ultraviolet (UV) rays and environmental factors. Furthermore, melanin, which is present in epidermis, is acknowledged to be the main pigment affecting the skin color, and the factors such as the abundance of melanocytes that enable melanin synthesis, melanogenic activity, melanin type, and size, abundance and distribution of melanosomes are considered to be the factors that determine the skin color.

Two different forms of melanin, namely pheomelanin (yellow/red) and eumelanin (brown/black), are considered to be the main reason upon which the skin color variations over the earth depend. Melanin is synthesized at the melanosome, which is an organelle similar to lysosome. Melanogenesis, which indicate melanin production, is regulated by the activity of three different enzymes named tyrosinase, Tyrosinase- Related Protein 1 (TRP1 ) and Tyrosinase-Related Protein 2 (TRP2). Tyrosinase the main determining enzyme in melanogenesis.

Melanin plays an important role in providing protection against skin damages. However, excess melanin accumulated in different skin regions result in development of apparent pigmented areas which constitute an esthetic problem. Abnormal melanin production and excessive hyperpigmentation due to exposure to excessive ultraviolet radiation may cause skin problems such as inflammation, age spots, melasma and freckles by excessive production of reactive species.

Skin whitening raw materials have become the most important component of cosmetic and hygiene products in the recent years. Therefore it is important that skin whitening raw materials especially possess hypopigmentation activity (anti-melanogenic activity). Numerous researchers and research institutions are engaged in studies to find an effective and safe anti-melanogenic and/or safe skin whitening raw materials. Skin whitening raw materials often function by inhibiting the melanogenesis and therefore called as anti-melanogenic agents.

In general, skin whitening products are preparations that provide depigmentation effect on the skin. Removal of hyperpigmentation induced skin blemishes is achieved by means of reducing the abundance of melanin in the skin and therefore, active components are applied to achieve the following actions:

i. inhibition of tyrosinase enzyme (inhibiting or retarding the catalytic action of tyrosinase using the skin whitening agent),

ii. inhibiting tyrosinase release,

iii. inhibiting intermediate steps/products in melanin synthesis,

iv. blocking transport of melanosomes to keratinocytes,

v. destruction of the available melanin in the skin, or

vi. destruction of melanocytes.

Combined approaches towards multiple activities can be applied to achieve increased effectiveness. Hydroquinone derivatives are used worldwide for skin whitening and removal of blemishes. A 2% hydroquinone used to be clinically considered as standard. However, FDA prohibited this drug in 2006 due to associated skin cancer risk. Arbutin, a glycoside attached derivative of hydroquinone, have been developed as a hydroquinone substitute. However, arbutin had started pull out of the market due to its low activity. As new skin lightening agents, gallic acid, which is obtained from gallnut, and kojic acid, which is obtained from Japanese rice wine, is now commercially available on the market. Use of kojic acid have also been prohibited in some countries such as Switzerland due to potential risks of developing liver and thyroid tumor. Therefore, there is need for a reliable skin whitening raw material with high activity and no associated health risks. Today, importance of natural sources such as polyphenols in meeting user demands are increasing as safe and effective raw materials.

Patens regarding the use of active agents obtained by plant extraction in relation to natural raw material production is summarized below:

Patent document no. US2016037491 1 relates to a cosmetic skin whitening product obtained from plant extracts containing curcumin and resveratrol.

Patent document no. W0120171014597 is a cosmetic formulation containing Schisandra chinensis seed extracts, with melanin generation reducing effect.

KR2017000291 1 (A) relates to a cosmetic composition which contains Caragana sinica extract with melanin synthesis reducing effect. KR20160147487 (A) relates to a cosmetic preparation which contains Vinca minor extract with melanin synthesis inhibiting effect.

TW200637606 (A) relates to a skin whitening product containing Pilea cavaleriei extract.

KR20160054164 (A) relates to a skin whitening product containing moringa extract.

KR20160091301 (A) relates to a pigmentation removing product containing Brassocattleya marcella koss extract.

Lack of sufficient activity, stability problems and restrictions regarding plant cultivation has led the cosmetics industry to seek new active agents and production methods. Plant based extracts have been used for some time in cosmetic products for skin whitening, anti-aging, anti-photoaging protective effects. Patents related to use of plant cells as cosmetic raw material is summarized below:

WO20161 13659 (A1 ) relates to useing Leontopodium alpinium plant cells as anti-aging agents. Patent applications WO 2009/151302 and KR 2009-01 18877 relate to anti-aging and antioxidant compositions obtained from undifferentiated plant cells of ginseng cambium and Taxus genus plants. Patent application EP 1985280 discloses the use of undifferentiated plant cells of Rosaceae family plants, especially Malus domestica apple, to provide protection against internal and external stressors for skin stem cells and to treat skin and hairs which demonstrate aging effects. Patent application EP 1699423 discloses use of freeze dried, salt resistant, undifferentiated plant based cells in rejuvenating the skin appearance.

DE-A-102 009 027 361 and EP 2,266,529 discloses use of undifferentiated plant cells of especially Malus genus plants in providing protection and treatment especially against UV radiation and treatment of hair keratin fibers.

Patent application EP 0909556 discloses use of undifferentiated cells of a plant from Roscaeae family in treatment of various conditions including skin conditions as a bradykinin antagonist.

Patent application WO201 1 121051 (A2) discloses use of undifferentiated cells of argan plant in treatment of skin aging, inflammation and wound healing.

Patent application EP2436759 discloses the undifferentiated cells cambia of Solanaceae family and the anti-aging activity they exhibit.

Patent application EP2436758 discloses the undifferentiated cambium cells of Ginkgoaceae family and the enhanced anti-oxidant activity they exhibit. Patent application EP2436757 discloses the undifferentiated cambium cells of Asteraceae family and the enhanced anti-inflammatory effect they exhibit, while W02010137879 discloses the undifferentiated cambium cells of Salicaceae family and the enhanced anti-inflammatory activity they exhibit. EP1064932 (A1 ) relates to use of undifferentiated cells for anti-odor purposes. Patent application EP1244464 discloses use of cell extracts of undifferentiated cells of plants from Leontopodium genus as ultraviolet filtering agents. Other purposes of use or plants are neglected. Patent application FR2837385 relates to production of phytoalexins and use of undifferentiated plant cells in cosmetic compositions.

Patent application EP 1 174120 relates to use of freeze dried extracts of undifferentiated cells of Iridaceae family to stimulate the immune function of skin.

Patent application WO 2005/072697 relates to use of protective and regenerating properties of freeze dried undifferentiated plant cells as cosmetic or pharmaceutical components for skin pigmentation removal and/or whitening purposes. However, it is not related to use of plant cells containing a metabolite complex with anti-tyrosinase activity, obtained through elicitor applications, in contrast to our patent proposal.

Therefore the abovementioned disadvantages and lack of adequate solutions in the background art have made it necessary to make an innovation and a development in the technical field concerned.

OBJECT OF THE INVENTION

The invention, inspired by the current state of the art, is developed to overcome the abovementioned disadvantages. The main object of the invention is to provide a plant based raw material for cosmetic products, effective in removal of skin blemishes and skin whitening, with anti-oxidant and anti-tyrosinase activity.

Another object of the invention is to contain all of the secondary metabolites of the plant cells, in contrast to the products that are currently available on the market, which contain only one active agent. Therefore, blemishes and tone differences due to excess melanin deposits on the skin is removed by the synergic effect of all of the components in the raw material composition. An object of the invention is to provide a raw material for cosmetic products which does not contain potentially harmful ingredients such as hydroquinone, hydroquinone derivatives or kojic acid. Instead of these substances, a raw material which contains a composition of 6,7,4 trihydroxyisoflavone, 7,3,4 trihydroxyisoflavone, 7,8,4 trihydroxyisoflavone, glyceollin, p-coumaric acid, ferulic acid, naringin, hesperidin, genistein, daidzein, daidzin, glycitein metabolites, whose benefits have been proven scientifically. Skin whitening and blemish removal effect is provided to the skin by the abovementioned metabolite composition. The invention is a raw material for cosmetic products to achieve the aforementioned objects, with skin blemish removal and/or skin whitening effects, intended to be used in cosmetic products, wherein; it comprises plant cell homogenate and a secondary metabolite complex with anti-tyrosinase activity. The invention is a method for production of a raw material for cosmetic products to achieve the aforementioned objects, comprising; performing sterilization of the plant, obtaining in vitro plants, achieving callus production in in vitro plants, and obtaining Callus suspension culture process steps, wherein; it comprises the following steps:

• Application of an elicitor, comprising incubation steps of the mentioned cell suspensions with abscisic acid between 20-500 μΜ and methyl jasmonate between 20-500 μΜ

• Lysis step of the cells which have received the elicitor application.

The structural and characteristic properties of the invention will become apparent with reference to the detailed description and the accompanying figures, therefore, assessment should be made based on these figures and the detailed description.

FIGURES THAT WILL HELP UNDERSTANDING THE INVENTION

Figure 1 Shows the total phenolic content of the extract obtained from the Soybean cotyledon cells.

Figure 2 Shows the cytotoxicity of the Soybean cotyledon cell extract on human fibroblast cells.

Figure 3 Shows DPPH scavenging activity of the extract obtained from Soybean cotyledon cells.

Figure 4 Shows the effect of the extract obtained from Soybean cotyledon cells on the amount of melanin synthesis. Figure 5 Shows the tyrosinase inhibition activity of the extract obtained from Soybean cotyledon cells.

DETAILED DESCRIPTION OF THE INVENTION

The detailed description herein provides preferred embodiments of the invention only for exemplary purposes and without imposing limitations in any respect.

The inventions describes a raw material obtained from soybean cotyledon cells, with skin whitening and blemish removal properties, and a method for production of the mentioned raw material. It is obtained from the cotyledon tissue of the mentioned soybean (Glycine max) plant.

The invention employs a method based on elicitor application in order to increase the abundance of 6,7,4 trihydroxyisoflavone, 7,3,4 trihydroxyisoflavone, 7,8,4 trihydroxyisoflavone, glyceollin, p-coumaric acid, ferulic acid, naringin, hesperidin, genistein, daidzein, daidzin, glycitein metabolites, which are produced by soybean cells and have tyrosinase inhibition activity. In the method, exerting stress on the cells using elicitors, resulting in a higher than normal secondary metabolite production. Soybean cotyledon cells with enriched secondary metabolites, obtained through this method are able to mitigate the skin blemishes and remove the tone differences in skin color. In addition to the soybean (Glycine max), the cells from Cydonia oblonga, Linum tauricum, Paeonia turcica, Muscari massayanum, Muscari armeniacum, Rosmarinus officinalis, Allium akaka, Allium tuncelianum, Citrus aurantium, Citrus limon, Crocus specious subsp xhantolaimos, Lathyrus undulates, Galanthus plicatus subsp. Byzantinus, Crocus olivieri subsp. Istanbulensis, Cochicum chalcedonicum, Hypericum avicularifolium subsp., Santalum album, Actinidia deliciosa, Glycine max, Viola tricolor, Diospyros kaki, Vaccinium myrtillus, Rosa canina, Rosa damascena, Oryza sativa, Glycyrrhiza glabra, Salvia anatolica, Fritillaria alburyana, Tulipa karamanica can also be used in the method of the invention.

Skin whitening and blemish removal composition of the invention comprises plant cell homogenate, secondary metabolite complex with anti-tyrosinase activity, glycerol and xanthan gum. Preferred amount by weight Usable amount by weight

Raw material content

(%) (%)

Plant cell homogenate 19,9 10-40

Secondary metabolite complex with

0,01 0,001 -0,1 anti-tyrosinase activity

Glycerol 79,98 50-90

Xanthan gum 0,02 0,01 -0,05

Table 1 : Contents and usage ratios of the raw materia of the invention

Plant cell homogenate:

When plant tissues are damaged mechanically, they form cell accumulations known as callus via meristematic action mechanism. Callus cells exhibit totipotency, which is the ability to give rise to any cell or organ of the plant or even forming a new plant. Plant cell culture method allows controlled growth of totipotent plant cells under sterile laboratory conditions and to perform various applications in order to obtain cell specific secondary metabolites. Preferably, soybean cotyledon is used as the plant cell homogenate of the invention. In vitro soybean seeds are sterilized in order to obtain in vitro soybean plants. Cell suspensions are created from the callus obtained from the cotyledon (embryonic leaf) explants of the in vitro soybean plants. Soybean cotyledon cell suspensions are harvested after elicitor application and homogenized under pressure. The obtained homogenate constitutes the most important part of the invention.

Secondary Metabolite Complex:

In order to obtain metabolites with anti-tyrosinase activity, elicitor application is performed after the plant cell suspensions reach the desired volume, as discussed. The obtained metabolite complex with anti-tyrosinase activity contains 6,7,4 trihydroxyisoflavone, 7,3,4 trihydroxyisoflavone, 7,8,4 trihydroxyisoflavone, glyceollin, p-coumaric acid, ferulic acid, naringin, hesperidin, genistein, daidzein, daidzin, glycitein metabolites. Natural phenolics consist of 6 main groups called phenolic acid, flavonoids, stilbenes, coumarins, lignans and tannins. Phenolic acids comprise hydroxybenzoic acis and hydroxycynnamic acids; flavonoids comprise flavonols, flavonons, flavons, flavanons (catetchin), antocyanides and isoflovonoids; and tannins comprise hydrolysable tannins and condensate tannins.

Various reports have been published on the potential effects of the phenolic compounds on preventing skin disorders or relieving symptoms of skin disorders and accelerating the healing. Numerous activities of phenolics on the skin such as cell regeneration, stimulating collagen and elastin synthesis, burn and wound healing, protecting against UV radiation and decreasing melanin production, in addition to antioxidant, anti-inflammatory and antimicrobial activities have been reported by studies.

It has been reported that 7,8,4 trihydroxyisoflavone and 7,3,4 trihydroxyisoflavone with 1 1 ,2 μΜ and 5,23 μΜ doses respectively inhibits tyrosinase with 50%. Furthermore, the study have found that respective doses of 7,8,4 trihydroxyisoflavone, 7,3,4 trihydroxyisoflavone and genistein components that inhibits the 50% of the cellular melanin formation are 12.23 μΜ, 7.83 μΜ and 57.83 μΜ, and that these components have more effective melanin suppressing activities in comparison to that of arbutin.

6,7,4 trihydroxyisoflavone, a metabolite found in soy, have shown to exhibit 6 times more inhibition compared to the kojic acid.

Glyceollin, a fitoalexin produced in soybean under stress conditions, have been reported to inhibit melanin biosynthesis and tyrosinase activity via cAMP suppression in B16 cells. Genistein is a primary isoflavon and known to accelerate antioxidant enzyme activities such as superoxide dismutase, catalase and glutathione reductase in various mice organs. Moreover, genistein has been reported to inhibit tyrosin kinase and topoisomerase.

Among soybean isoflavonoids, daidzein, glicytein and daidzin have been reported to exhibit anti-tyrosinase activity with 203, 218 and 267 μΜ tyrosinase inhibition IC₅o values respectively.

Studies comparing tyrosinase inhibition activity of p-coumaric acid with that of kojic acid and arbutin have shown that it is a strong inhibitor. It has also been reported that its melanogenesis preventive effect is stronger than molecules which it resembles structurally, such as 3- (4-hydroxyphenyl) propionic acid, cinnamic acid and caffeic acid, p-coumaric acid acts by competing with tyrosine for active regions on the tyrosinase enzyme, due to its chemical similarity with tyrosine and DOPA.

Hesperidin has been shown by conducted studies to suppress melanogenesis via Erk1 /2 mediated MITF degradation.

IC₅o value of naringin regarding anti-tyrosinase activity has been shown to be 1 ,9 mM. Ferulic acid is a phenolic acid naturally available in plants. Due to its chemical structure similar to that of tyrosine, it has been shown to exhibit tyrosinase activity and inhibit melanin synthesis by this way.

As stated above, soybean cotyledon cells containing secondary metabolites are produced in the invention. The total amount of all of the metabolites (6,7,4 trihydroxyisoflavone, 7,3,4 trihydroxyisoflavone, 7,8,4 trihydroxyisoflavone, glyceollin, p-coumaric acid, ferulic acid, naringin, hesperidin, genistein, daidzein, daidzin, glycitein metabolites) available in the metabolite complex is at least 0.01 % of the amount of plant cells (dry weight).

In the production method of the invention for a raw material for cosmetic products, plants are sterilized and in vitro reproduced, and then the plant is wounded using forceps and scalpel to cause callus stimulation. An elicitor is applied to the suspension culture comprising the obtained callus and the cells are lysed using a homogenizer. Following the addition of glycerol and xanthan gum to the lysed cells and metabolites, the resulting mixture is processed at a homogenizer and packed inside light-proof packings.

The method for production of the raw material of the invention for cosmetic products is described below in detail: In order to ensure rapid and successful callus growth, soybean plants are reproduced using tissue culture method after being transferred to an in vitro environment. For sterilization, after shaking soybean seed in 70% alcohol for 1 minute, they are left in a 10% sodium hypochlorite solution, containing 2 droplets of Tween-20 (detergent), for 10 minutes and rinsed 3 times with distilled water and dried. Sterile seeds are incubated in a dark environment inside jars, each containing 75 ml MS nutrient medium (pH: 5.8) with 30 g/l sugar and 7 g/l agar addition. After germination of the seeds, they are incubated under 25±2Ό temperature inside a cli mate chamber with photoperiodicity set to 16 hours light and 8 hours dark, and they are transferred to subculture using hormone-free MS medium for 3-4 week intervals. Therefore, in vitro soybean plants are obtained.

Contents of the mentioned MS nutrient medium is as followings:

Macro Elements (mg/L)

MgS0₄.7H₂0: 370

KH₂P0₄: 170

KN0₃: 1900

CaCI₂.2H₂0: 440

Micro Elements (mg/L)

H₃B03: 6.2

MnS0₄.H₂0: 15.6

ZnS0₄.7H₂0: 8.6

Na₂Mo0₄.2H₂0: 0.25

CuS0₄.5H₂0: 0.025

CoCI₂.6H₂0: 0.025

Kl: 0.83

FeS0₄.7H₂0: 27.8

Na₂EDTA: 37.3

Sucrose: % 3

Gelrite: % 0,4

In order to obtain callus, cotyledon explants of the in vitro soybean plant is wounded by a scalpel and transferred to a culture in MS medium containing 2,4 D (2mg/l) + NAA (2mg/l) + KIN (0.5mg/l). Explants transferred to the culture are kept in a dark environment at 25±2 Ό until callus growth is obser ved, while the explants are transferred to a fresh medium once in every 4 weeks. Once the transparent and fragile calluses reach a sufficient amount, they begin the suspension forming phase. Liquid form of the nutrient medium adjusted for the callus formation is used for cell suspension culture. In order to cell suspension, 2.5-3 g of callus of 40-45 days is transferred into a 250 ml flask containing 50 ml liquid nutrient medium and incubated at 25±2 Ό at 1 10 rpm in dark until a cell suspension containing separated single cells or groups of several cells are obtained. After about 4 weeks, the suspension is screened through a sterile filter (30 mesh) in order to remove larger callus masses. Screened suspension is inoculated with a ratio of 1 :4 by adding fresh nutrient medium and transferred to subcultures after being split into flasks and then incubation is continued at 1 10 rpm. Growth parameters (cell count, wet weight, dry weight, pellet cell volume (PCV), settled cell volume (SCV), cell vitality) of the cells inside the suspension is determined on a weekly basis.

Based on the cell vitality graph, cell suspension is transferred to a subculture at certain periods and the amount of suspension is increased. When the volume of soybean cotyledon cells reproduced inside flasks is 1 liter, volume of the suspension is increased to 2 liters by adding a nutrient medium with 1 :1 inoculation ratio and then transferred to a wave-shaking bioreactor with 20 liters maximum operating capacity. Cells are supplied with fresh nutrient media inside the bioreactor at 1 week periods and transferred to subculture and then the suspension volume is increased to 10 liters. Once the suspension is obtained, application of abiotic elicitor is required in order to produce secondary metabolites with anti-tyrosinase and anti-melanogenic activity in soybean cotyledon cells. Accordingly, once the soybean cotyledon cells reaches the desired volume after 10 days following the last subculture, abscisic acid added with the help of a medium transferring unit until a final concentration of 200 μηι achieved and the cells are left for incubation for 24 hours. At the end of the 24 hours, methyl jasmonate is added until a final concentration of 250 μηι is achieved and then the cells are left for an additional incubation for 7 days. After the incubation, cells inside the reactor are screened through a filter inside the cabin in order to remove the nutrient medium. The nutrient medium is removed by washing the cells on the filter. By following these processes, soybean cotyledon cells containing a 0.01 % metabolite complex (6,7,4 trihydroxyisoflavone, 7,3,4 trihydroxyisoflavone, 7,8,4 trihydroxyisoflavone, glyceollin, p-coumaric acid, ferulic acid, naringin, hesperidin, genistein, daidzein, daidzin, glycitein metabolites) with anti-tyrosinase activity is obtained. At the end of the screening process, a mixture containing 19.99% weight soybean cotyledon cells, 0.01 % secondary metabolite complex with anti-tyrosinase activity, 79.98% glycerol, and 0.02% xanthan gum is transferred to a high pressure homogenizer. The mixture is homogenized under 2000 bars pressure and packed inside 1 liter packs.

In vitro safety and activity tests

Obtaining extract from the soybean cotyledon cell homogenate containing a secondary metabolite complex with anti-tyrosinase activity:

In order to determine the biological activity of the soybean cotyledon cells containing a secondary metabolite complex with anti-tyrosinase activity, the cells were homogenized and freeze dried. Freeze dried soybean cotyledon cells were extracted in 10% ethanol for 48 hours under room temperature until 1 mg/ml concentration was reached and then, they were centrifuged at 14000 rpm for 10 minutes. After the centrifuge, supernatant was sterilized by screening through a 0.2 micrometer filter. Obtained soybean cotyledon cell extract was used in in vitro safety and activity tests. Biological activities of the soybean cotyledon cell extract is determined by analyzing in vitro cytotoxicity, antioxidant activity, total phenolic content, secondary metabolite analysis by LC-MS/MS analysis, effect on melanin production and tyrosinase inhibiting activity.

Determining the amount of total phenolic substance:

After harvesting, soybean cells, which had been washed with distilled water, were lysed under high pressure (2000 bars) and freeze dried. Freeze dried soybean cells were extracted inside 80% methanol for 48 hours at 25 ^eC. Following the incubation, cells inside methanol were centrifuged at 14000 g and used in determination of supernatant total phenolic substance and LC-MS/MS analysis. Amount of total phenolic substance was determined using Folin Ciocalteu method.

Gallic acid is used as the standard. The method is based on the principle of transferring electrons from phenolic components to phosphomolybdic/phosphoungustic acid complexes in alkali media. Formation of a blue colored complex can be identified at 765 nm spectrophotometrically. Result of the total phenolic content test of the extract is given in Figure 1 . In Figure 1 , Total phenolic content of the extract obtained from soybean cotyledon cells is given. Determination of phenolic substance content using LC-MS/MS analysis:

Using LC-MS/MS analysis, amounts of 6,7,4 trihydroxyisoflavone, 7,3,4 trihydroxyisoflavone, 7,8,4 trihydroxyisoflavone, glyceollin, p-coumaric acid, ferulic acid, naringin, hesperidin, genistein, daidzein, daidzin, glycitein metabolites inside the extract was determined. It has been found that the total amount of all of the metabolites inside the metabolite complex is more than 0.01 %. Amount of secondary metabolites with anti-tyrosinase activity is given in Table 2.

Table 2. Secondary metabolites contained in the soybean cotyledon cell extract and their amounts.

Analysis of the effect of the extract on cell proliferation:

Human fibroblast cells were added to 96 well plates with 8x10⁴ cell/well ratio. After 24 hour incubation, nutrient media inside the wells have been removed and replaced with nutrient medium suspensions which contain certain doses of the extract. After incubating the cells with the extracts for 24±0.5 hours, extract-nutrient solution was removed from the wells and an MTT-nutrient suspension with 5 mg/mL final concentration have been added to the wells and plates have been left for incubation for 3±0.1 hours. At the end of the duration, MTT-nutrient solution was removed from the plate and DMSO was added to each well to allow formazan salts to be dissolved. Plates are left at the plate shaker for 15 minutes. At the end, OD (optical density) value of the plates have been read at 570 nm. Using the numerical data obtained by the reading, by comparing the viability of the cells, which had been exposed to various doses of the substance, with the control cells, it was found weather a dose is toxic or proliferative.

In order to make the experimental setup blind, it was designed to include wells that only contain nutrient medium, wells with control cells that haven't been exposed to any substance and wells with cells that have been exposed to different deses of the substance. No toxicity of the extract with the tested doses on the fibroblast cells have been observed. Figure 2 shows the cytotoxicity of the Soybean cotyledon cell extract on human fibroblast cells. Determining antioxidant activities of the extract:

Antioxidant activities of the extract have been determined using DPPH scavenging activity method. The method is based on a reaction of proton transfer from the antioxidant to DPPH free radical. Antioxidant scavenging activity can be determined by measuring the loss of absorbance at 517 nm. Antioxidant activity of the extract, which was determined based on % DPPH scavenging activity, is given in Figure 3.

Impact of the extract on melanin amount:

B16-F10 cells have been left for incubation for 24 hours after they had been planted to culture plate in order to measure melanin amount. At the end of this duration, the cells have been incubated for 24 hours with a-msh (100 nM). Later on, after addition of soybean cotyledon cell extract/kojic acid, or cell culture only, the cells have been left for incubation for 24 hours. At the end of the duration, cell pellet was dissolved inside NaOH with 1 % DMSO and absorbance is measured at 475 nm. The impact of the extract on melanin amount of B16-F10 cells is given in Figure 4. Extract have decreased melanin formation in melanoma cells with a higher ratio compared to kojic acid. Impact of the extract, obtained from soybean cotyledon cells, on melanin amount is given in Figure 4. Tyrosinase suppressing activity of the extract

Anti-tyrosinase (skin whitening) activity is analyzed by measuring the suppression of dopachrome formation. For this purpose, B16-F10 cells (melanoma cells), after being placed on culture plates, have been left for incubation for 24 hours. At the end of the duration, the cells have been incubated for 24 hours with a-msh (100 nM). Later on, after addition of extract/kojic acid, or culture medium only, the cells have been left for incubation for 24 hours. After washing, cell pellet is lysed using lysis tampon. Supernatant obtained from the lysate is left for incubation with L-DOPA at 37 ^eC for 30 minutes. After the incubation, absorbance of the microplate have been measured at 475 nm. Dopachrome amount was calculated after the measurement. Anti-tyrosinase activity of the extract is given in Figure 5. The extract has been found to exhibit higher anti-tyrosinase activity compared to the kojic acid. Tyrosinase inhibition activity of the extract, obtained from soybean cotyledon cells, is given in Figure 5.

Claims

1. A raw material for cosmetic products, with blemish removal and/or skin whitening effects, intended to be used in cosmetic products, wherein; it comprises plant cell homogenate and a secondary metabolite complex with anti-tyrosinase activity.

2. A raw material for cosmetic products according to claim 1 , wherein; the mentioned plant comprises an individual or a combination among Glycine max, Cydonia oblonga, Linum tauricum, Paeonia turcica, Muscari massayanum, Muscari armeniacum, Raphanus sativus, Rosmarinus officinalis, Allium akaka, Allium tuncelianum, Citrus aurantium, Citrus limon, Crocus specious subsp xhantolaimos, Lathyrus undulates, Galanthus plicatus subsp. Byzantinus, Crocus olivieri subsp. Istanbulensis, Cochicum chalcedonicum, Hypericum avicularifolium subsp., Santalum album, Actinidia deliciosa, Glycine max, Viola tricolor, Diospyros kaki, Vaccinium myrtillus, Rosa canina, Rosa damascene, Oryza sativa, Glycyrrhiza glabra, Salvia anatolica, Fritillaria alburyana, Tulipa karamanica.

3. A raw material for cosmetic products according to claim 1 , wherein; it comprises 6,7,4 trihydroxyisoflavone, 7,3,4 trihydroxyisoflavone, 7,8,4 trihydroxyisoflavone, glyceollin, p-coumaric acid, ferulic acid, naringin, hesperidin, genistein, daidzein, daidzin, glycitein metabolites as the mentioned secondary metabolite.

4. A raw material for cosmetic products according to claim 1 or 3, wherein; it comprises an amount of metabolite complex which is at least 0.01 % of the dry weight of the amount of plant cells.

5. A method for production of the raw material according to claim 1 , comprising:

a. Sterilization of the plant,

b. Obtaining in vitro plants,

c. Achieving callus production in in vitro plants,

d. Obtaining callus suspension culture process steps, wherein; it comprises • Application of an elicitor, comprising incubation steps of the mentioned cell suspensions with abscisic acid between 20-500 μΜ and methyl jasmonate between 20-500 μΜ

• Lysis step of the cells which have received the elicitor application process steps.

A method for production of a raw material according one of the previous claims, wherein; it comprises a process step where the cells are lysed with a homogenizer.

A method for production of a raw material according one of the previous claims, wherein; it comprises a process step where glycerol is added to lysed cells and target metabolites.

A method for production of a raw material according one of the previous claims, wherein; it comprises a process step where xanthan gum is added to lysed cells and target metabolites.

A method for production of a raw material according one of the previous claims, wherein; it comprises a process step where the mixture comprising lysed cells, metabolites, glycerol and xanthan gum is processed with a homogenizer.