WO2023089462A1

WO2023089462A1 - Medicinal products based on exosomes derived from plants, humans, algae, and medicinal mushrooms to treat the skin diseases

Info

Publication number: WO2023089462A1
Application number: PCT/IB2022/060893
Authority: WO
Inventors: Maria BEIHAGHI; Reza SAHEBI; Samaneh ATTARANDOWOM; Houman TEHRANI; Ilia TEHRANI; Kamelia KOHAN NEZHAD; Ali NOUROLLAHI; Mohammad reza BEIHAGHI
Original assignee: Individual
Current assignee: Individual
Priority date: 2022-11-13
Filing date: 2022-11-13
Publication date: 2023-05-25
Anticipated expiration: 2025-05-13

Abstract

The invention of medicinal products based on plant exosomes, algae, and medicinal mushrooms to treat skin diseases are beneficial compounds, which have been selected from the extracts and essences of the intended plant families in accordance with the disease target. They are made with purified exosomes from the above-listed plants, spirulina algae, and shiitake mushrooms. Plant-derived exosomes have a structure similar to exosomes isolated from mammals and act as extracellular messengers in intercellular communication. Moreover, these vesicles are capable of transferring mRNAs, microRNAs (miRNAs), bioactive lipids, and proteins to human and animal cells.

Description

Medicinal Products based on Exosomes Derived from Plants, Humans, Algae, and Medicinal Mushrooms to Treat the Skin Diseases

A61K-A61Q-A61P-A61N

PLANT-BASED EXOSOME COMPOSITIONS AND USE THEREOF FOR REJUVENATING SKIN

WO/2020/180311

A topical composition for regulating skin conditions is described. The composition comprises an effective amount of isolated exosomes having increased levels of heat shock stress-response molecules and a carrier. The isolated exosomes are obtained from a plant's leaf flesh conditioned by growing the plant under conditions that include a heat shock of the plant at a temperature of about 33°C to about 45°C for about 1 hour to about 3 hours. Uses of the exosome-containing compositions for regulating human skin include inducing increased skin integrity by cell renewal, enhancing water content or moisture of the skin, reducing transepidermal water loss, skin flaking, and scaling, improving skin thickness, enhancing skin tensile properties, reducing the appearance of dermal fine lines and wrinkles, improving skin texture, reducing skin pores size, enhancing skin smoothness, improving skin age spots, improving skin tone, and improving the appearance of scars and skin abrasions.

EXOSOME COMPOSITIONS AND METHODS FOR PREPARATION AND USE THEREOF FOR REGULATING AND CONDITIONING SKIN AND HAIR

2993227

Improved stem cell exosome-containing compositions are provided, along with methods for their preparation and use for regulating skin condition. The compositions provided contain isolated stem cell exosomes having increased levels of heat shock stress-response molecules. Uses of the exosome-containing compositions for regulating human skin include inducing increased skin integrity by cell renewal, enhancing water content or moisture of the skin, reducing transepidermal water loss, skin flaking, and scaling, improving skin thickness, enhancing skin tensile properties, reducing the appearance of dermal fine lines and wrinkles, improving skin texture, reducing skin pores size, enhancing skin smoothness, improving skin age spots, improving skin tone, and improving the appearance of scars and skin abrasions.

A composition comprising an exosome and/or extracellular vesicle derived from stem cell as an active ingredient and its application for reinforcing or improving skin barrier

KR20190060646A

Provided is a composition for reinforcement and functional improvement of the skin barrier, comprising at least one member selected from the group consisting of a stem cell-derived exosome and/or extracellular vesicle, and fractions containing the exosome and/or extracellular vesicle isolated from a stem cell culture medium, as an effective ingredient. The composition of the present invention can improve the water loss index of the stratum corneum, such as transepidermal water loss (TEWL), and increase skin hydration. Therefore, the composition of the present invention can be effectively used as a cosmetic composition, an external preparation for skin, and a pharmaceutical composition for reinforcement and functional improvement of the skin barrier.

COMPOSITION FOR REDUCING PORES, COMPRISING STEM CELL-DERIVED EXOSOMES AS ACTIVE INGREDIENT

WO/2019/231133

The present invention provides a composition for reducing pores, comprising stem cell-derived exosomes as an active ingredient, and a beauty method for reducing skin pores using the composition. Treating the skin with the composition for reducing pores comprising stem cell-derived exosomes as an active ingredient, according to the present invention, has an excellent skin beauty benefit of reducing skin pores, particularly skin pores of the face, improving the appearance of the face.

Method for applying stem cell exosome to facial mask

CN113384518A

The invention discloses a method for applying stem cell exosomes to a facial mask, which comprises the following components in parts by weight: 1-5 parts of cogongrass extract, 2-4 parts of chamomile, 1-3 parts of trehalose, 5-15 parts of avocado, 2-5 parts of calendula, 4-6 parts of peppermint oil, 10-15 parts of honeysuckle, 5-10 parts of radix ophiopogonin, 1-3 parts of cysteine, 1-3 parts of serine, 1-3 parts of thymine, 1-2 parts of cytosine and 1-2 parts of silk. The method for applying the stem cell exosome to the facial mask extracts the lalang grass rhizome extract and the chamomile, obtains extract liquid, and mixes the extracted liquid with the secretion body dry ice powder, can increase the water replenishing capacity and the water locking capacity of the facial mask to the skin, simultaneously mixes trehalose in the raw materials, increases the calming effect of the facial mask to the skin on the basis of the original raw materials, enables material molecules to be in a stable state, is convenient for the skin tissue to fully absorb internal nutrient substances, and promotes the metabolism of the skin tissue.

It was determined based on the findings in the research conducted by An Y and his colleagues. Exosomes derived from stem cells can be used to repair skin wounds, reduce immune responses and inflammation, strengthen angiogenesis, reproduce skin cells, and regenerate collagen. Also, in the experiments conducted on diabetic patients, it caused wound healing and can also be used as a drug carrier.

In 2017, Kim and colleagues found that human umbilical cord blood-derived mesenchymal stem cells play an important role in skin wound healing, and studies show that exosomes derived from mesenchymal stromal cells (MSCs) activate several signaling pathways that promote wound healing. and cell growth are effective. They also play a significant role in the synthesis and penetration of skin collagen and cause skin rejuvenation. Therefore, by observing the increase in the expression of collagen I and elastin, the effect of exosome in the treatment of human skin can be observed. The research results showed that by absorbing exosome in human skin, it enhances the synthesis of collagen I and elastin in the skin, which are necessary for skin rejuvenation.

Shiqi Hu and colleagues found in 2016 that human dermal fibroblasts (HDFs), which constitute the main cell population of the dermis, gradually lose their ability to produce collagen and renew the intercellular matrix with age. Exosomes derived from bone marrow mesenchymal stem cells increase the expression of procollagen type I and significantly decrease the expression of MMP-1 through the reduction of tumor necrosis factor alpha (TNF-α) and the upregulation of transforming growth factor beta (TGF-β) and increases skin collagen deposition and is used to treat skin aging.

In 2015, Zhang J and his colleagues used mesenchymal stem cells derived from human induced pluripotent stem cells (hiPSC-MSCs) as a promising alternative for stem cell transplantation therapy, repairing damaged tissues.

Yang GH, and his colleagues in 2021 conducted studies on the effect of exosomes on skin defects such as aging, atopic dermatitis, and wounds and found that exosomes can be used as non-commercial components of skin treatment products.

In 2021, Shi and colleagues found that exosomes enriched with fibrin gel are a promising strategy for promoting chronic wound healing and complete skin regeneration. In this project, exosomes were purified from platelets and combined with injectable fibrin sealant (TISSEEL) and used to heal wounds created in rabbit ears.

It should be noted that currently various drugs based on exosomes are commercially available in the market, but so far there is no report on the production of pharmaceutical and cosmetic composition based on exosomes from the plants in question in the mentioned invention, the blood serum of each person specifically. , algae and medicinal mushrooms have not been reported. Therefore, the main goal of this invention is to produce medicinal compounds based on plant exosomes with immunogenic properties and without any allergic effects, blood serum of each person completely specifically, edible shiitake mushroom and spirulina algae for skin treatments of eczema, blemishes and melasma, sagging and skin aging, cellular regeneration of human skin, as well as increasing the water or moisture content of the skin, preventing skin flaking, improving skin thickness, increasing skin elasticity, removing skin moles, removing skin acne, improving skin texture, reducing pore size skin, increasing skin smoothness, improving skin aging spots, improving skin color and improving the appearance of wounds and healing wounds in diabetic people, for this purpose, by identifying the native species of Iran, medicinal and health compounds based on plant exosomes and Algae and medicinal mushrooms were selected that are suitable for experimental research in the future.

Exosomes are secreted from different cells, including stem cells, containing different molecules such as growth factors, proteins, broken nucleic acids, and microRNAs (miRNAs). Mesenchymal stem cells (MSCs) contain exosomes that carry out actions the same as the mesenchymal stem cells, including repair of tissue damage, inhibition of inflammatory responses, and regulation of the immune system. Using exosomes does not have risks such as aneuploidy and transplant (graft) rejection. To this end, they can be exploited for the treatment of illnesses such as autoimmune disease and cancer. Plant-derived exosomes have a structure similar to exosomes isolated from mammals and operate as extracellular messengers in intercellular communication. Moreover, exosomes are capable of delivering mRNAs, microRNAs (miRNAs), bioactive lipids, and proteins into animal cells. Concerning the gained results, using exosomes obtained from plants and humans to treat hyperpigmentation and rejuvenation is a suitable approach for the future of the cosmetics industry. Additionally, plant-derived exosomes are safe for the environment due to their small size, low toxicity, and high absorption; as a result, they can act as the next generation of drug delivery systems for treating multiple diseases. Furthermore, since some individuals are allergic to herbal compounds, so medicinal compounds based on the exosomes from the subjects' blood serum are specifically used. Hence, making medicinal and cosmetic compounds based on exosomes for skin treatments, including eczema, blemishes & melasma, skin sagging & aging, cellular regeneration of human skin and enhancing the water content or moisture of the skin, preventing skin peeling, improving skin thickness, increasing skin elasticity, removing skin moles, removing skin pimples, improving skin texture, reducing the size of skin pores, increasing skin smoothness, improving skin aging spots, burn spots, improving skin color and improving the appearance of wounds and healing wounds in diabetic people are among the objectives of this invention.

Melanin is a critical component of the human hair, eye, and skin pigmentary system, which is produced by melanocytes through the melanogenesis process. Abnormal accumulation of melanin causes skin problems such as freckles, solar lentigo and melasma, as well as cancer and vitiligo. Hence, the regulation of melanogenesis is a vital strategy to treat hyperpigmentation disorders. For example, hydroquinone is a hydroxyphenol chemical compound, which interferes with the activity of tyrosinase and is utilized as a skin-lightening agent in the cosmetic industry. However, hydroquinone may lead to side effects such as contact dermatitis and exogenous ochronosis (EO). Tretinoin, also known as all-trans retinoic acid (ATRA), is one of the other chemical compounds that inhibits tyrosinase activity, but its use causes complications like edema and skin sensitivity. Regarding the restriction of using existing compounds, the identification of alternative anti-melanogenic agents from natural sources has been greatly welcomed. This is because cosmetics derived from medicinal plants exhibit milder, non-biodegradable, and less toxic effects relative to chemical compounds.

It is essential to balance between synthesis and breakdown of collagen for maintaining skin rejuvenation and regeneration. Type I Collagen (80 to 85%) and Type III Collagen (10 to 15%) make up the largest amount of collagen in the skin. With increasing age, the amount of type I collagen declines, but an enhancement is observed in the amount of type III collagen. In order to improve the appearance of aging skin, multiple anti-aging strategies, including cosmetics, chemical peels, phototherapy, and micro-needling, have been developed so far. Nevertheless, the generated improvement through these methods has been less durable; thus, finding approaches for the durability of anti-aging effects has today attracted much attention.

Exosomes are found in body fluids such as saliva, blood, urine, serum, etc. A collection of evidence represents that exosomes have a crucial role in intercellular communication in physiological and pathological conditions. Besides, they enjoy the capability of passing through the membrane and protecting the degradation of proteins and RNAs contained in them and have the potential of transferring various compounds to the cell. Because of the features such as receptor specificity, not stimulating the immune system, and engineering them as drug carriers, exosomes have been introduced as an agent for the transfer of genetic material and the treatment of diseases. Exosomes are secreted from different cells, including stem cells, containing different molecules such as growth factors, proteins, broken nucleic acids, and microRNAs (miRNAs). Exosomes have different contents on the basis of the cell origin from which they are released. Exosomes can penetrate into the axon and regulate the inner mechanism of nerve repair and interfere in the repair of peripheral nerves and the recovery of the damaged area. Mesenchymal stem cells (MSCs) contain exosomes that carry out actions the same as the mesenchymal stem cells, including repair of tissue damage, inhibition of inflammatory responses, and regulation of the immune system. Using exosomes does not have risks such as aneuploidy and transplant (graft) rejection. To this end, they could be exploited in treating diseases such as autoimmunity and cancer as pharmaceutical compounds and nanocarriers to transfer drugs and genes.

Furthermore, skin ulcer healing is an unsolvable and so serious problem, indicating an urgent clinical need. To overcome this problem, extensive studies in connection with using exosomes derived from adipose-derived stem cells (ADSCs) have been performed. For this purpose, the fabrication of pharmaceutical compounds based on ADSCs-EXOs will have high efficacy for healing wounds. ADSCs-EXO compounds modulate immune and inflammatory responses at the site of the created wounds, as well as cause the strengthening of angiogenesis, skin cell reproduction, and collagen regeneration, and prevent wound hyperplasia. ADSCs have high stability and are not rejected by the immune system rather than ADSCs-EXO drugs, and their dosage can be simply controlled. Moreover, the above-listed compounds can operate as a combined carrier and scaffold for treatment, resulting in skin repair without scarring.

Human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) can greatly contribute to skin wound healing. Recent studies illustrate that exosomes derived from MSCs activate several signaling pathways, which are beneficial in healing wounds and cell growth. USC-CM (Umbilical cord blood-derived Stem Cell Conditioned Media) has a variety of growth factors associated with skin rejuvenation. The results of investigations demonstrate that USC-CM EXOs integrate into human dermal fibroblasts (HDFs) and are involved in cell migration and collagen synthesis. Besides, USC-CM EXOs are absorbed in human skin and boost the synthesis of type I collagen and elastin in the skin, which is critical for rejuvenating the skin.

With increasing age, human dermal fibroblasts (HDFs) gradually lose their capability of producing collagen and regenerating the intercellular matrix. Human dermal fibroblast-derived exosomes (HDF-XO) cause an enhancement in the type I procollagen expression and a substantial reduction in the MMP-1 expression through decreasing tumor necrosis factor-alpha (TNF-α) and the upregulation of transforming growth factor beta (TGF-β). Thus, among the clinical applications of HDF-XO-based medicinal composition, which can be noted, is the correction of facial lines (wrinkles) and reduction of skin aging rate.

Regarding the achieved findings, using exosomes obtained from plants, humans, algae, and edible mushrooms to treat hyperpigmentation and rejuvenation is a proper approach in the future of the cosmetics industry. Additionally, plant-derived exosomes are safe for the environment because of their small size, low toxicity, and high absorption; thus, they can operate as the next generation of drug delivery systems for treating a lot of diseases. While enjoying the excellent anti-melanogenesis and anti-aging (anti-wrinkle) properties, plant-derived exosomes can be utilized to decrease melanin and free radicals, thereby lightening and rejuvenating the skin. Nevertheless, exosomes possibly have a toxic effect on the skin. Thus, other additional tests such as an allergy skin test or an Ames test should be done as well. Few studies in conjunction with the application of plant exosomes to treat hyperpigmentation have been conducted so far, and many of the plants employed are indigenous to Korea and China.

Nowadays, diverse herbal compounds have been exploited in cosmetic formulations. However, utilizing plant-based cosmetic products to treat skin blemishes has encountered some problems due to the low solubility of plant compounds, their low affinity for targets, and their low and weak lightening effect in comparison with chemical compounds. This has led to the discovery of novel and promising technologies in promoting the efficacy of cosmetics with the natural-plant origin and using bioactive compounds and their delivery efficiency to the skin. For example, several nanotechnologies have been successfully carried out in the delivery of active ingredients to the skin. Among them, Nano Aloe Vera for more effective skin care, Nano Quercetin for delaying cell damage caused by UV rays, Nano Fullerene for collagen regeneration and protection against skin aging, Nano Lutein for maintaining antioxidant activity, and Nano-Resveratrol for protecting the skin against ultraviolet rays can be pointed out. Furthermore, it is found that the compounds gained from marine plants are involved in regulating skin pigmentation. Hence, due to the availability of an extensive range of plants with special medicinal properties in Iran, particularly for the skin treatments of eczema, blemishes & melasma, skin sagging & aging, the extraction of exosomes from more accessible plant sources with a wide distribution, spirulina algae, shiitake mushroom (which is a type of edible fungus) and, specifically, human serum is among the objectives of this invention.

Invention Objectives:

Multiple plants with special medicinal properties and without allergic effects are employed in Iran for the treatment of many skin diseases with easy access.
Plant-derived exosomes with high absorption and low toxicity, edible shiitake mushroom, and spirulina algae, are used as delivery systems and drug carriers to treat diseases.
Human blood serum is exploited exclusively for the extraction of exosomes and the application of exosomes in medicinal and cosmetic compounds.
Cosmetic products based on the aforementioned plant exosome, human serum, edible shiitake mushroom, and spirulina algae are produced for the treatment of hyperpigmentation.
Because of the excellent anti-melanogenesis and anti-aging properties of plant-derived exosomes, cosmetic products are generated for skin rejuvenation.
Plant exosomes are utilized to decline melanin and free radicals, and thus lighten and rejuvenate the skin.
This invention is an appropriate alternative to cosmetic creams for treating hyperpigmentation, skin treatments for eczema, blemishes and melasma, and skin sagging & aging, which will be a critical step towards upgrading the level of the health of communities.

Solution of problem

Medicinal products based on exosomes are limited to developed countries worldwide and the expense of their production to treat various diseases is very high. Therefore, the treatment of many incurable skin diseases with minimal costs can be achieved by using exosomes extracted from the extracts and medicinal compounds of plants, blood serum of people, medicinal mushrooms, and algae.

Anyway, the advancement of biotechnology knowledge to evaluate the interaction of medicinal compounds of plant extracts in question, blood serum of individuals, algae, and medicinal mushrooms to produce proper medicinal and cosmetic compounds with high performance based on exosomes is in line with the Islamic Republic of Iran’s 20-Year Vision Plan (1404 vision document). Hence, the medicinal products under study in this invention cause an enhancement in cell proliferation, synthesis, and penetration of skin collagen, and greatly contributed to skin rejuvenation. Thus, the effect of exosomes to treat human skin is evident by seeing increasing the expression of type I collagen and elastin. The results of the studies revealed that the absorption of exosomes in human skin enhances the synthesis of type I collagen and elastin in the skin, which are necessary for skin rejuvenation.

Hence, the current invention can be a benchmark for the improvement of wound healing, collagen formation, reduction of water loss in the stratum corneum (SC) area, skin treatments for eczema, blemishes and melasma, skin sagging & aging. As well as, it can be beneficially exploited as a cosmetic and medicinal combination to strengthen and improve the function and quality of the skin.

Medicinal compounds made respectively contain exosomes derived from the extract and essence of plants of the mint family, umbrellas, Rosaceae, borage, beech family (Fagaceae), Dwarf Pomegranate (Punica granatum L. var. nana), Laurus nobilis, berries, Amaryllidaceae, pistachios, frankincense, Cucurbitaceae, Carica papaya L., ginger (Zingiber officinale), Asparagaceae, chicory, Legume, Rutaceae, Asphodelaceae, blood serum of applicants, spirulina algae, and Shiitake mushroom based on cream, ointment, and salve, gel, serum, tonic, lotion, toner, detergents, cleansers, scrubs, masks. In this invention, some changes have been created in cosmetic ingredients. Besides, the combined exosomes of several medicinal plants and the blood of volunteers have been added, which has not yet been generated in any cosmetic product. Eventually, the exosomes derived from the extracts and essences of the studied plants and the blood serum of the subjects in the investigation were mixed with the intended cosmetic compounds. After creating a skin scratch on RATs, the gene expressions of Collagen I, Collagen III, Homo sapiens prolyl 4-hydroxylase subunit alpha 1, and Elastin were examined using tissue real-time PCR. Moreover, the techniques to evaluate the toxic properties of extracted exosomes and their survival rates, measuring the absorption of cells having exosomes, melanin content in cells through examining the activity of tyrosinase enzyme, measuring the bleaching effect in human models, measuring the size of exosomes by DLS method, shape and size range of exosomes by TEM method were explored. Furthermore, an allergy skin test was also performed to assess the toxicity effect of exosomes on the skin, and the results of the preliminary experiments demonstrated that the product in question can be a desirable choice for future empirical studies.

The procedure of the project and the devices employed in the preparation and approval of the made product are as follows:

The Choice of Medicinal Plants, Spirulina Algae and Shiitake Mushrooms

Initially, plants from the above-listed families, as well as spirulina algae and shiitake mushrooms, are chosen and gathered or purchased with respect to the target of the disease.

2- Preparation of Extracts and Essential Oils from the Plants under Study

At first, the fresh or dried plants are collected.
Water extraction from fresh plants or extraction from dried plants is carried out with BPS.
The target plants for the intended treatment are extracted by Hydro-Distillation (HD) method.
Exosomes derived from plants, and edible shiitake mushrooms are extracted in accordance with kit protocol.
The protein content of the extracted exosomes is assessed by the kit methods of Bicinchoninic acid (BCA), Western Blot, or Bradford assay.
Formulation for Making Medicinal and Cosmetic Compounds

The formulation, content, and composition of the constituent components vary regarding the type of product. For example, two phases of water and oil are typically utilized for the preparation of creams, lotions, and serums. These two components are kept together by an emulsifier. Moreover, antioxidants, preservatives, pH regulators, fragrances, additives, and active substances are also exploited in skin product formulations.

4- Design of Animal Experiments

20 Wistar rat samples were employed in order to conduct animal tests for evaluating the effect of medicinal products based on human and plant exosomes. After the extraction of exosomes, the medicinal products made based on exosomes were tested on 20 rat samples in two treatment and control groups, where the steps of the experiment are as follows:

4-1- Extraction Method of Exosomes

The extraction method of exosomes from human and plant cells is wholly identical, and a similar protocol was applied. Thus, 2 ml of the extracts of the examined plants and blood serum samples were separately transferred to micro-tubes. Then, it was centrifuged at 4 degrees for 10 minutes at 3000 rpm.

The exosome extraction kit with the brand name Exocib S, which was prepared and purchased from ANACELL (Supernatants), includes two solutions A and B, in which solution A was heated at a temperature of 37 degrees Celsius during the time interval of centrifugation in accordance with the sent instructions until no crystals remain in the solution and a completely transparent solution is achieved. To do so, some water is poured into a large petri dish, and a regular thermometer is placed in it, the container of the solution is held in the water for a few minutes with your hand, and it was vortexed after a few minutes so that a clear solution is obtained. According to the protocol of the manufacturer of the kit, a ratio of 5:1 (extract sample 5 and solution A in a ratio of 1) was taken from the serum samples in both groups, and the solution A in the exosome extraction kit using sampler in microliters and moved into the new microtube. After each dilution, the microtubes were vortexed for a few minutes. Then, they were transferred to a refrigerator with a temperature of 4 degrees and incubated for 12 hours. The specimens gained from the previous step were vortexed for 1 minute to make them homogenous. Next, centrifugation was performed again for 40 minutes at 4 degrees with a speed of 3000 rpm, and the upper solution was discarded. After that, 50 microliters of solution B were added to the remaining solution according to the standard protocol of the kit manufacturer, and the microtubes were transferred to a temperature of -20°C and stored.

4-2- Measuring Absorption of Cells with Exosome by Spectrophotometric Method

Taking into account that exosomes contain combinations of nucleic acids, DNA, RNA, and protein, so light absorption was measured at wavelengths of 260 to 280 nm using a spectrophotometer. The spectrophotometric technique is on the basis of the amount of transmitting or absorbing ultraviolet light by the substance. For DNA and RNA, the samples are exposed to ultraviolet light at a wavelength of 260 nm (260 A). As the absorption of light at 260 nm by the sample is more, the nucleic acid concentration in the sample containing the exosome will be higher. Besides, high light absorption at 280 nm represents an enhancement in protein content in the extracted product. Therefore, the spectrophotometry method was exploited to confirm the presence of exosomes in cosmetic products, and it was compared with medicinal and cosmetic products without exosomes.

4-3- Confirmation of the Quality and Structure of Exosomes Using Transmission Electron Microscopy (TEM)

An electron microscope was exploited to see the morphology and appearance of exosomes. For the preparation of the specimens to study via transmission electron microscopy (TEM), the exosome suspension was initially fixed using 1% glutaraldehyde and placed on a carbon-coated grid so that it is dried at room temperature. The grids were washed with PBS (phosphate buffered saline) sterile. Then, staining was done with 2% uranyl acetate (UA) dye for 10 minutes. Morphological studies associated with exosomes were performed using LEO field-emission scanning electron microscope model 912AB made in Germany at 120 KW voltage, and the images were ready to be examined. On the basis of table 1, the integrity of the exosome membrane was retained during the isolation processes, and the spherical structure was not damaged. Besides, the diameter of exosomes in this image is 100 nm or less.

4-4- Determination of Exosome Size and Percentage Utilizing Dynamic Light Scattering (DLS):

This technique operates as the measurement of the Brownian motion related to the hydrodynamic diameter based on Stokes-Einstein. The results of DLS are declared in three forms: intensity, volume, and number. In this study, the solvent used was phosphate buffer to specify the diameter size of exosomes by the DLS technique, and its refractive index is 1.33 with a viscosity of 1.64. The desired device is the Particle Size Analyze of Cordouan Company, Vasco3 model, France, where the size of existing particles is determined using light radiation and scattering. As illustrated in table 2, more than 80% of the extracted vesicles have a size of less than 100 nm, reflecting the presence of exosomes derived from plant samples, spirulina algae, and Shiitake medicinal mushrooms.

5- Evaluation of the Toxic Properties of the Extracted Exosomes and Their Survival Rates in Vitro

In order to examine the toxicity of exosomes on skin cells, they were kept in a CO2 incubator at 37 degrees after defrosting and culturing the cells in the desired medium, and the cells were passaged three days later. Then, the toxicity of the exosome was assessed on skin cells at different times of 24, 48, and 72 hours after incubation using an MTT assay. The results of the studies shown in Figure 5 indicate the cytotoxic effect of exosome relative to the control group (in comparison with DMEM culture medium) at different concentrations of 2.5, 5, 7.5, and 10 micrograms per milliliter on healthy skin cells (at a concentration of 7.5 micrograms per milliliter), and the survival rate of healthy cells incubated with exosome has reached 29.5% compared to DMEM culture medium as a control, and the IC50 value is 2.8 micrograms per milliliter.

6- Carrying out Clinical and Molecular Trials In Vivo

6-1- Examining Gene Expression at the RNA Level and Performing Real Time Tissue Testing

To study the gene expression of Collagen I, Collagen III, Homo sapiens prolyl 4-hydroxylase subunit alpha 1, and Elastin, the real-time PCR was initially conducted and analyzed using Pars Toos kit and ABI device based on the primers designed in . As indicated in Figure 7, the high gene expression of Collagen I and Collagen III relative to the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) housekeeping gene was examined, which is visible.

6-2- Examining the Tyrosinase Enzyme Expression

Melanin production in the skin and melanogenesis regulation is a vital strategy to treat hyperpigmentation disorders, which interfere with tyrosinase activity. Therefore, given that tyrosinase enzyme is of great importance in medicine and pharmaceuticals, and considering that increasing the expression of this enzyme declines the formation of skin pigments and reduces freckles and other skin spots, so investigating the expression of tyrosinase enzyme using SDS PAGE and western blotting using Bio-Metra electro-blotter was carried out. In table 5, the existence of the 58 (kDa) band corroborates the presence of tyrosinase enzyme in the epidermal layer of the skin of rats treated with medicinal compounds containing exosomes.

7- Performing Clinical Tests on Humans

For assessing the efficacy of exosome-based medicinal compounds on humans, about 50 healthy people and 50 people with skin eczema diseases, all kinds of skin pimples, hives, burns, skin spots, and skin cracks were chosen under the supervision of an allergy specialist. First, an allergy test was done to examine the toxicity effect of exosomes on the skin of subjects. After assurance of the non-allergenicity of the investigated subjects to medicinal compounds based on exosomes, clinical experiments on the remaining subjects were conducted, and macroscopic evaluation before and after the treatment was done. It is worth mentioning that some individuals were allergic to the herbal compounds employed; thus, medicinal compounds based on the exosomes from the subjects' blood serum were specifically used (table 6).

Advantage effects of invention

This invention provides our need in importing expensive drugs and limits the exorbitant cost spent on expensive chemical drugs.

2. The compounds used in this invention are based on green or phyto synthesis and have very few side effects compared to synthetic and chemical compounds, and also have less toxic properties compared to synthetic and chemical types.

3. Medicinal and cosmetic formulations used in this invention are environmentally friendly and biodegradable, and their effects are mild and non-aggressive.

4. These compounds usually have synergistic or synergistic effects in treatment.

5. It is expected that after conducting clinical tests, the herbal composition produced will increase the effect of other cosmetic creams in improving patients with skin diseases and repairing the epidermis layer and rejuvenating the skin.

6. By making cosmetic-sanitary compounds based on human exosomes, it is possible to produce highly effective and completely specific medicines for each person, and the level of allergenicity of cosmetic products can be reduced.

7. By producing the above-mentioned medicinal compound, which will not have any special chemical side effects and does not involve a lot of cost in case of commercialization, it can be used to heal wounds, treat skin patients, and obviously, in case of success, it will also save foreign currency costs for The introduction of expensive drugs with the commercial production of this drug also achieved the goal of gaining currency.

: Examining exosomes using electron microscope: Using TEM technique, the structure of exosomes was examined microscopically. The integrity of the exosome membrane is preserved during the separation processes and the spherical structure is not damaged. Also, in this image, the diameter of exosomes is 100 nm or less. TEM micrographs of isolated exosomes including membrane-enclosed spherical particles at 100, 200, and 500 nm magnification.

: Investigating the level of tyrosinase enzyme expression by western blotting method in order to determine the presence of tyrosinase enzyme and melanin production in the skin epidermis of mice treated with exosome medicinal compounds.

Examples

The production process of this invention in the industrial phase is as follows:

For production in the industrial phase, appropriate plant species are initially selected with regard to the purpose of the treatment. In the case of human exosomes, after taking blood and preparing the serum of any individual, the exosome can be extracted and exploited in medicinal and health compounds with proper formulation to enhance the efficacy of aforementioned medicinal compounds (All the above phases are carried out in a clean room and sterile conditions).

The guide for employing the invention in one of the industrial applications is as follows:

The choice of the suitable plant species with respect to the treatment goal

Sampling blood from the applicants and preparing blood serum from the applicants after performing the allergy test

Utilizing clean rooms and devices for the extraction, analysis, and expression of genes, making medicinal, cosmetic, and health compounds

Functional study of medicinal composition based on plant and human exosomes

Using medicinal compounds in the form of creams, ointments, gels, serums, tonics, lotions, toners, detergents, cleansers, scrubs, masks and etc.

Table1: Examining exosomes using DLS (Dynamic Light Scattering) method: The diameter of the particles in the suspension containing the isolated exosomes was determined using the VASCO3 model DLS device. The results of the DLS charts show that more than 80% of the extracted vesicles are less than 100 nm in size, which indicates the presence of the extracted exosomes in the examined plant species, algae and medicinal mushrooms.

Table 2: Investigating the toxic properties of extracted exosomes and their survival rate by MTT assay method on epidermal cells of the skin of mice treated with medicinal compounds containing exosome.

Table 3: Sequence of primers used for PCR and Real time PCR test

Table 4: Quantitative investigation of gene expression of Collagen I, Collagen III, Homo sapiens prolyl 4-hydroxylase subunit alpha 1 and Elastin genes using Realtime PCR test: NC in control and treated mice

This invention can be exploited in the pharmaceutical-medical industry (cancer and viral diseases) as an effective medicine to cure a variety of skin diseases, including eczema, blemishes & melasma, skin sagging & aging, cellular regeneration of human skin and enhancing the water content or moisture of the skin, preventing skin peeling, improving skin thickness, increasing skin elasticity, removing skin moles, removing skin pimples, improving skin texture, reducing the size of skin pores, increasing skin smoothness, improving skin aging spots, burn spots, improving skin color and improving the appearance of wounds and healing wounds in diabetic people.

Claims

The invention of medicinal products based on plant exosomes, algae, and medicinal mushrooms to treat skin diseases are beneficial compounds, which have been selected from the extracts and essences of the intended plant families in accordance with the disease target. They are made with purified exosomes from the above-listed plants, spirulina algae, and shiitake mushrooms. Plant-derived exosomes have a structure similar to exosomes isolated from mammals and act as extracellular messengers in intercellular communication. Moreover, these vesicles are capable of transferring mRNAs, microRNAs (miRNAs), bioactive lipids, and proteins to human and animal cells.
According to claim 1, medicinal compounds made respectively contain exosomes derived from the extract and essence of plants of the mint family, umbrellas, Rosaceae, borage, beech family (Fagaceae), Dwarf Pomegranate (Punica granatum L. var. nana), Laurus nobilis, berries, Amaryllidaceae, pistachios, frankincense, Cucurbitaceae, Carica papaya L., ginger (Zingiber officinale), Asparagaceae, chicory, Legume, Rutaceae, Asphodelaceae, blood serum of applicants, spirulina algae, and Shiitake mushroom based on cream, ointment, and salve, gel, serum, tonic, lotion, toner, detergents, cleansers, scrubs, masks.
the medicinal products in this invention cause an enhancement in cell proliferation in synthesis and penetration of skin collagen, and greatly contributed to skin rejuvenation. Moreover, the absorption of exosomes in human skin enhances the synthesis of type I collagen, type III collagen, and elastin, which are necessary for skin rejuvenation.
a plant-derived exosome for treating hyperpigmentation and rejuvenation is a suitable approach in the future of the cosmetics industry. Due to their small size, low toxicity, and high absorption, they are safe for the environment. Furthermore, the excellent anti-melanogenesis and anti-aging properties of plant-derived exosomes in regenerated human skin tissue, which is identical to the human epidermis, provide the grounds for future clinical trials.
the above-listed cosmetic compounds help to heal the wounds of diabetic patients and can be exploited as a drug carrier as well.
in order to confirm the authenticity of cosmetic ingredients made, after creating a skin scratch on RATs, the gene expressions of Collagen I, Collagen III, Homo sapiens prolyl 4-hydroxylase subunit alpha 1, and Elastin were examined using tissue real-time PCR. Moreover, the techniques to evaluate the toxic properties of extracted exosomes and their survival rates, measuring the absorption of cells having exosomes, the activity of tyrosinase enzyme, measuring the size of exosomes by DLS method, shape and size range of exosomes by TEM method was explored.