WO2020180265A1

WO2020180265A1 - Plant-based anti-aging composition and its production method

Info

Publication number: WO2020180265A1
Application number: PCT/TR2019/050278
Authority: WO
Inventors: Yıldız BODURLAR
Original assignee: Actv Bi̇yoteknoloji̇ Laboratuvar Sanayi̇ Ve Ti̇caret Anoni̇m Şi̇rketi̇
Priority date: 2019-03-06
Filing date: 2019-04-25
Publication date: 2020-09-10
Also published as: TR201903440A2

Abstract

The invention relates to a plant-based formulation to be used in the dermo-cosmetic industry, containing a cell stack having a phenolic compound complex as a result of MeJa application and a plant cell stack containing an increased mucilage content that is obtained increasing the expression of the MUM4 gene. Within the scope of the invention, individuals or combinations of Cydonia oblonga, Malva sylvestris, Linum tauricum, Paeonia turcica, Amygdalus communis, Persea americano, Muscari massayanum, Muscari armeniacum, Rosmarinus officinalis, Corylus avellane, Hypericum perforatum, Ocimum basilicum, Allium akaka, Allium tuncelianum, Aleo vera, Citrus aurantium, Citrus limon, Crocus specious subsp xhantolaimos, Lathyrus undulates, Galanthus plicatus subsp. byzantinus, Crocus olivieri subsp. istanbulensis, Cochicum chalcedonicum, Hypericum avicularifolium, 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 karamani may also be used as a plant.

Description

PLANT-BASED ANTI-AGING COMPOSITION AND ITS PRODUCTION METHOD

Technical Field

The invention relates to a plant-based composition obtained from plant cells having anti-aging, moisturizing and skin elasticity enhancing effect for use in the dermo-cosmetics industry and the method of production of the said plant-based composition.

The invention relates in particular to a herbal composition comprising a stack of quince cells with a complex of phenolic compounds having an anti-aging activity and a stack of quince cells with an increased mucilage content for use in the dermo-cosmetics industry, and to a method of production of the said composition.

Prior Art

Today, the phenomenon of skin aging is one of the issues that dermatological research and studies are most interested in. Skin aging is a complex biological process that occurs as a result of the combination of two different biological processes. The said processes are intrinsic aging or chronological aging, which is the skin aging due to time (such as genetic factors, hormones, cell metabolism) and extrinsic aging of the skin which is caused by environmental causes (such as radiation, chemicals, pollution). Along with aging, effects such as the decrease in skin elasticity, loss of skin tension, wrinkles and sagging are observed. Skin aging is mainly associated with a decrease in the level of dermal collagen, a component of the dermis layer. The collagen constitutes approximately 70% of the total skin weight and plays a very important role in the integrity of the skin structure. For this reason, the formation of new collagen fibers is of high importance for firm and healthy skin. Type 1 and Type 3 collagens respectively form 80% and 15% of the total collagen in the skin. Type 1 collagen is the main structural component of the extracellular matrix, which has an important function in maintaining the structure of the skin dermis. It is known that Type 1 and Type 3 collagen is found in the young skin in higher amounts compared to the aged skin and decreases significantly with age.

In the process of aging, in addition to decreasing collagen levels, collagen fibrils begin to cross link, mostly due to external effects such as exposure to UVA radiation. As a result of the cross- linking of collagen fibrils, the skin loses elasticity. While the amount of collagen that is cross- linked, insoluble in the elderly skin is high, the younger, healthier and firmer skin contains a higher level of newly formed, soluble collagen. Synthetic substances in the content of the products used to prevent skin aging and wrinkle can cause various side effects such as skin contact dermatitis, allergy, and irritation, and consequently, this becomes effective in the consumer's preference for these products less. Recently, the conscious consumer who wants to avoid these side effects is looking for cosmetic products with natural contents, so that there is a necessity to develop products of natural ingredients instead of synthetic substances. Since they are natural, instead of synthetic substance-based products, plant extracts which are rich in phytochemicals such as flavonoids, phenolic acid, saponin, and alkaloids, which have activity stimulating collagen synthesis, are widespread in anti-aging topical cosmetics. However, limited availability of plants due to seasonal reasons, limited inventory, problems in planting and plant protection, inefficient harvesting, global climate change, geographical differences, clone types, and pollution are important problems in the use of vegetable raw materials in the contents of dermo-cosmetic products.

On the other hand, in recent years, microRNA (miRNA)-based RNAi mechanism began to be used in different areas of plant biotechnology such as plant structure change, development of abiotic and biotic stress-tolerant plants, secondary metabolite synthesis, changing nutrient content of plants, extending the shelf-life of fruits. microRNAs (miRNAs) are non-coding RNA molecules of 21 to 24 nucleotides in length and generally act to suppress translation or cause mRNA to break down. The specific sequence-based gene silencing feature of microRNA (miRNA) by RNA interferons offers an alternative approach to plant biotechnology. The use of microRNA (miRNA) in plant biotechnology is increasing due to the identification of new miRNAs through the scientific studies carried out.

The mucilages, on the other hand, are the vegetable compounds used in cosmetic applications frequently as an antioxidant, binding agent, softener, emulsifier, gelling agent, granulation agent, lubricant, suspension agent and skin relaxing agent. Vegetable mucilages form a complex form of polysaccharides containing hydrocolloid and sugar - uronic acid. Due to polysaccharides containing large amounts of hydroxyl groups, the mucilages have a high water-holding capacity, so that they can be used as moisturizers in skin care products. However, considering the production processes and yields of the said plants, it does not seem possible for mucilage to be obtained from plants by conventional methods and meet the needs of the cosmetic sector. In this sense, the necessity of producing mucilages through biotechnological methods comes forth. Today, more than 60 genes responsible for mucilage biosynthesis, secretion, modification, and stabilization have been identified in studies carried out with mucilage biosynthesis. It is determined that the MUM-4 (mucilage-modified 4 or Rhamnose Biosynthesis 2; RHM2) enzyme involved in mucilage biosynthesis, and the TTG2 (T ransparent T esta Glabra 2) transcription factor responsible for the negative directional regulation of the said enzyme, were found to be the main regulatory factors in the mucilage synthesis. In other words, the increase or decrease of the expression of MUM-4 enzyme responsible for intracellular mucilage biosynthesis varies inversely proportional to the expression of the TTG2 transcription factor. The increase in the expression of TTG2 reduces the synthesis of MUM-4 and therefore the biosynthesis of mucilage.

In the patent research on the prior art, various documents concerning the use of undifferentiated plant cells in cosmetic compositions and/or applications for various biological activities have been found.

The patent application EP1699423 A1 is concerned with the use of dedifferentiated cells of halophilic plants as pigmentation removers, illuminators, and preservatives and regenerators of the epidermis.

The patent application W02004082643 A2 explains the cosmetic composition of use in skin cleansing which contains dedifferentiated and elicited cells of Boswellia serrata, and/or dedifferentiated and elicited cells of Negundo.

EP1064932 A1 relates to a cosmetic compound intended for the use of undifferentiated plant cells as anti-odor agents.

EP1244464 A1 concerns with the use of dedifferentiated cell extracts belonging to Leontopodium species in compositions of cosmetic products as the ultraviolet radiation filtration agent.

EP1985280 A2 relates in particular to the use of dedifferentiated plant cells from the fruits of Malus Domestics (apple) of the Rosaceae family plants for the protection of skin cells against different internal and external stress, and for the treatment of aging effects in skin or hair. However, unlike the present invention, this application is not intended to disclose a production process involving the application of methyl jasmonate (MeJa) and miRNA to dedifferentiated plant cells.

WO2013102882 A2 is intended for the use of dedifferentiated cells obtained from plants of Rosa species in cosmetics as an active agent for the aged skin and hair. However, the said invention does not contain a study on MeJa and miRNA elicitation processes involved in the production process.

W0201 1 121051 A2 relates to a preparation used in skin aging, inflammation and wound treatment obtained from non elicited and dedifferentiated cells of the argan tree. There is no use of elicitation techniques in this patent application, too.

EP2308462 A2 relates to an anti-aging or antioxidant composition containing a cell line derived from the cambium of Panax ginseng plant. However, the invention disclosed in that document originally bases on Panax ginseng plant and does not include any explanation for the use of the Cydonia oblonga plant. On the other hand, although the document contains an elicitation process with MeJa application to Panax ginseng cells, it does not involve the elicitation application, the production of the phenolic compound complex with anti-aging activity. Also, no application aimed at increasing the contents of the mucilage with artificial miRNA application is included in the document.

Although all of the patent applications mentioned in the literature search on the prior art used dedifferentiated plant stem cells, these documents do not disclose any study aimed at increasing the mucilage synthesis from the cells using artificial miRNA application and simultaneously increasing the phenolic compound synthesis with MeJa application.

As a result, since many problems and drawbacks are experienced in the said technical area, as mentioned above, the existing systems are inadequate in solving these problems and drawbacks. This necessitates development and innovation in the technical field.

Brief Description of the Invention

The present invention relates to a plant-based composition and production method of the said plant-based composition that meets the above-mentioned necessities, eliminates all disadvantages and brings some additional advantages.

The main objective of the invention is to develop an anti-aging dermo-cosmetic raw material for use in dermo-cosmetic products instead of synthetic substances as in the prior art. Accordingly, the invention comprises the phases of enhancing the synthesis of phenolic compounds by the application of MeJa for anti-aging activity from dedifferentiated Cydonia oblonga (quince) cells and increasing the mucilage synthesis through miRNA transfer for enhancing the effect of moisture and skin elasticity. Thus, these two effects are provided together.

Another objective of the invention is to prevent problems concerning plant use in the dermo- cosmetics sector. In this way, plant cell cultures can be used as raw materials of cosmetic products to obtain highly efficient and high-quality extracts. At the same time, because only a few tissue particles are used in this method, excessive plant use as in the conventional methods is prevented.

A further object of the invention is to obtain stacks of quince cells comprising the phenolic compound complex which exhibits effective anti-aging activity through the use of elicitor. Within the scope of the invention, the most effective MeJa dose, the incubation period with MeJa, the growth phase of MeJa is determined and thus the most effective MeJa application is made to the quince cells so that a complex consisting of phenolic compounds with anti-aging activity is obtained. The resulting complex shows anti-aging activity by stimulating the synthesis of new collagen. After MeJa application, the metabolite composition having anti-aging activity including high amounts of chlorogenic acid, cryptochlorogenic acid, procyanidin B2, quinic acid, trans ferric acid, quercetin-3-galactoside, quercetin-3-glucoside, coumarin, and caffeic acid is obtained in the plant cells. The resulting metabolite complex has an anti-wrinkle activity by stimulating the synthesis of new collagen. With the invention, the amount of metabolites synthesized from the quince cells has been significantly increased by the elicitor application.

Another object of the invention is to increase the expression (upregulation of expression level) of the MUM4 gene, which is one of the genes responsible for the synthesis of mucilage, to obtain quince cells enriched with mucilage, thereby increasing the water-holding capacity of the skin, thereby increasing the moisturizing and elasticity enhancing activity of the invention. With the invention, transcript expression of TTG2 is suppressed by micro-RNAs (miRNA) at a post- transcriptional level, and the expression of MUM-4 and mucilage biosynthesis are increased.

The structural and characteristic features of the invention and all advantages thereof will be more clearly understood by means of the following detailed description and figures. Therefore, the evaluation should be made considering the said detailed description and figures.

Brief Description of the Figures

Figure 1 A Shows a graph illustrating the expression of TTG2 gene in mir172 transfected quince cells.

Figure 1 B Shows a graph illustrating the expression of MUM4 gene in mir172 transfected quince cells.

Figure 2 Shows the graph illustrating the amount of mucilage in mir172 transfected quince cells.

Figure 3 Shows the graph illustrating in vitro cytotoxicity of the extract obtained from quince cells with MeJa application.

Figure 4 Shows the graph illustrating in vitro cytotoxicity of the extract obtained from the mir172 transfected quince cells.

Figure 5 Shows the graph illustrating the effect of extracts obtained from quince cells with mir172 transfect and MeJa application on the expression of COL1 A1 gene.

Figure 6 Shows the graph illustrating the effect of extracts obtained from quince cells with mir172 transfect and MeJa application on the synthesis of Pro-collagen type 1 -C.

Detailed Description of the Invention

In this detailed description, the preferred structures of the invention are explained only for a better understanding of the subject matter and without any restrictive effect.

The invention relates to the plant-based composition that has the anti-aging, moisturizing effects and that increases the skin elasticity for use in the dermo-cosmetics industry and the production method of the said plant-based composition. Two major raw materials are used in the plant-based composition of the invention. These are the cell stacks of the quince ( Cydonia oblonga) containing a phenolic compound complex with anti-aging activity, and cell stacks of the quince ( Cydonia oblonga) enriched in mucilage content. In a preferred structure of the invention, quince cells ( Cydonia oblonga) are used. For the same purpose, in different applications, the cells obtained by individual or combinations of plant types such as Malva sylvesths, Linum tauricum, Paeonia turcica, Amygdalus communis, Persea amehcano, Muscari massayanum, Muscari armeniacum, Rosmarinus officinalis, Corylus aveiiane, Hypericum perforatum, Ocimum basilicum, Allium akaka, Allium tuncelianum, Aleo vera, Citrus aurantium, Citrus limon, Crocus specious subsp xhantolaimos, Lathyrus undulates, Galanthus plicatus subsp. byzantinus, Crocus Olivieri subsp. istanbulensis, Cochicum chalcedonicum, Hypericum avicularifolium, Santalum album, Act i nidi a deliciosa, Glycine max, Viola tricolor, Diospyros kaki, Vaccinium myrtillus, Rosa canina, Rosa damascena, Oryza sativa, Glycyrrhiza glabra, Salvia anatolica, Fritillaria alburyana, Tulipa karamanica may also be used.

Plant tissues, which have suffered mechanical damage, form cell stacks with meristematic effect mechanism to repair the damage. These cell stacks are known as callus in the prior art. Callus cells are totipotent and have the ability to transform into all cells and organs of the plant and create a new plant. The plant cell culture technique allows a variety of applications in accordance with the aim of reproducing the totipotent plant cells in controlled, sterile laboratory conditions and obtaining the secondary metabolites specific to the cells. In this sense, in vitro quince plants are obtained by sterilizing the quince seeds in order to obtain the quince cell stacks within the plant- based formulation of the invention. The cell suspensions are formed from the callus obtained from leaf explants of in vitro quince plants. To stimulate the synthesis of the phenolic composition having anti-aging activity including chlorogenic acid, cryptochlorogenic acid, procyanidin B2, quinic acid, trans ferric acid, quercetin-3-galactoside, quercetin-3-glucoside, coumarin, and caffeic acid, elicitor applications are performed to suspensions of quince leaf cell. With the elicitor applied, after the incubation period of quince cell suspensions is complete, the cells are harvested. After the harvested cells have been washed, they are homogenized under high pressure to break down the cell stacks and homogenize the anti-aging phenolic compounds together with the cell stacks. The resulting the quince cell stack containing anti-aging phenolic compound complex stimulates the new collagen synthesis so that it has a wrinkle-free effect.

Another important raw material of the anti-wrinkle plant-based composition of the invention is the quince cell stack enriched in mucilage content. As mentioned above, instead of quince, the individuals or combinations of plant types such as Malva sylvestris, Linum tauricum, Paeonia turcica, Amygdalus communis, Persea americano, Muscari massayanum, Muscari armeniacum, Rosmarinus officinalis, Corylus aveiiane, Hypericum perforatum, Ocimum basilicum, Allium akaka, Allium tuncelianum, Aleo vera, Citrus aurantium, Citrus limon, Crocus specious subsp xhantolaimos, Lathyrus undulates, Galanthus plicatus subsp. byzantinus, Crocus Olivieri subsp. Istanbulensis, Cochicum chalcedonicum, Hypericum avicularifolium, 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 may also be used. Also in the stimulation of mucilage synthesis, quince cell suspensions are obtained. After the resulting quince cell suspensions have been reached to sufficient volume, protoplast isolation from the cells is carried out in order to make miRNA transfer. In order to suppress the expression of the TTG2 gene to stimulate the mucilage synthesis in the obtained protoplast cells, artificial-mir172 is transferred through PEG-liposome. After the incubation period with artificial miRNA is completed, the cells are harvested and passed through the washing process. After washing, the cell stacks are homogenized under high pressure so that the synthesized mucilage within the cell is released and the cell stacks and the mucilage can be homogenized. Following the miRNA transfer, the expression of the MUM4 gene, which is one of the genes responsible for the synthesis of mucilage, is increasing in the cells of the quince protoplast. In this way, more than 3.4 times the mucilage synthesis from the seed shell is performed from the quince cells. As a result, the quince cell stack enriched in mucilage content increases the moisture level and elasticity of the skin by increasing the water retention capacity.

The production process of anti-wrinkle plant-based composition is given below. Accordingly, first of all, in vitro quince plants are created. At this stage, in order to make the development of callus more successful and in a short time, quince plants are transferred to in vitro by using tissue culture method. Quince seeds are preferably rinsed in 70% alcohol for 1 minute, kept in preferably in 20% NaOCI solution, preferably in 10 minutes with preferably in 2 drops of Tween-20 (detergent), then rinsed with distilled water 3 times, and then sterilized. Sterile seeds were incubated in darkness in jars of 75 ml MS nutrient environment with pH 5.8 containing preferably 30 g/l added sugar and 7 g/l Agar. After germinated, the seeds were incubated at the temperature of 22 to 25 in the climate room, preferably set to 16 hour s light/8 hours dark photoperiod, and taken to sub-culture every 3 to 4 weeks using the hormone-free MS environment. This phase is common both for the quince cell stack containing the phenolic compound complex mentioned above and for the enriched quince cell stack containing the mucilage content. In Table 1 , Table 2 and Table 3, macro element quantities, micro element quantities, vitamin quantities in the MS nutrient medium are respectively given.

Table 1 : Macro elements and their amounts in MS nutrient medium

Table 2: Micro elements and their amounts in MS nutrient medium

Table 3: Vitamins and their amounts in MS nutrient medium

Following the production of in vitro plants, the process of forming callus and cell suspensions is started. In order to obtain callus, leaf explants of in vitro quince are wound with the help of a scalpel and cultivated in MS medium containing preferably 1 mg / L NAA and 0,5 mg / L BAP. The explants taken into the culture are kept in the dark environment at 25 Ό preferably until callus formation is observed and transferred to fresh medium preferably every 4 weeks. When the transparent and friable callus reaches a sufficient amount, the formation of suspension is initiated. The liquid form of the nutrient medium determined for callus formation is used for cell suspension culture. In order to achieve cell suspensions, preferably 2,5 - 3 g of 40 - 45 days of callus is incubated as it is put in a conical flack of 250 ml preferably containing 50 ml of liquid medium, and then separated preferably at 20Ό to 25 Ό at 1 10 rpm in dark until it becomes a cell suspension containing a single cell or several cell groups. Preferably, approximately after 4 weeks, the suspension is carried through a sterile sieve (30 mesh) in order to remove large thick masses of callus from the suspension. The suspension passing through the wire mesh is inoculated in a ratio of 1 :4 and divided into conical flacks by adding fresh nutrient medium to the sub-cultures and continued to be incubated at 1 10 rpm. The growth parameters of the cells in the resulting suspension (cell count, wet weight, dry weight, packed cell volume (PCV), settled cell volume (SC V), cell viability are determined weekly. According to the cell viability graph, the cell suspension is periodically divided into sub-cultures and the amount of suspension is increased. While the quince leaf cells, which were grown in the conical flacks, were preferably in 1 -liter volume, the suspension volume is increased to 2 liters with the addition of nutrient medium with 1 :1 inoculation ratio and is transferred to the wave - shaking bioreactor having the maximum working volume preferably 20 liters. In the bioreactor, the suspension volume is increased to 10 liters by adding fresh nutrient medium in 1 week periods preferably to cells and sub-culturing them. The said phases are common both for the quince cell stack containing the phenolic compound complex mentioned above and for the enriched quince cell stack containing the mucilage content. After this phase, the next phase aims to obtain a stack of quince cells containing the phenolic compound complex. In line with this aim, MeJa application is performed to quince cell suspensions. When quince cell suspensions in the bioreactor bag reached preferably 10 liters volume of 50% PCV density, elicitor application is started. By adding 50 pm MeJa to the quince cell suspension, the cells are left to incubation for 24 hours. After Meja application is completed, the cells are harvested and then homogenized preferably under 1200 bar pressure, in order to homogenize the phenolic compound complex with anti-aging activity in the quince cells and quince cell stacks. As a result, a stack of quince cells containing phenolic compound complex with anti-aging activity is obtained.

Along with the next process step, the process steps to obtain the quince cell stack enriched in mucilage content are performed. In accordance with this aim, artificial miRNA design and transfer of it to quince cells are implemented. Today, the method of 573’ RACE (rapid propagation of cDNA ends) is commonly used to form full-length complementary DNA (cDNA) containing axons. The said method requires 30 base length sequence information of the respective transcript. A full- length cDNA can be obtained with the use of this array known as the genetic sequence data (GSD). In addition, at least 30 base sequences of the target transcript, for which the sequence will be determined with RACE 573’ method, are required. Since there is no sequence information for the quince plant, 30 base length consensus sequence information is obtained from other plants with genomes of Rosaceae family (apples and peaches). The following method is used to design the proper primers for the consensus sequence and this sequence data (GSD).

The ortholog sequences of TTG2 are downloaded from tree plants belonging to Rosaceae family (Apple; Malus domesticus, and Peach: Prunus persica ) from the“Plaza Dicot 3.0” databases containing functional genomics and transcriptomic sequences belonging to dicotyl plants. The protected exon sequences of P. persica and M. domesticus are aligned with pair-wise alignment software. The consensus sequence for the Rosaceae family was determined from the sequences that showed similarity over 90%, and the Gene Specific Primer (GSP) for the RACE reaction for these quince plants was designed with the Primer3 web tool. The forward and reverse primers containing 73 base pair lengths and the synthetic quince miR A-172 (Cob-miR172) sequence can be synthesized as:

Forward-Primer-(5’3’)

T GT AAG AAT CTT GAT GAT GCT GCAAT GAT GAT CACATT CGTT AT CT ATTTTTTT GCAGCAT CAT CAAG ATT CT

Reverse- Primer-(5’3’) AAT GAG AAT CTT GAT GAT GCT GCAAAAAAAT AG AT AAC

G AAT GT GAT CAT CATT GCAGCAT CAT CAAG ATT CT

For the production of artificial miRNA, E.coli strain containing pENTR-AtMIR390a-B/c carrier plasmid is used. For short and long term storage and isolation of the plasmid, E. coli DB3.1 strain, which is in the form of agarose stab, is amplified in solid B (Luria broth) nutrient medium and single colony selection is made. The antibiotics Chloramphenicol (25 pg / ml.) and Kanamycin (50 pg / ml) are added to the agarose nutrient medium, thus due to the resistance genes of pENTR-AtMIR390a-B/c plasmid in E. coli DB3.1 strain, the selection of single colony is made.

In the preparation of B solid nutrient medium preferably in 1 -liter volume, preferably in 5 g peptone, 10 g yeast extract, 10 g Sodium Chloride (NaCI) and 12 g bacteriological Agar are added and the prepared medium is sterilized with autoclave preferably at 1210 for 20 minutes. After the sterilization, the cooled nutrient medium is taken to a water bath of 600 and antibiotics Chloramphenicol (25 pg/ml) and Kanamycin (50 pg/mL) are added. The solidification of B nutrient medium is which is preferably spilled into 10 ml volume of Petri dishes in sterile conditions waited, then E. coli DB3.1 samples, which are in the form of agarose stab by sterile pipette (or core), are preferably incubated at 370 for 16 hours in a shak ing oven. Bacteria, which are harvested and colony-selected after incubation, are grown in liquid B (Luria broth) nutrient medium for isolation of the plasmid and storage at -800 for a long time .

In the preparation of the B liquid nutrient medium, the bacteria in the single colony in the petri dish are selected by means of a sterile pipette and transferred to the liquid B (Luria broth) nutrient medium and left for incubation in order to effect the reproduction of the original plasmid-bearing bacteria. Liquid B (Luria broth) nutrient medium is prepared by adding preferably 4 g peptone, 4 g yeast extract, 4 g Sodium Chloride (NaCI) and preferably 400 ml distilled water on the compounds. Then the medium is sterilized with autoclave preferably at 121 O for 20 minutes. The antibiotics (Chloramphenicol and Kanamycin) are added to the medium which is left to cool down in a sterile cabinet, and then the liquid nutrient medium is poured into the falcon tube of 5 ml. Bacteria selected from the petri dish through sterile pipette are planted in the nutrient medium in the falcon tube and the samples are preferably left to incubate at 370 for 16 hours in shaking oven. For long periods of storage, 500 pL from the bacteria culture which is replicated in liquid B medium and 50% glycerol is added to 2 ml cryotubes and stored at -80 O. For plasmid purifications, Plasmid DNA isolation kit is used.

After plasmid isolation, the preparation of the binding buffer for forward and reverse oligonucleotides and the reaction stages are initiated.

In the preparation of the binding buffer, the synthesized forward and reverse oligonucleotides are diluted with deionized sterile water for the final concentration preferably 100 pm. 5 binding buffers (60 mM T ris-HCI, 500 mM NaCI, 60 mM MgCh, 10 mM DTT) are prepared, each preferably 1 ml. The binding reaction of forward and reverse oligonucleotides is carried out in a PCR tube using a PCR device as indicated below:

The reaction is held at 94 O for 5 minutes in a PC R device and reduced 0.05 O per second and brought to 200. Then, oligonucleotides mapped to h ydrogen bonds are diluted as follows:

The ligation phase of the double-stranded oligonucleotides to be cloned in isolated plasmids formed as a result of the binding reaction of forward and reverse oligonucleotides.

Before the transformation of double-chain oligonucleotides to quince suspension cells, the ligation of plasmids (carrier expression vector) is performed. The reaction is performed as follows:

When the reaction is complete, the transfer of oligonucleotides containing the double-chain mature quince miRNA sequence to the plasmid is performed. Artificial miRNA preparation phase to be transferred to the protoplast cells of the quince is completed. Following the transfer of miRNA to vector, the isolation of the protoplast cells that are necessary for transformation is carried out. The cells are transferred from pipe to filtration unit under aseptic conditions through a peristaltic pump to perform protoplast isolation from the quince cell suspensions replicated in the bioreactor. After the filtration phase, the solution containing preferably 0.33% Cellulase R10, 0.033% Pectolyase Y-23, and CPW13M containing 5 mM MES (KN03 101 mg, CaCI 2H₂0 1480 mg, MgS0₄7H₂0 246 mg, KH₂P0₄ 27,2 mg, Kl 0,16 mg, Mannitol %13, BSA %0,1 , ph:5,8) is added the cell pellet (g/10 ml), and the cells are transferred back to the bioreactor bag. After 15 hours of incubation, preferably with the enzyme mixture, the cells are transported through peristaltic pump under aseptic conditions, preferably under vacuum from 64, 45 and 30 mM meshes respectively. The obtained filtrate is removed from the enzyme mixture on the filter paper. These cells are washed 3 times on filter paper with CPW13M medium. Cells are filtered and separated on filter paper. PEG-liposome mediated transformation method is used in miRNA transfer to protoplast cells.

Protoplast cells are suspended in the mixing unit in MMg buffer (0,4 M Mannitol, 15 mM CaCI₂, 4 mM MES) in a final concentration of preferably 1 x10⁶/ml, and left to incubation for 30 min at room temperature. After incubation, 50 mI Cob-mir172 is added to every 0,5x10⁶ cells on the protoplast cells. In order to ensure the transfer of miRNA to the cells, the protoplast-MMg buffer mixture in the mixing unit is added with 550mI PEG/Ca solution for each 0,5x10⁶ cells (4 g PEG 4000, 0,4 M Mannitol, 100 mM CaCI₂) and is left to gently mix at room temperature for 15 minutes. At the end of said period of time, 2.24 ml W5 solution (154 mM NaCI, 125 mM CaCI₂, 5 mM KCI, 2 mM Mes) is added to the mixing unit for 0,5x10⁶ cells and the unit is gently mixed. After this phase, the protoplast cells in the mixing unit are filtered under aseptic conditions. For each 0,5x10⁶ cells, 3 ml W5 solution is added on the obtained cells and the protoplasts are transported to the bioreactor bag. After miRNA transfer, the cells in the bioreactor bag, preferably at 2513, are left to incubate for 6 hours, preferably in the dark, in order to ensure proper time for the expression change of the relevant genes. After incubation, the cells in the bioreactor are harvested and washed. At the end of the process, the quince protoplast cell stack with rich mucilage content is obtained.

In order to determine the effectiveness of miRNA transfer and MeJa applications, RNA isolation was performed using kit from cells after the incubation process of miRNA transfer to protoplast cells is completed. cDNA synthesis was performed from the obtained RNAs and the expression level of the genes was measured using the SYBR green method using the primary pairs designed with each gene. The expression levels of the target genes (TTG2 and MUM-4) were compared to the expression level of the reference gene (ACTB), and normalized according to the 2 ^DD0T method, and fold changes are determined. In addition, the experiments were carried out, preferably, by three biological repetitions. Gene expression differences between control group and the transfected group were determined by Student's test (p < 0.05) and RT-qPCR analysis showed that transfected group that has the expression level of the TTG2 gene was transferred by COB - miR172 is reduced by 4 times compared to the control group. The said change is given in Figure 1 .A. On the other hand, the expression level of the MUM-4 gene in the transferred group with cob-miR172 is increased by about 20 times compared to the control group. The graph showing the said increase is given in Figure 1 .B. According to RT-qPCR results, the results of the regulation of the TTG2 transcription factor and the MUM-4 enzyme in the group transferred with Cob-miR172 indicate that the selection of the suppression of the expression of the TTG2 gene as the correct target for expressing the MUM-4 gene.

When miRNA transfer to the protoplast cells and incubation period after the said transfer was completed, the cells were lyophilized. The amount of mucilage from lyophilized cells was determined using the ruthenium red (RR) dyeing method. The RR dyeing method is based on the fact that due to the RR coating is connected to the pectin, the main component of the mucilage, thereby opening the supernatant color containing RR and measuring this color change as spectrophotometric. As the mucilage content of the test material is increased, the supernatant color is tinted and this difference can be determined by absorption drop. The RR experiment was carried out using the following process. Accordingly, as a positive control, mucilage isolated from quince seeds was used and, preferably, the standard series was formed with 5 different doses. Preferably 0,02% 500mI Ruthenium Red solution was added on the tubes. After vortexing briefly, it is allowed to incubate for 5 minutes at room temperature. The tubes were preferably centrifuged for 15 minutes at 14000 g. The supernatant was carefully withdrawn and transferred to the wells of the 24-hole plate. In 534 nm, absorbances were measured and the mucilage contents of the control and Cob-mir172 transfer groups were determined by taking the average of all absorbances obtained from repeated experiments in 3 different time periods. According to the results, the amount of mucilage in the lyophilizates (1 mg/ml) obtained from Cob-mir172 transferred cells showed similar absorbance with approximately 0.25 mg/ml mucilage. The mucilage of lyophilizates obtained from the non-transferred control group yielded similar absorbance with approximately 0.2 mg/ml_ mucilage. According to this, approximately 25% of mucilage increase was achieved in the cells transferred with mir172 compared to the control group. The absorbances obtained by RR dyeing method of quince cell lyophilizes which were transferred with Cob-mir172 and which were not, are given in Figure 2. As a result, the expression of the TTG2 transcription factor was suppressed to increase the expression of MUM4, one of the enzymes responsible for mucilage synthesis, and the aim of increasing the mucilage synthesis was carried out. After the transfer of cob-mir172, it was found that the amount of mucilage (25%) obtained from the quince cells was about 3.4 times higher than the amount of mucilage (7.3%) obtained from the seed shell.

In accordance with the objective of determining phenolic compound contents of quince cell suspensions by LC-MS/MS analysis, quince cell suspensions were washed with distilled water after harvesting after MeJa application and were decomposed under high pressure (preferably 1500 bar) and lyophilized. Lyophilized quince cells were extracted in 80% methanol, preferably for 48 hours at 25 TT After incubation, LC-MS/MS a nalysis was performed after centrifugation of the cells in methanol, preferably at 14000g, and transferred from supernatant microfiltration. As a result of LC-MS/MS analysis, it was determined that the total phenol content of the cells with MeJa application which forms the anti-aging phenolic compound complex is increased by 21 .5% compared to the control group. These results are given in Table 4. In this way, the amount of phenolic compounds which have anti-aging activity has been increased with Meja application and a more effective product has been obtained.

Table 4: Phenolic compound quantities in quince cells after MeJa application according to control group

After this stage, in vitro safety and efficacy tests of the product are given below. In order to carry out these in vitro safety and efficacy tests, extracts of cells obtained after miRNA transfer and MeJa application were obtained. And after the harvesting of quince cell suspension containing a complex of phenolic compounds with anti-aging activity, and quince cell suspensions enriched with miRNA transfer in mucilage content were homogenized and lyophilized under high pressure to be used in in vitro tests. Extraction was carried out separately from both lyophilized cell stacks and for this purpose, the concentration of lyophilized cell stacks was extracted at room temperature in 10% ethanol for 48 hours preferably to be 20 mg/ml in concentration, and then centrifuged at 14000 rpm for 10 minutes. Following the said centrifuge, the supernatant was passed through the microfilter of preferably 0.2 micrometers and sterilized. The resulting cell extracts were used in in vitro safety and efficacy tests. In vitro cytotoxicity of quince cell suspension extract containing anti-aging phenolic compound complex and quince cell suspension extract enriched with miRNA transfer and mucilage content, the effects of collagen synthesis by pro-collagen type 1 -C Elisa method on the expression of COL1 A1 gene were determined by RT- qPCR analysis.

The next test phase is the determination of in vitro cytotoxicities of the extracts. Human fibroblast cells were preferably seeded to 1 x 10⁵cells/well in 24-well plates. After the 24-hours incubation, the nutrient mediums in the wells were poured and replaced with the nutrient mediums containing the specific doses of extractions. After the extraction of the cells provided preferably 24 ± 0.5 hours incubation, the extraction-nutrient medium solution in wells was removed and the final concentration of the wells was added to the MTT-nutrient medium suspension having 5 mg/ml_ concentration, and the plates were preferably left to the incubation for 3 ± 0.1 hours. At the end of the specified period, MTT-nutrient medium solution was poured and DMSO was added to each well to dissolve the formazan salts formed by living cells. Plates are preferably left in the plate shaker for 15 minutes. The OD (Optical Density) values of the plates were then read at 570 nm. By using the numerical data obtained as a result of the reading, it was determined whether extraction doses were toxic or proliferative by comparing the viability amounts of the cells exposed to certain doses of the substance in comparison to the control cells. The tested doses of the extracts did not reveal any toxicity on fibroblast cells. Cytotoxicity results are given in Figure 3 and Figure 4. At the same time, the extract obtained from the miRNA transferred quince cells has a proliferative effect on fibroblast cells depending on the dose. A dose of extract of % 0,01 provided proliferation of % 13, dose of % 0,02 provided % 24, dose of % 0,03 provided % 38 and the dose of 0.1 % provided 49%.

The effects of the extracts on the gene levels was investigated with human normal fibroblast cells. For RNA analysis, normal aged human fibroblast cells (p 17) were cultured to 25 cm² flasks with 2x10⁶ cells per each and were left to incubate for 24 hours. After the cells were clamped to the culture flask, the doses of the quince cell extract with MeJa application and the quince cell extract with miRNA transfer were applied to the growth medium and the cells were left to incubate for 24 hours. After incubation, the cells were removed from the culture medium with trypsin / EDTA and centrifuged. Pellets obtained after centrifugation were used for RNA isolation. RNA isolation was carried out in accordance with the recommended procedure in the kit (PureLink RNA mini kit, Life Technologies). cDNA synthesis was performed using obtained RNAs according to the procedure recommended by the manufacturer of the kit for use in RT-qPCR studies (PrimeScript First Strand cDNA synthesis kit, Takara). The obtained cDNAs were used in RT-qPCR studies. Using the primary pairs designed for the COL1 A1 gene, the expression level of genes was measured using SYBR green (LightCycler 480 SYBR green master, Roche) method. The expression level of the target gene (C0L1 A1 ) is proportioned to the expression level of the reference gene (ACTB) and is normalized according to the method 2^_MCT (Schmittgen and Livak, 2008) and the fold change is calculated. The expression of the COL1 A1 gene was found to be increased 1 .5 times in cells treated with quince cell extract with MeJa application compared to the control group and 5 times more in cells with mir172-transferred quince cell extract. It is determined that the extract obtained from both applications stimulates expression of the collagen gene in fibroblast cells. The results are given in the chart mentioned in Figure 5.

PICP sandwich immunoassay kit was used in the determination of Pro-collagen type I C-peptide synthesis (Procollagen Type I C-Peptide EIA kit, Takara). First of all, human normal fibroblast cells (CRL-2076) were planted on 6 wells with 2x 10⁴ cells/wells and preferably treated for 24 hours after incubation with certain doses of extracts. A 100 mI Antibody-POD conjugate solution was then transferred to each well of the PICP plate with 96 wells, then 20 mI sample (nutrient media of extractable cells) or standard solution was transferred. The plate was closed with foil and incubated for 3 hours at 370. At the end of th e time, the liquid content in the wells was removed by vacuum and the wells were washed 4 times with 400 mI_ washing solution. After each washing, the plate is turned upside down and is lightly shot on the paper towel to be emptied. For substrate incubation, 100 mI_ substrate solution (TMBZ) was transferred to each well and the plate was kept at room temperature for 15 minutes. At the end of the period, 100 mI_ stopping solution was added to each well and the plate was gently hit and mixed to ensure the mixture of the solutions. Absorption of the plate was measured in 450 nm wavelength in a microplate reader. The standard chart was formed by the absorptions of standards. The value of PICP included in the samples was determined by the standard chart. According to the results of the experiment, the dose of 0.01 % of the extract obtained from the miRNA transferred quince cells increased Pro- Collagen Type 1 -C synthesis by 7%, 0.02% dose by 9%, 0.03% dose by 12% when compared with the control group. MeJa applied quince cell extract, the dose of 0.01 % of the extract obtained by MeJa application on quince cells increased Pro-Collagen Type 1 -C synthesis by 12%, 0.02% dose 16%, 0.03% dose 21 % compared to control group. The graph of increasing synthesis is given in Figure 6. As a result, according to the data obtained from two different applications, the extract of the quince cell extract stimulates the synthesis of Pro-Collagen Type 1 C in fibroblast cells, depending on the dose.

Claims

1. The invention is a plant-based composition to be used in the dermo-cosmetics industry; characterized in that it comprises a plant cell stack containing the phenolic compound complex obtained by MeJa application and/or a plant cell stack containing mucilage contents obtained by artificial miRNA transfer.

2. The invention is a plant-based composition according to Claim 1 , wherein; said plant cell characterized in being individual of combinations selected from Cydonia oblonga, Malva sylvestris, Linum tauricum, Paeonia turcica, Amygdalus communis, Persea americano, Muscari massayanum, Muscari armeniacum, Rosmarinus officinalis, Corylus aveiiane, Hypericum perforatum, Ocimum basilicum, Allium akaka, Allium tuncelianum, Aleo vera, Citrus aurantium, Citrus limon, Crocus specious subsp xhantolaimos, Lathyrus undulates, Galanthus plicatus subsp. byzantinus, Crocus Olivieri subsp. istanbulensis, Colchicum chalcedonicum, Hypericum avicularifolium., 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 karamani.

3. The invention is a plant-based composition according to Claim 1 or Claim 2, characterized in comprising plant cell stacks containing 50% phenolic compound complex by weight.

4. The invention is a plant-based composition according to Claim 1 or Claim 2, characterized in comprising plant cell stacks containing 50% mucilage by weight.

5. The invention is a plant-based composition production method according to Claim 1 , wherein; comprises the process steps below;

• Stimulation of callus from the explants of in vitro plants under aseptic conditions,

• Obtaining suspension culture from the said callus,

• Elicitor application of the obtained suspension cultures,

• The transfer of artificial miRNA to these suspension cultures.

6. The invention is a plant-based composition production method according to Claim 5, wherein; comprising the process step of the homogenization of the cells that are applied with elicitor and miRNA.

7. The invention is a plant-based composition production method according to any one of the preceding claims, characterized in the use of plasmid in the production and reproduction of artificial miRNA.

8. The invention is a plant-based composition production method according to any one of the preceding claims, characterized in the use of the PEG-liposome mediated transformation method in miRNA transfer.