PROCESS FOR OBTAINING ENRICHED COFFEA CANEPHORA EXTRACT FROM PLANT STEM CELLS, ENRICHED EXTRACT, USE OF ENRICHED COFFEA CANEPHORA EXTRACT IN SKIN CARE FORMULATIONS AND FORMULATION CONTAINING THE ENRICHED EXTRACT

Description

1 / 21 PROCESS FOR OBTAINING ENRICHED EXTRACT OF Coffea canephora FROM PLANT STEM CELLS, ENRICHED EXTRACT, USE OF ENRICHED EXTRACT OF Coffea canephora IN SKIN CARE FORMULATIONS AND FORMULATION CONTAINING THE ENRICHED EXTRACT. BRIEF DESCRIPTION

[001] This patent application relates to a “PROCESS FOR OBTAINING ENRICHED COFFEA CANEPORA EXTRACT FROM PLANT STEM CELLS, ENRICHED EXTRACT, USE OF ENRICHED COFFEA CANEPORA EXTRACT IN SKIN CARE FORMULATIONS AND FORMULATION CONTAINING THE ENRICHED EXTRACT”, which refers to obtaining an enriched Coffea canephora extract from the lysate of undifferentiated, totipotent and homogeneous plant cells obtained from leaf explants, together with previously enriched Murashige and Skoog culture medium, which is subsequently used in the preparation of pharmaceutical formulations for skin care, such as formulations with anti-aging action; tissue regeneration; anti-inflammatory action; antioxidant action; collagen production stimulation; Neovascularization; preventative and restorative treatment of skin blemishes.To obtain plant stem cells from Coffea canephora, which will subsequently be lysed, a biotechnological process is applied consisting of: dedifferentiation, induction, and multiplication of plant stem cells. The present invention also relates to the enriched extract obtained, its use, and a liposomal formulation comprising the enriched extract obtained. FIELD OF APPLICATION

[002] The present invention belongs to the section of human needs, to the field of medical science or hygiene, more specifically, to the field of preparations for medical or personal hygiene purposes, such as cosmetic preparations, since it refers to obtaining Coffea canephora extract from lysate prepared with undifferentiated, totipotent and homogeneous plant cells, obtained from leaf explants and enriched with previously enriched Murashige and Skoog medium, Petition 870230023068, dated 03 / 17 / 2023, page 10 / 46 2 / 21 for use in the preparation of skin care formulations; to the extract itself with bioactivity on the skin; to the use of the extract for the preparation of skin care formulations and to a formulation itself. CONVINCING

[003] The use of plant extracts in skin treatment products has been growing. This use is often based on ancestral knowledge; however, with advances in science and industry, increasingly high-quality and effective technological products whose active ingredients are natural extracts are being found.

[004] In recent years, coffee has been explored by the cosmetics industry due to its bioactive compounds. Among these bioactive compounds, chlorogenic acid stands out, which has beneficial effects such as protection against ultraviolet radiation, anti-inflammatory and antimicrobial action, in addition to having positive effects that help improve the skin (BAGDAS et al., 2014: Pharmacologic overview of systemic chlorogenic acid therapy on experimental wound healing).

[005] Coffee has antioxidant potential and contains compounds described as important cell protectors (ANDRADE, 2011: Evaluation of extraction techniques and antioxidant potential of extracts obtained from coffee husk and grounds (Coffea arabica L.)). These effects are observed due to the presence of phenolic compounds that act in scavenging free radicals, contributing to the prevention of cellular aging (FERNÁNDEZ et al., 2009: Superoxide dismutase and catalase: tissue activities and relation with age in the long-lived species Margaritifera margaritifera).

[006] The use of coffee, in its various forms, and its bioactive compounds by the cosmetic industry has been growing over the years.The use of coffee grounds, for example, in cosmetic products is widespread, given that this residue is essentially composed of polysaccharides, oligosaccharides, lipids, aliphatic acids, amino acids, proteins, alkaloids (such as caffeine and trigonelline), minerals, phenolic compounds, lignin, melanoidins, and volatile compounds (HERMANN et al., 2019: Evaluation of the use of coffee grounds for use in cosmetic products).

[007] Another example is the use of green coffee, which is recognized as a source of antioxidant phenolic compounds that help combat stress caused by Petition 870230023068, dated 03 / 17 / 2023, page 11 / 46 3 / 21 free radicals, thus promoting human health. The topical use of compounds present in green coffee has been recommended for the prevention of photoaging and natural aging, and as a sun protection agent (BUZANELLO, 2019: Development of green coffee nanoemulsions for pharmaceutical purposes).

[008] Coffea canephora is a widely known and commercially available coffee species, especially as the main raw material for instant coffee (DOS SANTOS, 2017: Development of a cosmeceutical formulation for the eye area with standardized green coffee extract). Compared to Arabica coffee, C. canephora stands out due to its higher caffeine, chlorogenic acid and solids content, as well as its lower price (VIGNOLI et al., 2014: Roasting process affects differently the bioactive compounds and the antioxidant activity of Arabica and Conilon coffees).

[009] Given the widespread use of coffee and its bioactive compounds, the present invention describes a process for obtaining an enriched extract of Coffea canephora prepared from the lysate of undifferentiated, totipotent, and homogeneous C. canephora cells, derived from calluses of C. canephora leaf explants, with Murashige and Skoog culture medium enriched with sucrose, 2,4-dichlorophenoxyacetic acid (2,4-D), Gamborg's vitamins, L-cysteine, malt extract, casein, and benzylaminopurine (BAP), which is lyophilized and encapsulated in liposomes, to be subsequently used in the preparation of pharmaceutical formulations for skin care, such as formulations with anti-aging action; tissue regeneration; anti-inflammatory action; antioxidant action; collagen production stimulation; neovascularization; prevention and repair of skin blemishes.

[010] The enriched extract obtained from the process described in the present invention has numerous primary and secondary metabolites with a complex and indeterminate composition with anti-aging, tissue-regenerating, anti-inflammatory, antioxidant, collagen production-stimulating and neovascularization properties, as well as preventive and reparative properties for skin blemishes, especially on the face.

[011] The extract obtained from the lysate of undifferentiated, totipotent and homogeneous C. canephora cells, derived from calluses of C. canephora leaf explants, associated with the previously enriched Murashige and Skoog culture medium, Petition 870230023068, dated 03 / 17 / 2023, page 12 / 46 4 / 21 was partially characterized in relation to its chemical composition, and the presence of total phenolics, total flavonoids, caffeic acid, chlorogenic acids, rosmarinic acid, catechin gallate, rutin, sucrose, and the amino acids Glycine, Lysine, Histidine, Arginine, Asparagine, Glutamine, Serine, Threonine, Tyrosine, Alanine, Valine, Leucine, Isoleucine, Proline, and Phenylalanine could be verified.

[012] In addition to being partially characterized in relation to its chemical composition, the extract was subjected to numerous biological tests and assays to evaluate and prove its possible therapeutic effects and benefits. Chemical and in vitro (cell culture) tests were performed to verify antioxidant activity and the ability to stimulate fibroblast migration and proliferation. Tests of the product's anti-inflammatory activities, such as inhibition of the production of superoxide anion, nitric oxide, inflammatory cytokines such as IL-6 and TNF-α, and inhibition of NF-κB (nuclear factor kappa B), were also performed.

[013] Based on the results obtained, it was found that the extract obtained from the lysate of undifferentiated, totipotent, and homogeneous C. canephora cells, derived from calluses of C. canephora leaf explants, associated with the previously enriched Murashige and Skoog culture medium, possesses numerous compounds with therapeutic potential. Based on the good results obtained, a liposomal formulation was developed capable of incorporating the obtained hydrophilic extract and transporting it to the dermis, where fibroblasts, for example, responsible for collagen production, and blood vessels are found.

[014] The formulation containing the extract was characterized, verifying the formation of liposomes and the incorporation of the extract by particle size analysis tests for dynamic light scattering, atomic force microscopy and transmission electron microscopy. In addition, the preparation was tested on animals in order to verify its effectiveness on tissue repair capacity. In the tests, it was observed that the animals treated with the formulation showed a greater speed of repair of the damaged tissues, in relation to the control group. In biochemical studies carried out with biopsies of the injured tissues treated with the product, a decrease in inflammatory infiltrate was observed, especially in neutrophils and macrophages, and a decrease in Petition 870230023068, dated 03 / 17 / 2023, page 13 / 46 5 / 21 cytokines TNF-α and IL-6 (i.e., less inflammation), an increase in IL-10 (which is anti-inflammatory), and an increase in Transforming Growth Factor beta (TGF-β), a protein that controls fibroblast proliferation, and vascular endothelial growth factor (VEGF), which plays a key role in angiogenesis. Furthermore, an increase in collagen production was observed in animals treated with the formulation.

[015] This same formulation was also clinically tested in humans for the treatment of melasma. In the test, 23 patients with melasma were treated daily with the formulation for 60 days. At the end, a significant clinical improvement in quality of life was observed, particularly in psychological health and social interaction; a significant reduction in the area affected by melasma in patients after 60 days of treatment; and an improvement in the severity of melasma at the end of the study, demonstrating the effectiveness of the treatment in reducing the spots.

[016] Thus, the present invention describes a biotechnological process that makes it possible to obtain an enriched extract of Coffea canephora prepared from undifferentiated, totipotent and homogeneous C. canephora cells, derived from calluses of C. canephora explants; and previously enriched Murashige and Skoog culture medium; its use for obtaining compositions for skin care, in addition to an effective and safe formulation for this purpose.

[017] It is worth highlighting that the present invention was developed entirely biotechnologically. The entire process of obtaining the product went through important points through in-depth knowledge of plant biotechnology. The somatic cells of coffee, which are already differentiated cells, for example the cells of the leaves used, underwent a process of cellular dedifferentiation, where the somatic cells ceased to be cells with pre-programmed genetic information and were induced to regress to their meristematic and totipotent state, now called plant stem cells. This process, although used in other plant species, is unique to each species, where the composition of the culture medium, supplementation with different chemical agents and with plant growth regulators, such as 2,4-D and BAP in specific concentrations for each species are key points for Petition 870230023068, dated 03 / 17 / 2023, page 14 / 46 6 / 21 to dedifferentiate these already differentiated cells. The enriched extract of Coffea canephora, prepared from undifferentiated, totipotent, and homogeneous plant cells derived from calluses of C. canephora explants and previously enriched Murashige and Skoog culture medium, stands out because it is not merely an extract obtained directly from the plant through the simple collection of leaves or other parts thereof. To obtain it, the leaves undergo profound physiological and biochemical changes culminating in the production of cells with the totipotent capacity to produce all the compounds present in the C. canephora plant—the so-called plant stem cells from which the bioactive compounds to be used in the intended formulation, as well as in other skin care formulations, were extracted. BACKGROUND OF THE INVENTION

[018] Currently, prior art is available that refers to the use of plant extracts from different parts of coffee species, as well as the metabolites produced by these plants, by the cosmetic and pharmaceutical industries. However, none of them anticipates what is described in the present application, which refers to a process for obtaining an enriched extract of Coffea canephora obtained from undifferentiated, totipotent and homogeneous C. canephora cells, derived from calluses of C. canephora leaf explants; and previously enriched Murashige and Skoog culture media; its use for obtaining compositions for skin care, as well as an effective and safe formulation for this purpose, as observed below.

[019] The prior art BR112013002195A2, entitled Use of green coffee and probiotic for regulating skin pigmentation, refers to a composition containing green coffee beans and microorganisms or enzymes capable of promoting the hydrolysis of chlorogenic acids to generate phenolic acids for the prevention and / or treatment of skin hyperpigmentation, skin color imperfections, and other skin disorders characterized by abnormal pigmentation. The prior art does not refer to the preparation of an enriched extract prepared from undifferentiated, totipotent, and homogeneous C. canephora cells derived from calluses of leaf explants. Petition 870230023068, dated 03 / 17 / 2023, page 15 / 46 7 / 21 of C. canephora; and Murashige and Skoog culture medium previously enriched or a liposomal formulation containing it, as well as not suggesting any process that would allow obtaining such an extract.

[020] The prior art BR112013002326B1, entitled Cosmetic Use of a Mixture of Roasted Coffee Beans, describes the cosmetic use of an effective amount of at least one mixture of roasted and unroasted coffee beans or extracts thereof, characterized in that it is used as an active agent for the preparation of an oral composition for the treatment and / or prevention of skin pigmentation disorders and / or skin pigmentation imperfections, said extracts being of aqueous-alcoholic or alcoholic extraction from coffee beans. The prior art does not anticipate the present invention, firstly by employing coffee beans, while the present invention uses cells from callus of leaf explants of C. canephora, therefore, different parts of the plant; secondly, the prior art describes obtaining a composition for oral use, while the present invention claims the use of the extract obtained in the preparation of compositions for topical use.

[021] The prior art BR112013002216B1, entitled Cosmetic Use of Roasted Coffee Beans, describes the cosmetic use of an effective amount of roasted coffee beans or an extract thereof, characterized in that it acts as an active agent for the treatment and / or prevention of skin pigmentation disorders associated with hyperpigmentation, wherein said active agent is comprised in an oral composition. The extract employed is derived from selected coffee beans of the species Coffea arabica, Coffea robusta, Coffea canephora or Coffea iberica. Thus, like the aforementioned prior art, this does not anticipate the present invention, initially by employing coffee beans, while the present invention utilizes cells derived from callus of leaf explants of C.canephora, therefore, different parts of the plant; secondly, the prior art describes obtaining a composition for oral use, while the present invention claims the use of the extract obtained in the preparation of compositions for topical use.

[022] Previous US9095532B2, entitled Composition for anti-aging or antioxidant composition containing plant stem cell line derived from cambium or Panax ginseng Petition 870230023068, dated 03 / 17 / 2023, page 16 / 46 The patent application 8 / 21, including wild ginseng and ginseng as active components, describes an anti-aging or antioxidant composition containing as an active ingredient a plant stem cell line derived from Panax ginseng, including wild ginseng and ginseng, an extract thereof, a lysate thereof, or a culture of these cells. The patent application states that the composition minimized side effects compared to existing anti-aging and antioxidant agents and is therefore safe for the skin. Furthermore, the described composition demonstrates an antioxidant effect of inhibiting reactive oxygen species caused by exposure to UV radiation, which is the main cause of skin aging, and can effectively reduce or inhibit aging-related factors.Although the prior art mentions the use of a plant stem cell line, the species employed is different, as is the procedure used to obtain it, despite the prior art also aiming to obtain compositions for skin care.

[023] The prior art US11033587B2, entitled Administration of cells and cellular extracts for rejuvenation, describes methods and agents for improving the appearance or restoring the health of cells and tissues, preferably aged or damaged skin. Although the invention aims at the production of compositions for skin care, the active ingredients used are of animal origin, more specifically, from fish; thus, in some embodiments, the invention refers to compositions of cells, eggs, cell extracts, egg extracts and extract components, such as purified nucleic acids, polypeptides, lipids, carbohydrates or other natural products. Therefore, the prior art does not anticipate the present invention.

[024] The prior art AU2008301352B2, entitled Plant stem cell line derived from cambium of herbaceous plant with storage root and method for isolating the same, refers to a method for isolating and optimizing the proliferation of homogeneous cell lines, which have the ability to divide and are obtained from the meristematic tissue of selected plants from among: ginseng, Codonopsis lanceolata, Ostericum koreanum Kitagawa, Platycodon grandiflorum, Pueraria thunbergiana, Aralia contonentalis Kitagawa, Ledebouriella seseloides, Angelica gigas Nakai, carrot, sweet potato, maca and cassava. Although the prior art describes methods of Petition 870230023068, dated 03 / 17 / 2023, page 17 / 46 9 / 21 isolation and optimization of homogeneous cell line proliferation, it does not describe any specific method for C. canephora, it should be noted that different plant species require different procedures for optimization and proliferation of homogeneous cell lines. Furthermore, the prior art does not suggest obtaining extracts with therapeutic activity for skin care prepared from undifferentiated, totipotent, and homogeneous C. canephora cells derived from calluses of C. canephora leaf explants; and previously enriched Murashige and Skoog culture media.

[025] The prior art WO2012171106A1, entitled Skin cosmetic compositions comprising Malus domestica extract and Argania spinosa sprout extract for improving skin appearance, refers to a composition comprising an epidermal stem cell activator and a dermal stem cell activator that have the potential to improve skin appearance, said activators being Malus domestica and Argania spinosa. Given that the prior art does not suggest the use of extracts with therapeutic activity for skin care prepared from undifferentiated, totipotent, and homogeneous C. canephora cells, derived from calluses of C. canephora leaf explants; and from previously enriched Murashige and Skoog culture medium, it does not anticipate the present invention.

[026] Based on the prior art found in the current state of the art, it is noted that none of them describes the technical advances provided by the present invention, such as a highly reproducible process for obtaining an enriched, homogeneous, and effective extract from undifferentiated, totipotent, and homogeneous C. canephora cells derived from calluses of C. canephora leaf explants and the previously enriched Murashige and Skoog culture medium. Furthermore, it should be highlighted that the present invention employs plant cells derived from undifferentiated C. canephora calluses associated with the previously enriched Murashige and Skoog medium, and that, therefore, it is an enriched extract, which has important differences compared to a natural extract obtained from cells derived from C. canephora calluses without the dedifferentiation process and association with the aforementioned culture medium. In addition, no prior art was found that Petition 870230023068, dated 03 / 17 / 2023, page 18 / 46 10 / 21 anticipated or even suggested obtaining such an enriched extract, which corroborates the inventiveness of the matter. OBJECTIVE OF THE INVENTION

[027] The present invention aims to provide: an enriched extract of Coffea canephora, prepared from a lysate of undifferentiated, totipotent and homogeneous plant cells, associated with previously enriched Murashige and Skoog medium, to be used in pharmaceutical and cosmetic formulations for skin care; the process used in the production of the enriched extract; and a formulation containing the enriched extract; in addition to aiming to promote and stimulate the production of products based on plant actives. OF THE INVENTION

[028] The present invention relates to obtaining an enriched Coffea canephora extract, prepared from the lysate of undifferentiated, totipotent, and homogeneous plant cells, together with previously enriched Murashige and Skoog culture medium, which is subsequently used in the preparation of pharmaceutical formulations for skin care, such as formulations with anti-aging action; tissue regeneration; anti-inflammatory action; antioxidant action; collagen production stimulation; neovascularization; prevention and repair of skin blemishes. To obtain the cells used in the lysate, cells from Coffea canephora leaves undergo a biotechnological process consisting of dedifferentiation, induction, and multiplication of plant stem cells. The present invention also relates to the enriched extract obtained, its use in the preparation of skin compositions, and a liposomal formulation comprising the enriched extract obtained. ADVANTAGES OF THE INVENTION

[029] The present invention has the following main advantages: ^ To provide a highly reproducible process for obtaining an enriched extract of Coffea canephora from undifferentiated, totipotent, and homogeneous plant cells with a high capacity for producing secondary metabolites, which guarantees obtaining a homogeneous extract with activity. Petition 870230023068, dated 03 / 17 / 2023, page 19 / 46 11 / 21 biological, associated with previously enriched Murashige and Skoog culture medium; To provide an enriched extract of Coffea canephora, prepared from undifferentiated, totipotent, and homogeneous plant cells derived from calluses of C. canephora leaf explants and previously enriched Murashige and Skoog culture medium, to be used in pharmaceutical and cosmetic formulations for skin care, due to its anti-aging, tissue-regenerating, anti-inflammatory, antioxidant, collagen-producing, neovascularizing, preventive, and reparative actions against skin blemishes; To provide a liposomal formulation comprising an enriched extract of Coffea canephora, prepared from undifferentiated, totipotent, and homogeneous plant cells derived from calluses of C. canephora leaf explants and previously enriched Murashige and Skoog culture medium, whose delivery of active ingredients to the dermis and epidermis is facilitated and efficient due to the high permeability of the liposomes prepared and present in the formulation; To provide a liposomal formulation comprising a safe and fast-acting enriched extract of Coffea canephora, associated with pathways that cause aging, such as inflammatory and oxidative pathways; To provide a liposomal formulation comprising a safe and fast-acting enriched extract of Coffea canephora, whose results are observed from the first month of use, demonstrating effectiveness in improving skin rejuvenation, in addition to antioxidant, regenerative, anti-inflammatory effects, promoting collagen synthesis, brightening and reducing melasma. DESCRIPTION OF THE FIGURES

[030] In order to facilitate a better understanding of the present invention, reference will be made to the figures described below: FIG. 1: In vivo effects of the liposomal formulation (liposomal stem cell extract formulation from C. canephora (LSCECC)) demonstrating the ability to reduce Petition 870230023068, dated 03 / 17 / 2023, page 20 / 46 12 / 21 the area and intensity of melasma spots. View of the right cheekbone in Wood's light and external white light at time 0 (D0), 30 days (D30) and 60 days (D60); FIG. 2: In vivo effects of the liposomal formulation (liposomal stem cell extract formulation from C. canephora (LSCECC)) demonstrating tissue repair and wound healing capacity. Wound healing effects of LSCECC over 21 days of treatment. (A) Macroscopic images of wounds on the indicated days after injury. (B) Percentage of wound closure at 2, 5, 7, 10, 14, and 21 days after daily topical application of LSCECC or control vehicle. Data are expressed as percentage reduction in wound area from original size (day 0). Values ​​represent mean ± SD (n = 20 wounds / group). * Significant (p < 0.05) compared to the vehicle control; V FIG. 3: APCI-MS representation for C. canephora stem cell extracts in (A) negative ionization and (B) positive ionization; FIG. 4: Decrease in nitric oxide and superoxide anion production in vitro. Effect of SCECC on the production of (A) nitric oxide (NO) and (B) superoxide radical (O2·-). RAW 264.7 macrophages were exposed to different concentrations of SCECC and stimulated with LPS (1 pg / mL). L-NIL 30 μM and Tempol 10 μM were used as positive controls. Results are expressed as mean ± SD. # Significant (p < 0.05) compared to the baseline control group. * Significant (p < 0.05) compared to LPS-induced cells by one-way ANOVA followed by Tukey's post-hoc test; FIG. 5: In vitro decrease in the production of inflammatory cytokines IL-6 and TNF-α. Effect of SCECC on the concentration of pro-inflammatory cytokines IL-6 (A) and TNF-α (B). RAW 264.7 macrophages were exposed to different concentrations of SCECC in the presence or absence of LPS. Results are expressed as mean ± SD. # Significant (p < 0.05) compared to the baseline control group. * Significant (p < 0.05) compared to LPS-induced cells by one-way ANOVA followed by Tukey's post-hoc test; FIG. 6: In vitro inhibition of NF-κB production. Effect of SCECC on NF-κB inhibitory activity. HEK 293 cells were exposed to different concentrations Petition 870230023068, dated 03 / 17 / 2023, page 21 / 46 13 / 21 of SCECC and incubated for 6 h with or without TNF-α (2 ng mL-1). Results were expressed as the percentage of NF-kB inhibitory activity and are expressed as mean ±SD. * Significant (p < 0.05) compared to the control with TNF-α-induced cells by one-way ANOVA followed by Tukey's post hoc test; ^ FIG. 7: Positive effects on fibroblast proliferation and migration. Influence of SCECC on fibroblast proliferation and migration. (A) Percentage of total cell count in the scraping area after 14 h of incubation (37°C at 5% CO2) without (control) or with SCECC at 10, 50 and 100 pg mL-1 and PDGF 2 ng mL-1 (positive control). Bars represent the mean ±SD of three independent experiments. # Indicates significant differences compared to untreated cells (control) (p<0.05); *Compared to PDGF (p < 0.05). (B) Representative microscope image illustrating the cell wound immediately after injury (zero scratch 0 h) and after 14 h post-treatment, as indicated in each panel. (C) Effect of SCECC on fibroblast proliferation. Results were expressed as absorbance at a wavelength of 450 nm and are expressed as mean ±SD. *Indicates significant differences compared to untreated cells (control) (p<0.05). DETAILED DESCRIPTION OF THE INVENTION

[031] The present invention relates to the process of obtaining enriched Coffea canephora extract, prepared from the lysate of undifferentiated, totipotent and homogeneous plant cells, together with previously enriched Murashige and Skoog culture medium; to the enriched extract itself, to its use in the preparation of skin compositions and to a liposomal formulation comprising the enriched extract obtained.

[032] To obtain the enriched Coffea canephora extract, well-expanded C. canephora leaves with a medium intense green color and no signs of lesions, diseases, or insect infestation were selected from 10 individuals (plants). These leaves were washed in running tap water three times for 5 minutes each time, using neutral commercial detergent. After washing, the leaves were immersed in ethanol at Petition 870230023068, dated 03 / 17 / 2023, page 22 / 46 14 / 21 70% (v / v) for 2 minutes and then in a commercially available sodium hypochlorite solution containing 2.0-2.5% active chlorine for 20 minutes. Finally, the leaves were rinsed 3 times with autoclaved distilled water in a laminar flow chamber (sterile environment), ready to be placed in vitro to begin the cell dedifferentiation stage, i.e., the induction of calluses (aggregates of plant stem cells).

[033] For callus induction, Murashige and Skoog half-strength culture medium (half the total nutrient concentration recommended by the manufacturer) was used. For this purpose, the culture medium was supplemented with sucrose (30 g / L), 2,4-dichlorophenoxyacetic acid (2,4-D) (9.06 μM), Gamborg's vitamins (10 mL / L), L-cysteine ​​(0.08 g / L), malt extract (0.4 g / L), casein (0.1 g / L), Benzylaminopurine (BAP) (4.44 μM) and a gelling agent based on a bacterial substrate consisting of glucuronic acid, rhamnose and glucose (Phytagel®) (2.8 g / L). The culture medium had its pH adjusted to 5.7 before autoclaving at 121°C for 20 minutes at 1.5 atm. Explants of approximately 2 cm were removed from the disinfected leaves using a scalpel and blade and inoculated into the culture medium with the adaxial side (upper part of the leaf) facing the culture medium to provide better absorption of nutrients and growth regulators (2,4-D and BAP).The explants were inoculated into test tubes containing 10 mL of culture medium. After inoculation, the tubes were kept in a BOD (biochemical oxygen demand) type growth chamber in the dark at a temperature of 24 ± 2°C for 30 days until the full development of plant stem cells (calluses) from the explants.

[034] In a laminar flow chamber, friable calluses, 45 days old, obtained from the leaves were transferred to Erlenmeyer flasks containing Murashige and Skoog half-strength culture medium, sucrose (30 g / L), 2,4-dichlorophenoxyacetic acid (2,4-D) (9.06 μM), Gamborg's vitamins (10 mL / L), L-cysteine ​​(0.08 g / L), malt extract (0.4 g / L), casein (0.1 g / L), and benzylaminopurine (BAP) (4.44 μM). Prior to the callus transfer, the culture medium pH was adjusted to 5.7 before autoclaving at 121°C for 20 minutes at 1.5 atm. Each Erlenmeyer flask received 60 mL of culture medium and approximately 500 mg of cell mass from Petition 870230023068, dated 03 / 17 / 2023, page 23 / 46 15 / 21 calluses were obtained from C. canephora leaf explants. The cultures were maintained in a growth chamber at 24 ± 2°C in the dark using an orbital shaker at 110 rpm for 14 days. The cultures were subcultured every 14 days for two months to increase cell biomass. After each subculture, the culture medium was filtered in a laminar flow hood and discarded, with the cells retained on the membrane. 500 mg of the filtered mass was transferred to a new liquid culture medium, prepared under the same conditions described above. In the last subculture, both the culture medium and the plant stem cells were collected, from which the extract was prepared.

[035] For the extraction of bioactive compounds, the cell suspensions (culture medium containing plant stem cells) were ground once for 5 minutes in a blender to disintegrate the cell membranes and release the compounds. Then, the resulting mixture was placed under ultrasound for 16 minutes to completely break down the membranes. All the material obtained was lyophilized for 72 hours. After lyophilization, the resulting material was resuspended in ultrapure water (10% w / v), homogenized in a vortex mixer for 5 minutes, and centrifuged at 10,000 rpm at 4°C. The supernatant obtained was named enriched Coffea canephora extract.

[036] Alternatively, other parts of Coffea canephora can be used as starting material for the preparation of plant stem cells, such as: stem, roots, flowers, fruits or seeds.

[037] After obtaining the enriched Coffea canephora extract, a liposome-forming solution was prepared using the following components: soy lecithin, in a proportion of 10% w / w of the solution; phosphatidylcholine, in a proportion of 10% w / w of the solution; cholesterol, in a proportion of 2% w / w of the solution; polyethylene glycol 400, in a proportion of 5% w / w of the solution; sorbitol, in a proportion of 10% w / w of the solution; xanthan gum, in a proportion of 0.1% w / w of the solution; phenoxyethanol, in a proportion of 1% w / w of the solution; distilled water, in sufficient quantity to make 100% w / w of the solution. For this purpose, lecithin, phosphatidylcholine, cholesterol and polyethylene glycol were levigated, and sorbitol was incorporated to them using a mortar and pestle until complete homogenization. Next, distilled water was added while stirring for 5000 minutes. Petition 870230023068, dated 03 / 17 / 2023, page 24 / 46 Mixing at 16 / 21 rpm, using Turrax-type equipment, until complete dispersion. Then, phenoxyethanol was added and xanthan gum was pulverized under agitation at 1000 rpm. After complete homogenization, the solution was stored under refrigeration at 4°C until use.

[038] To obtain the liposomal formulation containing the enriched and previously prepared extract, the following were added to a container: 10% w / w of the enriched Coffea canephora extract formulation; 20% w / w of the previously prepared liposome-forming solution formulation; 1% w / w of phenoxyethanol and sufficient distilled water to make 100% w / w of the formulation. The mixture was then stirred at 15,000 rpm in a Turrax-type apparatus for 15 minutes, after which the stirring speed was reduced to 8,000 rpm for 5 minutes, and then the resulting formulation was left to stand.

[039] The enriched extract of Coffea canephora obtained from plant stem cells claimed is characterized by being a lysate, with a concentration of 8 to 10 mg / mL, prepared from undifferentiated, totipotent and homogeneous Coffea canephora cells, derived from calluses of Coffea canephora leaf explants; and Murashige and Skoog culture medium enriched with sucrose (30 g / L), 2,4-dichlorophenoxyacetic acid (2,4-D) (9.06 μM), Gamborg's vitamins (10 mL / L), L-cysteine ​​(0.08 g / L), malt extract (0.4 g / L), casein (0.1 g / L), benzylaminopurine (BAP) (4.44 μM) and comprising caffeic acid, sucrose, rosmarinic acid, catechin gallate and rutin.

[040] The enriched extract of Coffea canephora can be used to obtain pharmaceutical and cosmetic formulations for skin care, more specifically, to obtain formulations with anti-aging action; tissue regeneration; anti-inflammatory; antioxidant; collagen production stimulation; neovascularization; prevention and repair of skin blemishes, for topical use and in cream form.

[041] A proposed formulation containing an enriched extract of Coffea canephora obtained from plant stem cells, tested in the development of the present invention, showed excellent results in in vivo tests, as observed in Figures 1 and 2, and comprises: the enriched extract of Coffea canephora, prepared at Petition 870230023068, dated 03 / 17 / 2023, page 25 / 46 17 / 21 from undifferentiated, totipotent, and homogeneous Coffea canephora cells, derived from calluses of Coffea canephora leaf explants and Murashige and Skoog culture medium enriched with sucrose (30 g / L), 2,4-dichlorophenoxyacetic acid (2,4-D) (9.06 μM), Gamborg's vitamins (10 mL / L), L-cysteine ​​(0.08 g / L), malt extract (0.4 g / L), casein (0.1 g / L), and benzylaminopurine (BAP) (4.44 μM), with a concentration of 8 to 10 mg / mL, in a proportion of 10% w / w of the formulation; encapsulated in a liposome-forming solution, in a proportion of 20% w / w of the formulation; Phenoxyethanol, in a proportion of 1% w / w of the formulation, and distilled water, in sufficient quantity to make up 100% w / w of the formulation.The liposome-forming solution shall comprise: soy lecithin, in a proportion of 10% w / w of the solution; phosphatidylcholine, in a proportion of 10% w / w of the solution; cholesterol, in a proportion of 2% w / w of the solution; polyethylene glycol 400, in a proportion of 5% w / w of the solution; sorbitol, in a proportion of 10% w / w of the solution; xanthan gum, in a proportion of 0.1% w / w of the solution; phenoxyethanol, in a proportion of 1% w / w of the solution; distilled water, in sufficient quantity to make 100% w / w of the solution.

[042] Below are presented and discussed some tests carried out with the enriched extract of Coffea canephora.

[043] Chemical composition of the enriched extract of Coffea canephora obtained from plant stem cells (SCECC).

[044] The SCECCs were analyzed in negative (Fig. 3A) and positive (Fig. 3B) APCI ionization modes with monitoring of the m / z mass range from 100 to 1000. Processing and comparison of the spectra obtained by MS-MS from extracts with the literature led to the tentative identification of five compounds (Table 1), using the negative ionization mode, suggesting two flavonoids, two phenolic acids, and one sugar. The flavonoids identified were rutin and catechin gallate. Catechin gallate showed m / z 441 (MH)- for the deprotonated molecule and ion fragment at m / z 289 (MH-152)-, suggesting the presence of the galloyl unit. In turn, rutin showed m / z 609 (MH)- and the ion fragment at m / z 301 indicated the deprotonated quercetin aglycone molecule. Petition 870230023068, dated 03 / 17 / 2023, page 26 / 46 18 / 21

[045] The phenolic acids provisionally identified were caffeic and rosmarinic acids. Caffeic acid shows an m / z 179 (MH)-e the main fragment observed m / z 135 is related to the neutral loss of CO2 (44 Da) due to the α-elimination mechanism, which is a typical charge migration fragmentation in deprotonated compounds. Rosmarinic acid showed an m / z 359 (MH)-e the main fragments are m / z = 197, the deprotonated 3,4-dihydroxyphenylactic acid at / z = 179 for the deprotonated caffeic acid molecule. In addition, the disaccharide sucrose m / z 341 (MH)- was also tentatively identified, showing main fragments at m / z 179 and 161, both generated from the breaking of glycosidic bonds. Compound [MH] - Ion Fragment Caffeic acid 179 161,135 Sucrose 341 179,161 Rosmarinic acid 359 197, 179, 161 Catechin gallate 441 289 Rutin 609 301 Table 1. Compounds identified in the enriched extract of Coffea canephora obtained from plant stem cells (SCECC). [M - H]-: deprotonated molecule.

[046] The amino acid profile in SCECC.

[047] In Table 2 below, the 15 identified amino acids are observed, with asparagine and glycine being the amino acids identified in the greatest quantity. Petition 870230023068, dated 03 / 17 / 2023, page 27 / 46 19 / 21 Amino acids SCECC Glycine 10.54 Lysine 5.45 Histidine 4.40 Arginine 2.97 Asparagine 13.89 Glutamine 7.46 Serine 7.20 Threonine 4.60 Tyrosine 1.72 Alanine 7.96 Valine 5.85 Leucine 3.90 Isoleucine 3.12 Proline 5.90 Phenylalanine 2.65 Table 2. Quantification of amino acids in simple medium suspension extracts (ESMS) and compound medium suspension extracts (ESMC) of C. canephora. *Results expressed as residues of each specific amino acid per 100 total amino acid residues.

[048] Total flavonoids and phenolic content in the enriched extract of Coffea canephora obtained from plant stem cells (SCECC).

[049] SCECC has a lower flavonoid content compared to phenolic compounds. The flavonoid content was 7.65 ± 0.05 mg quercetin equivalent (QE) per 100 mL of SCECC, while the total phenolic compounds were 974.12 ± 0.06 mg gallic acid equivalent (GAE) per 100 mL of SCECC, indicating the high content of phenolic compounds in SCECC, as observed in Table 3. Total flavonoids (mg 100 mL-1)# Phenolic content (mg 100 mL-1)* Petition 870230023068, dated 03 / 17 / 2023, p. 28 / 46 20 / 21 SCECC 7.65 ± 0.05 974.12 ± 0.06 Table 3. Total flavonoids and phenolic content in the enriched extract of Coffea canephora obtained from plant stem cells (SCECC). # Total flavonoid content is expressed in mg of quercetin equivalent (QE) / 100 mL of SCECC. *Total phenolic content was expressed in mg of gallic acid equivalents (GAE) / 100 mL of SCECC. Tests were performed in triplicate and expressed as mean ± SD.

[050] The in vitro antioxidant activity of the extract, measured by ABTS free radical scavenging activity and iron reducing antioxidant power (FRAP), is shown in Table 4. Antioxidant activity (IC50 pg mL-1) ABTS FRAP SCECC# 58.15 ± 12.4 a* 15.52 ± 0.21 a Chlorogenic acid 6.32 ± 0.21 b 4.71 ± 0.17 b Table 4. In vitro antioxidant activity of the enriched extract of Coffea canephora obtained from plant stem cells (SCECC). * Different letters in the same column correspond to significant differences (p<0.05) calculated by one-way ANOVA followed by Tukey's post-hoc test. Tests were performed in triplicate (n=3) and expressed as mean ± SD. # Coffea canephora stem cell extract.

[051] The results of in vitro anti-inflammatory activity tests in culture of the enriched extract of Coffea canephora obtained from plant stem cells (SCECC) demonstrated: a decrease in the production of nitric oxide (Fig. 4A) and superoxide anion in vitro (Fig. 4B); a decrease in the production of the inflammatory cytokines IL-6 (Fig. 5A) and TNF-α (Fig. 5B) in vitro; inhibition of NF-kB production in vitro (Fig. 6); and positive effects on fibroblast proliferation and migration (Fig. 7).

[052] Based on the results obtained, it was found that the extract obtained from the lysate of undifferentiated, totipotent, and homogeneous C. canephora cells, derived from calluses of C. canephora leaf explants, associated with the previously enriched Murashige and Skoog culture medium, has numerous compounds with potential Petition 870230023068, dated 03 / 17 / 2023, page 29 / 46 21 / 21 therapeutic applications, justifying its use in the preparation of pharmaceutical formulations for skin care, such as formulations with anti-aging, tissue-regenerating, anti-inflammatory, antioxidant, collagen-stimulating, neovascularization, preventive, and reparative actions for skin blemishes. In this context, a liposomal formulation was developed capable of incorporating the obtained hydrophilic extract and transporting it to the dermis, where fibroblasts, responsible for collagen production, and blood vessels are located, for example, corroborating the technical advances provided by the present invention. Petition 870230023068, dated 03 / 17 / 2023, pp. 30 / 46

Claims

1 / 4 CLAIMS 1. PROCESS FOR OBTAINING ENRICHED EXTRACT OF Coffea canephora FROM PLANT STEM CELLS characterized by starting with the selection of well-expanded leaves, with a medium intense green color without signs of lesions, diseases or insect attack of C. canephora, followed by washing in running water under a tap 3 times for 5 minutes each time, using neutral commercial detergent;After washing, the leaves were immersed in 70% (v / v) ethanol for 2 minutes and then in a sodium hypochlorite solution containing 2.0-2.5% active chlorine for 20 minutes. They were then rinsed three times with autoclaved distilled water in a laminar flow chamber, and the callus induction stage was ready. For this, half-strength Murashige and Skoog culture medium was used, supplemented with sucrose (30 g / L), 2,4-dichlorophenoxyacetic acid (2,4-D) (9.06 μM), Gamborg's vitamins (10 mL / L), L-cysteine ​​(0.08 g / L), malt extract (0.4 g / L), casein (0.1 g / L), benzylaminopurine (BAP) (4.44 μM), and a gelling agent based on a bacterial substrate consisting of acid. glucuronic acid, rhamnose and glucose (2.8 g / L), and pH adjusted to 5.7 before autoclaving at 121°C for 20 minutes at 1.5 atm;After preparing the culture medium, explants of approximately 2 cm should be removed from disinfected leaves and inoculated into tubes containing the culture medium with the adaxial side facing the culture medium; after inoculation, the tubes should be kept in a BOD-type growth chamber, in the dark at a temperature of 24 ± 2°C, for 30 days until full callus development; In a laminar flow chamber, friable calluses, after 45 days, were transferred to Erlenmeyer flasks containing half-strength Murashige and Skoog culture medium, supplemented with sucrose (30 g / L), 2,4-dichlorophenoxyacetic acid (2,4-D) (9.06 μM), Gamborg's vitamins (10 mL / L), L-cysteine ​​(0.08 g / L), malt extract (0.4 g / L), casein (0.1 g / L), Benzylaminopurine (BAP) (4.44 μM), obtaining a cell suspension with a concentration of 8 to 10 mg / mL;Therefore, the cultures prepared in the flasks should initially be kept in a growth chamber at a temperature of 24 ± 2°C in the dark on an orbital shaker at 110 rpm for 14 days, Petition 870230023068, dated 03 / 17 / 2023, page 31 / 46 2 / 4, being subcultured every 14 days for two months; in each subculture, the culture medium is filtered in a laminar flow chamber and discarded; the filtered mass is then resuspended at a concentration of 8 to 10 mg / mL; in the last subculture, the culture medium and plant stem cells are collected for the extraction of bioactive compounds, by grinding the solution for 5 minutes in a blender and then placing the resulting mixture under ultrasound for 16 minutes and lyophilizing the material obtained for 72 hours; After lyophilization, the resulting material is resuspended in ultrapure water (10% w / v), homogenized in a vortex mixer for 5 minutes, and centrifuged at 10,000 rpm at 4°C to finally obtain the enriched Coffea canephora extract.

2. PROCESS FOR OBTAINING ENRICHED EXTRACT OF Coffea canephora FROM PLANT STEM CELLS, according to claim 1, characterized by alternatively employing other parts of Coffea canephora as starting material for the preparation of plant stem cells, such as: stem, roots, flowers, fruits or seeds.

3. ENRICHED EXTRACT OF Coffea canephora OBTAINED FROM PLANT STEM CELLS, as defined in claim 1, characterized by consisting of a lysate, with a concentration of 8 to 10 mg / mL, comprising caffeic acid, sucrose, rosmarinic acid, catechin gallate and rutin; prepared from undifferentiated, totipotent and homogeneous Coffea canephora cells, derived from calluses of Coffea canephora leaf explants; and Murashige and Skoog culture medium enriched with sucrose (30 g / L), 2,4-dichlorophenoxyacetic acid (2,4-D) (9.06 μΜ), Gamborg's vitamins (10 mL / L), L-cysteine ​​(0.08 g / L), malt extract (0.4 g / L), casein (0.1 g / L), benzylaminopurine (BAP) (4.44 μM).

4. USE OF ENRICHED EXTRACT OF Coffea canephora OBTAINED FROM PLANT STEM CELLS, as defined in claim 3, characterized by being for obtaining pharmaceutical and cosmetic formulations for skin care. Petition 870230023068, dated 03 / 17 / 2023, page 32 / 46 3 / 4 5. USE OF ENRICHED EXTRACT OF Coffea canephora OBTAINED FROM PLANT STEM CELLS, according to claim 4, characterized by being for obtaining formulations with anti-aging action; tissue regeneration; anti-inflammatory action; antioxidant action; collagen production stimulation; neovascularization; prevention and repair of skin blemishes.

6. USE OF ENRICHED EXTRACT OF Coffea canephora OBTAINED FROM PLANT STEM CELLS, according to any one of claims 4 or 5, characterized by being for obtaining formulations for topical use.

7. USE OF ENRICHED EXTRACT OF Coffea canephora OBTAINED FROM PLANT STEM CELLS, according to any one of claims 4, 5 or 6, characterized by being for obtaining cream-type formulations.

8. FORMULATION CONTAINING ENRICHED EXTRACT OF Coffea canephora OBTAINED FROM PLANT STEM CELLS, as defined in any of claims 3 or 4, characterized by comprising: enriched extract of Coffea canephora, prepared from undifferentiated, totipotent and homogeneous Coffea canephora cells, derived from calluses of Coffea canephora leaf explants and Murashige and Skoog culture medium enriched with sucrose (30 g / L), 2,4-dichlorophenoxyacetic acid (2,4-D) (9.06 μM), Gamborg's vitamins (10 mL / L), L-cysteine ​​(0.08 g / L), malt extract (0.4 g / L), casein (0.1 g / L) and benzylaminopurine (BAP) (4.44 μM), with a concentration of 8 to 10 mg / mL, in a proportion of 10% w / w of the formulation; encapsulated in liposome-forming solution, in a proportion of 20% w / w of the formulation; phenoxyethanol, in a proportion of 1% w / w of the formulation and distilled water, in sufficient quantity to make up 100% w / w of the formulation.

9. FORMULATION CONTAINING ENRICHED EXTRACT OF Coffea canephora OBTAINED FROM PLANT STEM CELLS, according to claim 8, characterized by the liposome-forming solution being Petition 870230023068, dated 03 / 17 / 2023, p. 33 / 46 4 / 4 comprising: soy lecithin, in the proportion of 10% w / w of the solution; phosphatidylcholine, in the proportion of 10% w / w of the solution; cholesterol, in the proportion of 2% w / w of the solution; polyethylene glycol 400, in the proportion of 5% w / w of the solution; sorbitol, in the proportion of 10% w / w of the solution; xanthan gum, in the proportion of 0.1% w / w of the solution; phenoxyethanol, in the proportion of 1% w / w of the solution; Distilled water, in sufficient quantity to make up 100% of the solution (w / w). Petition 870230023068, dated 03 / 17 / 2023, pp. 34 / 46

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