PROCESS FOR OBTAINING AN EXTRACT OF ROSMARINUS OFFICINALIS, ROSMARINUS OFFICINALIS EXTRACT, USE OF AN EXTRACT, DERMOCOSMETIC OR PHARMACEUTICAL COMPOSITION AND METHOD OF TREATING AND / OR PREVENTING ALOPECIA

Description

1 / 24 PROCESS FOR OBTAINING AN EXTRACT OF ROSMARINUS OFFICINALIS, ROSMARINUS OFFICINALIS EXTRACT, USE OF AN EXTRACT, DERMOCOSMETIC OR PHARMACEUTICAL COMPOSITION AND METHOD OF TREATING AND / OR PREVENTING ALOPECIA Field of Invention

[0001] The present invention relates to a method for obtaining an extract from the leaves of Rosmarinus officinalis (rosemary) involving two steps of maceration, filtration, concentration and drying, ensuring an extract comprising hydroxycinnamic derivatives, rosmarinic acid, luteolin, neptin and carnosol, as well as the use of the extract in dermocosmetic and pharmaceutical compositions useful in the prevention and treatment of androgenetic alopecia, wherein the extract of the invention acts on multiple mechanisms of action, which allows its use in both men and women. Background of the Invention

[0002] Androgenetic alopecia (AGA), also known as baldness, is the most common type of progressive hair loss, having a characteristic pattern in both men and women. While in men hair loss is more prominent in the frontotemporal and vertex regions; women tend to have a decrease / thinning of hair density in the centro-parietal region of the scalp.

[0003] Caucasians are most affected by AGA, followed by Asians and African Americans, where the incidence in men is strongly linked to age progression; while in women there is a higher incidence after menopause.

[0004] Although the pathogenesis of AGA is not fully elucidated, it is known that factors such as stress, diet, and the individual's genetics and sensitivity to androgens contribute to its manifestation.

[0005] AAG is characterized by progressive miniaturization Petition 870240110548, dated 12 / 27 / 2024, page 23 / 58 2 / 24 of hair follicles are affected, leading terminal hair to transform into vellus hair (i.e., shorter and thinner strands). This occurs due to a change in the hair cycle, where the duration of the anagen phase (growth phase) of the hair gradually decreases and the telogen phase increases (resting phase, where the follicle prepares to release the hair strand).

[0006] Androgens are hormones important for the growth and development of the human body, with testosterone being the main and most active androgen present in men. Testosterone is converted by the enzyme 5-alpha-reductase type II into dihydrotestosterone (DHT). Elevated DHT production, high levels of the enzyme 5-alpha-reductase type II, and increased androgen receptors in the scalp region cause a reduction in hair follicles, replacing terminal hairs with vellus hairs.

[0007] Furthermore, systemic factors also influence the hair cycle, as it has been clinically observed that androgen blockade does not always result in the reversal of miniaturized hair follicles. Experiments with hair transplantation have demonstrated that the system for the hair cycle is probably located within the hair follicle itself, considering that the organ maintains its characteristic cyclical behavior after transplantation. Studies with transplanted hairs in mice showed an intrinsic cyclical pattern, but gradually acquired the host pattern, suggesting systemic influence. In light of this, important molecules for the hair cycle can be identified, such as insulin-like growth factor 1 (IGF1) and fibroblast growth factor 7 (FGF7), which are important for hair follicle development.Some other relevant signaling molecules identified are the TGF-β, WNT / β-catenin, BMP, prostaglandin, neurotrophin families, among others (CuevasDiaz Duran, R.; Martinez-Ledesma, E.; Garcia-Garcia, M.; Bajo Gauzin, D.; Sarro-Ramírez, A.; Gonzalez-Carrillo, C.; Rodríguez-Sardin, D.; Fuentes, A.; Cardenas-Lopez, A. The Biology and Genomics of Human Hair Follicles: A Focus). Petition 870240110548, dated 12 / 27 / 2024, p. 24 / 58 3 / 24 on Androgenetic Alopecia. Int. J. Mol. Sci. 2024, 25, 2542. (https: / / doi.org / 10.3390 / ijms25052542).

[0008] Hair plays an important cultural role in an individual's social life, representing beauty, vitality, youthfulness, and personal care. The manifestation of androgenetic alopecia, therefore, affects people's quality of life, lowering their self-esteem and causing shame, impacting their mental health.

[0009] Options approved by the Food and Drug Administration The FDA recommends only two treatments for androgenetic alopecia: minoxidil and finasteride. Treatment with both requires continuous use, with results only visible after at least 4 to 6 months of consistent use.

[0010] Minoxidil is a vasodilator initially used to control high blood pressure. When applied, minoxidil is converted to minoxidil sulfate by sulfotransferase enzymes, which potentiates potassium channels in smooth muscle, causing its relaxation. The result will be an increase in the supply of oxygen and growth factors.

[0011] Finasteride is a competitive and specific inhibitor of 5-alpha-reductase type II. Inhibition of this enzyme prevents the peripheral conversion of testosterone to the androgen DHT, decreasing its concentration in tissues. The use of finasteride by women with androgenetic alopecia is off-label, as there is a risk of fetal malformation in pregnant women.

[0012] However, both treatments can bring with them adverse effects such as contact dermatitis, tachycardia and hypertrichosis with topical minoxidil; itching, skin irritation, hypertrichosis and hypervolemia with oral minoxidil; and decreased libido, erectile dysfunction and decreased ejaculate volume with finasteride.

[0013] Despite this, other treatments not approved by The FDA has also shown that some drugs are effective for the treatment of AGA or are being studied, for example, spironolactone, cyproterone, flutamide, clascoterone, Petition 870240110548, dated 12 / 27 / 2024, page 25 / 58 4 / 24 JAK inhibitors, prostaglandin analogs, etc. Among alternative therapies, two molecules have been widely used for alopecia: alpha-estradiol and latanoprost.

[0014] Alpha-estradiol is a hormone with low affinity for estrogen receptors, and its mechanism of action involves weak inhibition of 5-alpha-reductase, prolonging the anagen phase of the hair follicle.

[0015] The use of latanoprost, a prostaglandin analogue, for the treatment of alopecia is due to the eyelash and eyebrow growth effect observed in glaucoma patients using the compound. Further research has indicated that PGF2 and PGE2 cause hair growth and prolong the anagen phase.

[0016] Botanical extracts have also been researched over the past few years.

[0017] However, despite the research efforts aimed at finding alternatives, it is still necessary to provide an alternative treatment for androgenetic alopecia that can be used in both men and women, that promotes a faster perception of effectiveness, leading to a higher rate of responsive patients, and that does not cause adverse reactions, especially those present in minoxidil and finasteride, or that have fewer adverse reactions. Summary of the Invention

[0018] The present invention solves the technical problem set forth by providing a process for obtaining an extract of Rosmarinus officinalis (rosemary), the extract, the use of the extract, a dermocosmetic or pharmaceutical composition and a method of treating androgenetic alopecia (AGA).

[0019] The present invention has the advantage of acting on multiple mechanisms of action, presenting a lower probability of adverse effects and enabling the use of the present extract in both men and women. Petition 870240110548, dated 12 / 27 / 2024, page 26 / 58 5 / 24

[0020] Furthermore, the extract of the present invention can contribute to a higher rate of responsive patients, since, experimentally, it does not present hormonal effects and offers the perception of rapid effectiveness, comparable to conventional treatments currently on the market.

[0021] The present invention will now be defined by its objects:

[0022] As a first object, there is a process for obtaining an extract of Rosmarinus officinalis, in which the extraction is done from the whole rosemary leaf and comprises the following steps:

[0023] a) 1st Extraction by maceration with agitation;

[0024] b) Filtration of product a), obtaining the drained liquid;

[0025] c) 2nd Extraction by maceration with agitation;

[0026] d) Filtration of product c), obtaining the drainedII;

[0027] e) Mixture of drained materials I and II;

[0028] f) Concentration; and

[0029] g) Drying.

[0030] In a second object, there is an extract of Rosmarinus officinalis, comprising mainly hydroxycinnamic derivatives, rosmarinic acid, luteolin, neptin, and phenolic diterpenes.

[0031] In another object, there is Rosmarinus officinalis extract for use in the treatment / prevention of alopecia and / or strengthening of hair strands, in which the extract comprises, as major compounds, hydroxycinnamic derivatives, rosmarinic acid, luteolin, neptin and phenolic diterpenes.

[0032] In a fourth object, there is the use of an extract of Rosmarinus officinalis is used in the manufacture of a dermocosmetic or pharmaceutical product for the treatment / prevention of alopecia and strengthening of hair strands, as well as the growth of eyelashes, eyebrows, body hair and / or beard.

[0033] As a fifth object, there is a dermocosmetic or pharmaceutical composition comprising an extract of Rosmarinus Petition 870240110548, dated 12 / 27 / 2024, page 27 / 58 6 / 24 officinalis and at least one dermocosmetically or pharmaceutically acceptable excipient.

[0034] A sixth object is a method of treatment and / or prevention of alopecia, comprising the administration of the dermocosmetic or pharmaceutical composition or extract of Rosmarinus officinalis.

[0035] Brief Description of the Figures

[0036] In the figures presented and the text that follows, the mention of the code PDI38, or ACH38 refers to the extract according to the present invention.

[0037] Figure 1 shows a flowchart summarizing the process of extracting the invention and parameters used in one embodiment.

[0038] Figure 2 shows the chromatographic profile of the Rosmarinus officinalis extract of the present invention by HPLC-DAD and the major compounds (Tr 10.37 min Nepetin-7-glucoside; Tr 11.48 min Rosmarinic Acid; Tr 12.39 min Luteolin-3-glucuronide; Tr 29.05 min Carnosol).

[0039] Figure 3 shows the comparative graph of the time course and hair growth during treatment with the extract of the present invention and reference treatments, in mouse models with testosterone-induced alopecia.

[0040] Figure 4 shows the time course graph and hair growth for the extract according to the present invention, compared with reference controls in the mouse model with alopecia.

[0041] Figure 5 shows representative images of hair growth in mice from each group on day 30.

[0042] Figure 6 shows photomicrographs of the skin of representative animals from the different groups after H&E staining. The anagen and telogen phases are indicated in each panel.

[0043] Figure 7 shows the relaxation / contraction, in pre-contracted preparations with noradrenaline (%), induced by A) ACH38 Petition 870240110548, dated 12 / 27 / 2024, page 28 / 58 7 / 24 (extract according to the present invention), B) minoxidil and C) gallic acid.

[0044] Figure 8 shows the relaxation / contraction, in pre-contracted preparations with KCl (%), induced by A) ACH38 (extract according to the present invention), B) minoxidil and C) gallic acid.

[0045] Detailed Description of the Invention

[0046] The present invention provides a process for obtaining an extract of Rosmarinus officinalis from the whole leaf of the plant comprising two extractions by maceration, followed by two filtrations, concentration of the two drained extracts obtained and drying, which process yields an extract with activity in the treatment of androgenetic alopecia and strengthening of keratin fibers, particularly of hair strands, eyelashes, eyebrows, body hair and / or beard, effective in both men and women, since the extract of the invention acts through multiple mechanisms of action. Definitions

[0047] Unless explicitly stated otherwise, the following terms used throughout this descriptive report and claims shall have the following definitions for the purpose of the present invention:

[0048] “Pharmaceutically acceptable excipient” means compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response or any other problem or complication, with a reasonable benefit / risk ratio. Examples of pharmaceutically acceptable compounds, materials, compositions and / or dosage forms can be found in pharmaceutical compendia such as the United States Pharmacopeia and its future editions or the Handbook of Pharmaceutical Excipients. 9th Edition (2020). Ed. Rowe et al. Pharmaceutical Press and American Pharmaceutical Association.

[0049] “Dermocosmetically acceptable excipient” means that which is useful in the preparation of dermocosmetic compositions and generally safe for use in contact with human keratinized tissue without Petition 870240110548, dated 12 / 27 / 2024, page 29 / 58 8 / 24 undue toxicity, allergic reaction or similar reactions, and not biologically or otherwise undesirable. A dermocosmetically acceptable excipient may be selected from diluents, emollients, humectants, thickeners, preservatives, colorants, dyes, sunscreen agents, moisturizing agents, film-forming / waterproofing agents, bioactive ingredients, pharmaceutical ingredients, pH adjusting agents, chelating agents, rheology modifying agents, fragrance agents, plant extracts, absorbents, vitamins, photostabilizing agents, sunscreen enhancers, antioxidants, exfoliating agents, liquid carriers, waxes, conditioners, lubricants, antibacterial agents, anti-aging agents and combinations thereof.

[0050] “At least” establishes the limit or minimum quantity required for something. For example, 'at least x%' means at least x% or more.

[0051] The terms “about”, “probably”, “around” and the like, when used for measurable values, mean a quantity comprising variations of ± 20%, ± 10% or ± 5%, more preferably ± 2%.

[0052] The term “effective amount” means an amount of the active ingredient included in the composition capable of providing a therapeutic / cosmetic effect, but not sufficient to cause adverse or toxic reactions, when the composition of the present invention is administered to an individual.

[0053] In the first object, there is a process for obtaining an extract of Rosmarinus officinalis, in which the extraction is done from the whole leaf of the plant and comprises the following steps:

[0054] a) 1st Extraction by maceration with agitation;

[0055] b) Filtration of the product from a), obtaining the drained I;

[0056] c) 2nd Extraction by maceration with agitation;

[0057] d) Filtration of the product from c), obtaining the drained II; Petition 870240110548, dated 12 / 27 / 2024, page 30 / 58 9 / 24

[0058] e) Mixture of drained materials I and II;

[0059] f) Concentration; and

[0060] g) Drying.

[0061] In one embodiment, the 1st and 2nd extractions are carried out with 50% ethanol, in a liquid:solid ratio of 10:1.

[0062] In another embodiment, the 1st and 2nd extractions are carried out by heating at 70 ± 5°C.

[0063] In one embodiment, drying is carried out by spray dryer.

[0064] In a second object, there is an extract of Rosmarinus officinalis comprising hydroxycinnamic derivatives, rosmarinic acid, luteolin, neptin and phenolic diterpenes.

[0065] In one embodiment, the extract comprises 15 to 25% hydroxycinnamic derivatives, 1 to 10% rosmarinic acid, 1 to 10% phenolic diterpenes, 0.1 to 1% luteolin and 0.1 to 0.5% neptin.

[0066] In a preferred embodiment, the extract comprises about 19% hydroxycinnamic derivatives, about 5% rosmarinic acid, about 5% phenolic diterpenes, about 0.5% luteolin and about 0.3% neptin.

[0067] In a preferred embodiment, the phenolic diterpene comprises carnosol.

[0068] In another object, there is Rosmarinus officinalis extract for use in the treatment / prevention of alopecia and / or strengthening of hair strands, in which the extract comprises hydroxycinnamic derivatives, rosmarinic acid, luteolin, neptin and phenolic diterpenes.

[0069] In a fourth object, there is the use of an extract of Rosmarinus officinalis is used in the manufacture of a dermocosmetic or pharmaceutical product for the treatment / prevention of alopecia and strengthening of keratin fibers, including hair strands, eyelashes, eyebrows, body hair and / or beard, particularly hair strands. Petition 870240110548, dated 12 / 27 / 2024, page 31 / 58 10 / 24

[0070] As a fifth object, there is a dermocosmetic or pharmaceutical composition comprising an extract of Rosmarinus officinalis and at least one dermocosmetically or pharmaceutically acceptable excipient.

[0071] In another embodiment, the dermocosmetic or pharmaceutical composition comprises a concentration of at least about 2.5% to 15% of the extract.

[0072] In one embodiment, the composition comprises a concentration of about 2.5%, 5% or 10% of the extract.

[0073] In one embodiment, the composition is for use in the treatment / prevention of alopecia and / or strengthening of hair strands.

[0074] In one embodiment, the composition is used in the treatment / prevention of alopecia and / or strengthening of hair strands, by acting on the activation mechanisms of the WNT / β-cathenin pathway, modulation of prostaglandins, inhibition of TGF-β, up-regulation of IGF-1 and VEGF genes and vasodilatory effect.

[0075] A sixth object is a method for treating and / or preventing alopecia, comprising the administration of the dermocosmetic or pharmaceutical composition or extract of Rosmarinus officinalis.

[0076] In one embodiment, the administration of the composition or extract is topical.

[0077] In one embodiment, the extract or composition comprising said extract does not act on the 5-alpha-reductase enzyme and does not produce androgenic effects. Examples

[0078] The following examples are not limiting and only provide some of the various ways in which the invention can be carried out.

[0079] Process for extracting the invention

[0080] A diagram illustrating the extraction process of the invention can be seen in figure 1. Petition 870240110548, dated 12 / 27 / 2024, page 32 / 58 11 / 24 Extraction

[0081] In a 3000L reactor, 40 kg of dried Rosmarinus officinalis leaves and 400L of 50% ethanol solution were added. The mixture was heated for 2 hours at 70°C ± 5°C with constant stirring. After 2 hours, the extract was filtered using a rotary filter, and another 400L of 50% ethanol solution was added to the resulting bagasse, and another extraction was performed using the same parameters as the first. The mixture was then filtered again using a rotary filter. The second resulting drained liquid was added to the first and homogenized.

[0082] In this stage, 618 kg of total drainage was obtained after the two extractions of the plant drug without grinding. Concentration

[0083] The total drained material from the first and second extractions, totaling 618 kg, was homogenized and concentrated in a Bernauer concentrator at a maximum temperature of 75°C. 30.2 kg of concentrated extract was obtained with a solids content of 28.7%. Drying

[0084] Finally, the concentrate was dried in a spray dryer under the following parameters: inlet temperature = 165°C; minimum outlet temperature = 90°C; air flow pressure control = 2.0 bar. 6.15 kg of dry extract were obtained according to the present invention.

[0085] Figure 2 shows the chromatographic profile of the extract according to the present invention by HPLC-DAD.

[0086] Comparative tests showed significantly lower results.

[0087] Testosterone-Induced Alopecia in Mice

[0088] Alopecia was induced in C57BL / 6 mice by topical application of 100 pL of testosterone solution (0.05% in 50% ethanol) to shaved skin on the back once daily for 30 days. The animals were Petition 870240110548, dated 12 / 27 / 2024, page 33 / 58 12 / 24 treated topically, daily, with reference medications and the extract according to the present invention at different doses. Vehicle (100 pL 50% ethanol) was applied to sham control animals that served as controls. Formulations

[0089] The required amount of testosterone was weighed and dissolved in 50% ethanol to prepare a 0.05% solution. The vehicle used for finasteride was ethanol:propylene glycol:water (8:1:1). The vehicle used for the other formulations, except the market comparator, was propylene glycol:ethanol (20:80). The formulation with the market comparator was prepared using 0.25% carboxymethylcellulose as a vehicle. All formulations were prepared daily immediately before treatment. Treatment

[0090] Each day, 30 minutes after testosterone application, groups of animals were treated with the test and reference formulations, once daily (groups 1 to 15) or twice weekly (groups 16 to 18), as specified in the experimental design. Observations

[0091] Hair growth was evaluated in each mouse on days 0, 7, 12, 15, 18, 21, 24, and 30 after alopecia induction, and classified according to the following scale:

[0092] 0 no hair growth observed

[0093] 1 less than 20% growth observed

[0094] 2 from 20% to less than 40% growth observed

[0095] 3 from 40% to less than 60% growth observed

[0096] 4 from 60% to less than 80% growth observed

[0097] 5 from 80% to 100% growth observed

[0098] Histopathology Petition 870240110548, dated 12 / 27 / 2024, page 34 / 58 13 / 24

[0099] On days 0, 7, 12, 15, 18, 21, 24, and 30, ten animals from each group per time point were euthanized using CO2. Samples of the treated dorsal skin area were collected and preserved in 10% buffered neutral formalin. The samples were processed using the standardized paraffin fixation technique. The prepared blocks were sectioned at 3 to 5 µm thickness using a microtome and mounted on clean glass slides. The slides were stained with hematoxylin-eosin and observed under a light microscope. The stained tissues were analyzed for the number of follicles in the anagen (A) and telogen (T) phases, and the A / T ratio was determined. The H&E-stained slides were photographed with a digital photomicrograph, and representative images from each group are reported (Figure 6). Results

[0100] Mean ± SD values ​​of hair growth assessments were estimated in each group at different times. Significant differences between group means and the control were analyzed by one-way ANOVA followed by Dunnett's multiple comparison test using Graphpad Prism at 95% confidence levels. A P-value <0.05 was considered significant.

[0101] The time course of hair growth is shown in Figure 3. Figure 4 shows a comparison of hair growth for the extract of the invention and Figure 5 shows representative animals from each group after 30 days of treatment. Discussion

[0102] Hair growth classification:

[0103] Finasteride (5%, do) and Minoxidil (2%, do) showed significant efficacy when compared to the testosterone control vehicle (p<0.05) after 30 days of treatment.

[0104] Alpha-estradiol (0.03%, do) and the market comparator (30 mg / kg, po, do) did not show significant efficacy when compared to the testosterone control vehicle (p>0.05) after 30 days of Petition 870240110548, dated 12 / 27 / 2024, pages 35 / 58 14 / 24 treatment.

[0105] The extract according to the present invention showed significant dose-dependent efficacy (p<0.05) at 2.5%, 5% and 10% (do) when compared to the control vehicle on day 30. Efficacy was also observed from day 18 onwards for the highest dose and from day 21 onwards for the lowest doses. The efficacy (magnitude and rate of hair growth) of the extract according to the present invention at 5% and 10% was comparable to that of finasteride and minoxidil.

[0106] Carnosic acid demonstrated significant dose-dependent efficacy (p<0.05) of 1.25%, 2.5%, and 5% (do) when compared to the control vehicle on day 30. Efficacy was also observed from day 18 onwards for the medium and high doses and from day 21 onwards for the lower doses.

[0107] On day 30, the efficacy of the extract according to the present invention (5%, of) and carnosic acid (5%, of) was comparable to that of finasteride (5%, of)

[0108] On day 30, the efficacy of the extract according to the present invention (5%, twice a week) was not significantly different from the control vehicle (P<0.05) and was significantly lower (not higher) when compared to the once-daily dosage.

[0109] Carnosic acid (2.5%, twice weekly) showed significant efficacy when compared to the control vehicle and was comparable to once-daily dosing. Histopathology

[0110] At the end of 30 days of treatment, finasteride and minoxidil showed significantly (p<0.05) higher A / T ratios (P<0.05) when compared to the negative control.

[0111] The extract according to the present invention (once daily) showed a dose-dependent increase in A / T ratios and was significantly (p<0.05) different by 5% and 10% when compared to the negative control. Petition 870240110548, dated 12 / 27 / 2024, pp. 36 / 58 15 / 24 Vasodilation effect

[0112] The vasodilating effect of the extract according to the present invention was evaluated in an in vitro model of isolated rat aorta.

[0113] Eight naive male Wistar (Han) rats were euthanized by CO2 inhalation and then exsanguinated. A segment of the thoracic aorta was carefully excised. The artery was cleared of all fat and connective tissue and cut into 2.5 mm long rings. The rings were then mounted horizontally in 20 ml organ baths filled with Krebs solution, maintained at 37.7 ± 0.5°C and gassed with a mixture of 95% O2 and 5% CO2.

[0114] The method evaluated the effect of the test substance on pre-prepared samples.

[0115] The preparations were pre-contracted with 20 mM KCl (or 0.1 μM noradrenaline) until a stable tonic contraction was achieved. Six cumulative test concentrations (0.01, 0.1, 1, 10, 100, and 1000 μg / mL) or 7 cumulative comparison concentrations (minoxidil: 0.1, 0.3, 1, 3, 10, 30, and 1000 μM; gallic acid: 0.1, 0.3, 1.3, 10, 30, and 3000 μM) were then added to the bath, and the relaxations were recorded to plot a concentration-response curve. The preparations were washed, and a second concentration-response curve was evaluated on the same preparation (with a new pre-contraction agent).

[0116] Relaxation / contraction responses to the test substance were expressed as the percentage change in agonist-induced pre-contraction. Results Pre-contraction with noradrenaline

[0117] In isolated rat aortic rings pre-contracted with 0.1 μM noradrenaline, the vehicle (10-5% DMSO at 0.1%) had no substantial effect (-16 ± 4% at 0.1%). In contrast, at 1%, DMSO induced relaxation of aortic rings pre-contracted with 0.1 μM noradrenaline (Petition 870240110548, 12 / 27 / 2024, p. 37 / 58). 16 / 24 ± 6%).

[0118] The ACH38 extract according to the present invention (0.01 to 1 μg / mL) (Figure 7A) had no relaxing effects on aortic rings pre-contracted with 0.1 μm noradrenaline, compared to the vehicle control group (DMSO). In contrast, from 10 to 1,000 μg / mL, the extract according to the present invention induced relaxation in a concentration-dependent manner compared to the control group:

[0119] at 10 μg / mL: -23 ± 11% versus -11 ± 3% in the control group,

[0120] at 100 μg / mL: -43 ± 17% versus -16 ± 4% in the control group, and

[0121] at 1000 μg / mL: -80 ± 8% versus -43 ± 6% in the control group.

[0122] The extract according to the present invention was dissolved in DMSO and then diluted in Krebs solution (the same DMSO concentrations tested in the control group).

[0123] Minoxidil (0.1 to 10 μM) (Figure 7B) had no relaxing effects on aortic rings pre-contracted with 0.1 μM noradrenaline. At 30 and 1000 μM, minoxidil relaxed the preparations in a concentration-dependent manner (-24 ± 5% and -77 ± 3%, respectively). The minoxidil was dissolved in distilled water. Therefore, time-matched control preparations were not included for comparison.

[0124] Gallic acid, one of the substances present in the extract according to the present invention, (from 0.1 to 3 μM) (Figure 7C) had no relaxing effects on aortic rings pre-contracted with 0.1 μM of noradrenaline. At 10, 30 and 3,000 μM, gallic acid relaxed the preparations in a concentration-dependent manner (-23 ± 19%, -31 ± 23% and -87 ± 5%, respectively). It should be noted that the profile was different from one preparation to another. For two preparations (No. 4 and 21), an increase in tension was observed up to 30 μM. For all preparations, a marked relaxation was observed. Petition 870240110548, dated 12 / 27 / 2024, pp. 38 / 58 17 / 24 measured at 3000 μM. This dual effect had already been described in the literature: contraction at low concentrations and relaxation at high concentrations. Gallic acid was dissolved in distilled water. Therefore, control preparations with corresponding times for comparison were not included. Pre-contraction with KCl

[0125] In isolated rat aortic rings pre-contracted with 20 mM KCl, the vehicle (10-5% DMSO at 0.1%) had no substantial effect (-17 ± 2% at 0.1%). In contrast, at 1%, DMSO induced relaxation of aortic rings pre-contracted with 20 mM KCl (-42 ± 4%).

[0126] The ACH38 extract according to the present invention (0.01 to 100 μg / mL) (Figure 8A) had no relaxing effects on aortic rings pre-contracted with 20 mM KCl, compared to the vehicle control group (DMSO). In contrast, at 1000 μg / mL, the extract according to the present invention induced a relaxation of -71 ± 6% versus -42 ± 4% in the control group. The extract according to the present invention was dissolved in DMSO and then diluted in Krebs solution (the same DMSO concentrations tested in the control group).

[0127] Minoxidil (0.1 to 30 μM) (Figure 8B) had no relaxing effects on aortic rings pre-contracted with 20 mM KCl. At 1000 μM, minoxidil relaxed the preparations (-81 ± 10%). The minoxidil was dissolved in distilled water. Therefore, control preparations with corresponding time for comparison were not included.

[0128] Gallic acid (0.1 to 3 μM) (Figure 8C) had no relaxing effects on aortic rings pre-contracted with 20 mM KCl. At 10 and 30 μM, gallic acid slightly increased the tension developed by aortic rings already pre-contracted with 20 mM KCl (+9 ± 8% and +21 ± 11%, respectively). It should be noted that the profile differed from one preparation to another. For three preparations (No. 4, 10, and 28), an increase in tension was observed up to 30 μM, while for the other 3 preparations (No. 5, 15, and 21), the tension was not modified. At the highest concentration (3,000 μM), gallic acid relaxed Petition 870240110548, dated 12 / 27 / 2024, pp. 39 / 58 18 / 24 all preparations (-62 ± 7%). This dual effect has already been described in the literature: contraction at low concentrations and relaxation at high concentrations. Gallic acid was dissolved in distilled water. Therefore, control preparations with corresponding time were not included for comparison. Conclusion

[0129] The data suggest that the extract according to the present invention relaxed the aortic rings from 100 pg / mL when precontracted with 0.1 μM noradrenaline and from 1000 pg / mL when precontracted with 20 mM KCl. This shows that the extract of the invention induces vasodilation and this effect may be related to the presence of gallic acid in its composition. Hair growth through prostaglandin modulation.

[0130] Prostaglandins regulate a variety of physiological activities, such as inflammation, neurotransmitter release, etc., but they are also involved in hair growth. Two subgroups of prostaglandins, PGE2 and PGF2, have been shown to promote hair growth in mice and humans. Conversely, prostaglandin D2 inhibits hair follicle elongation and promotes the onset of the catagen phase, leading to hair follicle miniaturization in androgenetic alopecia.

[0131] In this study, the effects of the extract of the invention were evaluated on prostaglandins E2, F2 and D2 (PGE2, PGF2 and PGD2) released by human follicle dermal papilla cells (HFDPC) using a specific ELISA kit.

[0132] More specifically, the potential activity of the test compounds was evaluated in the basal release of PGE2 and PGF2 to verify the stimulatory effect, while their activity in the release of PGD2 was evaluated under stimulation conditions, in order to show the inhibitory effects.

[0133] Before evaluating the extract according to the present invention in the PGD2 release analysis, an assay of Petition 870240110548, dated 12 / 27 / 2024, pages 40 / 58 19 / 24 Development was carried out to determine the best experimental conditions for inducing PGD2 release by HFDPC. For this purpose, two potential inducers were tested at 3 concentrations:

[0134] IL-Ια at 0.1, 1 and 10 ng / ml, and

[0135] PMA at 0.1, 0.3 and 1 ng / ml.

[0136] It was then decided to evaluate the effects of the test compound under conditions stimulated by IL-Iα at 1 ng / ml. Cultures and treatments

[0137] Basal conditions - release of PGE2 and PGF2

[0138] Human dermal papilla cells (HFDPC) were cultured in 96-well plates and cultured for 24 h in culture medium. The medium was replaced with assay medium (L-glutamine 2mM, penicillin 50U / mL, streptomycin 50pg / mL, FCS 10%) and the cells were incubated for a further 24 h. The medium was then replaced with assay medium containing or not (control) the extract according to the present invention or reference medium and the cells were incubated for 48 h. All experimental conditions were performed with n=3, except for the control n=6. Stimulated conditions - PGD2 release - assay development

[0139] HFDPC cells were cultured in a 96-well plate and cultured for 24 h in culture medium. The medium was then replaced with assay medium containing or not (stimulated control) the test or reference compound (indomethacin at 1 μM) and the cells were pre-incubated for 4 h. After pre-incubation, the inflammatory inducers, IL-Iα (tested at 0.1, 1 and 10 ng / ml) or PMA (tested at 0.1, 0.3 and 1 ng / ml) were added and the cells were incubated for 48 h. A control without inducer (unstimulated control) was prepared in parallel. All experimental conditions were performed with n=3. Stimulated conditions - PGD2 release - effect of the test compound

[0140] HFDPC were grown in a 96-well plate and cultured for 24 h in culture medium. The medium was then replaced with medium Petition 870240110548, dated 12 / 27 / 2024, pp. 41 / 58 20 / 24 assay cells containing or not (stimulated control) the extract according to the present invention or reference extract (indomethacin and dexamethasone at 1 μM) were pre-incubated for 4 h. After pre-incubation, the inflammatory inducer IL-Iα at 1 ng / ml was added and the cells were incubated for 48 h. A control without inducer (unstimulated control) was prepared in parallel. All experimental conditions were performed with n=3, except for the stimulated control condition n=6.

[0141] For each test, at the end of the incubation period, the culture supernatants were collected to measure the amount of PGE2, PGF2 and PGD2 released.

[0142] Additionally, to analyze the effect of the test compound on the PGD2 release assay, after collecting the supernatants, a cell viability assay was performed using a standard MTT reduction assay. Effect on basal PGE2 release

[0143] Under non-stimulation conditions, basal PGE2 release by HFDPC was limited (~50 pg / ml), but treatment with reference PMA at 0.1 μg / ml greatly stimulated PGE2 release (~6800 pg / ml). These results were expected and validated the assay.

[0144] Under the experimental conditions of the assay, the extract according to the present invention induced a significant increase in PGE2 release, but only at the highest concentration tested (110% stimulation at 100μg / ml). Table 1: Effect of the extract according to the present invention on PGE2 release by HFDPC under basal conditions Treatment Basic Data Normalized Data Tested Compound PGE2 Concentration (pg / ml) Average (pg / ml) % control (% pg / ml) % Stimulation (% pg / ml) Petition 870240110548, dated 12 / 27 / 2024, pp. 42 / 58 21 / 24 (dilution factor adjusts from ml) ) ) 43 <39 control - 40 <48 5 100 11 - 0 11 - e 43 50 74 6373 682 1419 56 ** 56 ** PMA 0.1 pg / ml 7306 0 270 7 2 * 14097 2 * 6781 42 nn 10 pg / ml <39 <40 1 <84 2 s <-16 2 s Extract <39 <39 of 30 pg / ml 64 <53 7 <111 15 n <11 15 n invention 57 ss 103 ** ** 100 pg / ml 107 101 4 210 8 * 110 8 * 93 ns: >0.05, not significant ***: <0.001, extremely significant Effect on basal PGF2 release

[0145] Under non-stimulation conditions, basal PGF2 release by HFDPC was substantial (571 pg / ml), but treatment with reference PMA at 0.1 pg / ml resulted in an even stronger release (3172 pg / ml, 555% of the control). These results were expected and validated the assay. Petition 870240110548, dated 12 / 27 / 2024, pp. 43 / 58 22 / 24

[0146] Under the experimental conditions of the assay, the extract according to the present invention did not significantly modulate the release of PGF2. Table 2: Effect of the extract according to the present invention on the release of PGF2 induced by HFDPC under basal conditions Treatment Basic Data Normalized Data Tested Compound PGF2 Concentration (pg / ml) Average (pg / ml) without (pg / ml) % control without (%) p % Stimulation without (%) p control - 655 564 627 595 447 539 571 30 100 5 - 0 5 - PMA 0.1 pg / ml 2745 3321 3450 3172 217 555 38 ** * 455 38 ** * Extract of the invention 10 pg / ml 470 551 565 529 29 93 5 ns -7 5 ns 30 pg / ml 472 541 614 542 41 95 7 ns -5 7 ns 100 pg / ml 597 461 537 532 39 93 7 ns -7 7 ns ns: >0.05, not significant ***: <0.001, extremely significant Effect on PGD2 release Petition 870240110548, dated 12 / 27 / 2024, pages 44 / 58 23 / 24

[0147] No basal release of PGD2 was detected (<2 pg / ml) and IL-Iα stimulation at 1 ng / ml in HFPDC resulted in a strong and significant increase in PGD2 release. Indomethacin and dexamethasone, tested as alternative reference compounds, both completely inhibited IL-1α-induced PGD2 release.

[0148] The extract according to the present invention showed a dual effect on IL-Iα-induced PGD2 release with an inhibitory effect at 10 and 30 pg / ml (24% and 48% inhibition) and then a stimulatory tendency at 100 pg / ml (37% stimulation). Depending on the individual proportion of ingredients in the extract, the loss of inhibitory activity at high concentrations may be related to the stimulatory effect of apigenin in the extract.

[0149] The extract according to the present invention showed a stimulatory effect on PGE2 release (at 100 pg / ml). It also significantly decreased IL-1α-induced PGD2 release, but only when tested at moderate concentrations (10 and 30 pg / ml). Table 3: Effect of the extract according to the present invention on the release of IL-Ια-stimulated PGD2 by HFDPC Treatment Basic Data Normalized Data MTT Feasibility Compound Tested Concentration PG D2 (pg / ml) Average (pg / ml) without (pg / ml) % stimulated control without (%) p % Relative inhibition without (%) p % stimulated control unstimulated control - < 2 < 2 < 2 < 2 0 < 7 0 * * * 100 0 * * * 90 control - 30 29 1 100 5 - 0 5 - 100 Petition 870240110548, dated 12 / 27 / 2024, pages 45 / 58 24 / 24 25 26 29 34 28 < 2 * * Indomet 1 μM < 2 < 2 0 < 7 0 * >10 0 * 108 acina < 2 * 0 * < 2 * * Dexame 1 μM < 2 < 2 0 < 8 1 * >99 1 * 91 tasona 3 * * 24 Extract 10 μg / ml 25 18 22 2 78 8 * 24 8 * 101 14 * * da 30 18 16 1 55 4 * 48 4 * 97 invention μg / ml 15 * * 100 35 * * μg / ml 39 42 39 2 137 7 * -39 8 * 108 ns: >0.05, not significant * : 0.01 to 0.05, significant **: 0.001 to 0.01, very significant **: <0.001, extremely significant Petition 870240110548, dated 12 / 27 / 2024, pages 46 / 58

Claims

1 / 3 CLAIMS 1. PROCESS FOR OBTAINING AN EXTRACT OF ROSMARINUS OFFICINALIS characterized by the extraction being from its whole leaf and comprising the following steps: a) 1st Extraction by maceration with agitation; b) Filtration of the product of a), obtaining the drained liquid I; c) 2nd Extraction by maceration with agitation; d) Filtration of the product of c), obtaining the drained liquid II; e) Mixing of the drained liquids I and II; f) Concentration; g) Drying.

2. PROCESS, according to claim 1, characterized in that the 1st and 2nd extractions are carried out with 50% ethanol, in a liquid:solid ratio of 10:

1.

3. PROCESS, according to claim 1 or 2, characterized in that the 1st and 2nd extractions are carried out by heating at 70 ± 5°C.

4. ROSMARINUS OFFICINALIS EXTRACT characterized by comprising hydroxycinnamic derivatives, rosmarinic acid, luteolin, neptin and phenolic diterpenes.

5. EXTRACT, according to claim 4, characterized in that said extract comprises 15 to 25% of hydroxycinnamic derivatives, 1 to 10% of rosmarinic acid, 1 to 10% of phenolic diterpenes, 0.1 to 1% of luteolin and 0.1 to 0.5% of neptin; wherein the phenolic diterpene comprises carnosol.

6. EXTRACT, according to claim 5, characterized in that said extract comprises 19% hydroxycinnamic derivatives, 5% rosmarinic acid, 5% phenolic diterpenes, 0.5% luteolin and 0.3% neptin.

7. ROSMARINUS OFFICINALIS EXTRACT characterized as being for use in the treatment / prevention of alopecia and / or strengthening of hair strands, in which the extract comprises as major compounds hydroxycinnamic derivatives, rosmarinic acid, luteolin, neptin and phenolic diterpenes.

8. USE OF AN EXTRACT OF ROSMARINUS OFFICINALIS, Petition 870240110548, dated 12 / 27 / 2024, page 47 / 58 2 / 3 as defined in claim 4 or 7, characterized by being for the manufacture of a dermocosmetic or pharmaceutical product for the treatment / prevention of alopecia and / or strengthening of hair strands.

9. USE OF AN EXTRACT OF ROSMARINUS OFFICINALIS, as defined in claim 4 or 7, characterized by being for the manufacture of a dermocosmetic or pharmaceutical product for strengthening keratin fibers.

10. DERMOCOSMETIC OR PHARMACEUTICAL COMPOSITION, characterized by comprising an extract of Rosmarinus officinalis, as defined in any one of claims 4 to 7, and at least one dermocosmetically or pharmaceutically acceptable excipient.

11. COMPOSITION, according to claim 10, characterized by comprising a concentration of between 2.5% and 15% of the extract.

12. COMPOSITION, according to claim 11, characterized by comprising the extract in a concentration of 2.5%, 5% or 10%.

13. COMPOSITION, according to any one of claims 9 to 12, characterized by being for use in the treatment / prevention of alopecia and / or strengthening of hair strands.

14. COMPOSITION, according to claim 13, characterized by being used in the treatment / prevention of alopecia and / or strengthening of hair strands, by acting on the activation mechanisms of the WNT / β-cathenin pathway, modulation of prostaglandins, inhibition of TGF-β, upregulation of IGF-1 and VEGF genes and vasodilatory effect.

15. METHOD FOR TREATING AND / OR PREVENTING ALOPECIA, characterized by comprising the administration of the composition, as defined in any one of claims 10 to 14, or of the extract of Rosmarinus officinalis, as defined in any one of claims 4 to 7.

16. METHOD, according to claim 15, characterized by the extract or composition acting on the activation of the WNT / β-cathenin pathway, modulation of prostaglandins, inhibition of TGF-β, up-regulation of IGF- and VEGF genes and vasodilatory effect. Petition 870240110548, dated 12 / 27 / 2024, page 48 / 58 3 / 3 1 17. METHOD, according to claim 15, characterized in that the extract or composition does not act on the 5-alpha-reductase enzyme and does not produce androgenic effects. Petition 870240110548, dated 12 / 27 / 2024, pp. 49 / 58

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