Process of obtaining an extract of rosmarinus officinalis, extract of rosmarinus officinalis, use of an extract, dermocosmetic or pharmaceutical composition and method of treatment and / or prevention of alopecia

Abstract

The present invention relates to a method for obtaining an extract of the leaves of Rosmarinus officinalis (rosemary) involving two stages of maceration, filtration, concentration of the two drained products and drying, guaranteeing an extract comprising mainly hydroxycinnamic derivatives, rosmarinic acid, luteolin, neptin and carnosol, and the use of the extract in cosmetic compositions in the prevention and treatment of androgenetic alopecia, in which the extract of the invention acts through multiple mechanisms of action, and can be used in men and women.

Description

PROCESS OF OBTAINING AN EXTRACT OFROSMARINUS OFFICINALIS, EXTRACT OFROSMARINUS OFFICINALIS, USE OF AN EXTRACT, DERMOCOSMETIC OR PHARMACEUTICAL COMPOSITION AND METHOD OF TREATMENT AND / OR PREVENTION OF ALOPECIAField of the Invention

[0001] The present invention relates to a method for obtaining an extract from the leaves ofRosmarinus officinalis(rosemary) involving two stages of maceration, filtration, concentration and drying, ensuring an extract comprising hydroxynamic 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, in which the extract of the invention acts in multiple mechanisms of action, which allows the use in both men and women.Background of the Invention

[0002] Androgenetic alopecia (AGA), also known as pattern baldness, is the most common type of progressive hair loss, with 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 decreased / thinning hair density in the central - 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 understood, it is known that factors such as stress, diet, and the individual's genetics and sensitivity to androgens contribute to its manifestation.

[0005] AGA is characterized by the progressive miniaturization of hair follicles, leading to terminal hair becoming vellus hair (i.e., shorter, thinner strands). This occurs due to a change in the hair cycle, in which the duration of the anagen phase (growth phase) of the hair gradually decreases and the telogen phase (resting phase, wherein the follicle prepares to release the hair) increases.

[0006] Androgens are important hormones 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). Increased DHT production, increased levels of the enzyme 5-alpha-reductase type II, and increase of androgen receptors in the scalp cause hair follicle reduction, replacing terminal hair with vellus hair.

[0007] Furthermore, systemic factors also influence the hair cycle, as it has been clinically observed that androgen blockade does not always result in the reversion of miniaturized hair follicles. Hair transplant experiments have shown that the system for the hair cycle is likely located within the hair follicle itself, considering that the organ maintains its characteristic cyclical behavior after transplantation. Studies with hair transplanted into mice showed an intrinsic cyclical pattern, but gradually acquired the host pattern, suggesting systemic influence. In this regard, molecules important for the hair cycle, such as insulin-like growth factor 1 (IGF1) and fibroblast growth factor 7 (FGF7), can be identified, which are important for hair follicle development. Some other relevant signaling molecules identified are the families of TGF-β, WNT / β- catenin, BMP, prostaglandins, neurotrophins, among others (Cuevas-Diaz 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 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 coexistence in society, representing beauty, vitality, youthfulness, and personal care. The manifestation of androgenetic alopecia, therefore, impacts people's quality of life, lowering their self-esteem and causing shame, affecting their mental health.

[0009] Options approved by the Food and Drug Administration (FDA) for the treatment of androgenetic alopecia are only two: 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 administered, minoxidil is converted to minoxidil sulfate by sulfotransferase enzymes, which potentiate potassium channels in smooth muscle, causing them to relax. This results in increased oxygen delivery 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, reducing its concentration in tissues. The use of finasteride in women with androgenetic alopecia is off- label, as it poses a risk of fetal malformations in pregnant women.

[0012] However, both treatments can bring with them adverse effects such as contact dermatitis, tachycardia and hypertrichosis with topical minoxidil; pruritus, 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 have also proven effective for the treatment of AGA or are being studied, for example, spironolactone, cyproterone, flutamide, clascoterone, JAK inhibitors, prostaglandin analogues, 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 research efforts to find 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 efficacy, 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 minor adverse reactions.Summary of the Invention

[0018] The present invention solves the aforementioned technical problem by providing a process for obtaining an extract ofRosmarinus officinalis(rosemary), the extract, the use of the extract, a dermocosmetic or pharmaceutical composition and a method of treatment of 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.

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

[0021] Below, the present invention will be defined through its objects:

[0022] As a first object, there is a process of obtaining an extract ofRosmarinus officinalis,in which extraction is done from the whole rosemary leaf and comprises the following steps:a) 1stextraction by maceration with agitation;b) Filtration of product a), obtaining drained I;c) 2ndextraction by maceration with agitation;d) Filtration of product c), obtaining drained II;e) Mixture of drained materials I and II;f) Concentration; andg) Drying.

[0023] In a second object, there is an extract ofRosmarinus officinaliscomprising mainly thehydroxynamic derivatives, rosmarinic acid, luteolin, neptin and phenolic diterpenes.

[0024] In another object, there is theRosmarinus officinalisextractfor use in the treatment / prevention of alopecia and / or strengthening of hair strands, in which the extract comprises, as major compounds, hydroxynamic derivatives, rosmarinic acid, luteolin, neptin and phenolic diterpenes.

[0025] In a fourth object, there is the use of an extract ofRosmarinus officinalisfor 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.

[0026] As a fifth object there is a dermocosmetic or pharmaceutical composition comprising an extract ofRosmarinus officinalisand at least one dermocosmetically or pharmaceutically acceptable excipient.

[0027] In a sixth object there is a method of treatment and / or prevention of alopecia, comprising the administration of the dermocosmetic or pharmaceutical composition or of theRosmarinus extract officinalis.Brief Description of the Figures

[0028] 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.

[0029] is a flowchart summarizing the extraction process of the invention and parameters employed in an embodiment.

[0030] shows the chromatographic profile ofRosmarinus officinalisextract 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).

[0031] 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 of testosterone-induced alopecia.

[0032] shows the graph of the time course and hair growth for the extract according to the present invention compared to reference controls in the mouse model of alopecia.

[0033] shows representative images of hair growth in mice from each group on day 30.

[0034] 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.

[0035] [Figure 7] shows the relaxation / contraction, in preparations pre-contracted with noradrenaline (%), induced by A) ACH38 (extract according to the present invention), B) minoxidil, and C) gallic acid.

[0036] [Figure 8] shows the relaxation / contraction, in preparations pre-contracted with KCl (%), induced by A) ACH38 (extract according to the present invention), B) minoxidil, and C) gallic acid.Detailed Description of the Invention

[0037] The present invention provides a process for obtaining an extract ofRosmarinus officinaliswith the whole leaf of the plant comprising two extractions by maceration, followed by two filtrations, concentration of the two drained extracts obtained and drying, the process of which produces an extract with activity in the treatment of androgenetic alopecia and strengthening of keratin fibers, particularly in 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

[0038] Unless explicitly stated otherwise, the following terms used throughout this specification and claims will have the following definitions for the purpose of the present invention:

[0039] “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 and 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.

[0040] "Dermocosmetically acceptable excipient" means that which is useful in the preparation of dermocosmetic compositions and it is generally safe for use in contact with human keratinized tissue without undue toxicity, allergic response, or similar reactions, and without biologically or otherwise undesirable effects. 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.

[0041] "At least" establishes the limit or minimum amount required for something. For example, 'at least x%' means at least x% or more.

[0042] The terms “about,” “probably,” “around,” and the like, when used for measurable values, mean an amount comprising variations of ±20%, ±10%, or ±5%, more preferably ±2%.

[0043] 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 a subject.

[0044] In a first object, there is a process of obtaining an extract ofRosmarinus officinalis,in which extraction is done from the whole leaf of the plant and comprises the following steps:a) 1stextraction by maceration with agitation;b) Filtration of the product from a), obtaining drained I;c) 2ndextraction by maceration with agitation;d) Filtration of the product from c), obtaining drained II;e) Mixture of drained materials I and II;f) Concentration; andg) Drying.

[0045] In one embodiment, the 1stand 2ndextractions are performed with 50% ethanol, in a liquid:solid ratio of 10:1.

[0046] In another embodiment, the 1stand 2ndextractions are carried out at a temperature of 70 ± 5ºC.

[0047] In one embodiment, drying is accomplished by spray drying.

[0048] In a second object, there is an extract ofRosmarinus officinaliscomprising hydroxynamic derivatives, rosmarinic acid, luteolin, neptin and phenolic diterpenes.

[0049] In one embodiment, the 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.

[0050] In a preferred embodiment, the extract comprises about 19% of hydroxycinnamic derivatives, about 5% of rosmarinic acid, about 5% of phenolic diterpenes, about 0.5% of luteolin, and about 0.3% of neptin.

[0051] In a preferred embodiment, the phenolic diterpene comprises carnosol.

[0052] In another embodiment, there is the extract ofRosmarinus officinalisfor use in the treatment / prevention of alopecia and / or strengthening of hair strands, in which the extract comprises hydroxynamic derivatives, rosmarinic acid, luteolin, neptin and phenolic diterpenes.

[0053] In a fourth object, there is the use of an extract ofRosmarinus officinalisfor 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.

[0054] As a fifth object there is a dermocosmetic or pharmaceutical composition comprising an extract ofRosmarinus officinalisand at least one dermocosmetically or pharmaceutically acceptable excipient.

[0055] In another embodiment, the dermocosmetic or pharmaceutical composition comprises a concentration of at least about 2.5% to 15% of the extract.

[0056] In one embodiment, the composition comprises a concentration of about 2.5%, 5%, or 10% of the extract.

[0057] In one embodiment, the composition is for use in treating / preventing alopecia and / or strengthening hair strands.

[0058] 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-catenin pathway, modulation of prostaglandins, inhibition of TGF-β, up-regulation of IGF-1 and VEGF genes and vasodilatory effect.

[0059] In a sixth object, there is a method of treatment and / or prevention of alopecia, comprising the administration of the dermocosmetic or pharmaceutical composition or of the extract ofRosmarinus officinalis.

[0060] In one embodiment, administration of the composition or extract is topically.

[0061] 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

[0062] The following examples are not limitative and only provide some of the various forms in which the invention may be embodied.Invention extraction process

[0063] A diagram illustrating the extraction process of the invention can be seen inExtraction

[0064] In a 3000L reactor, 40 kg of dried leaves ofRosmarinus officinalisand 400 L 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 through a rotary filter, and another 400 L 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 through a rotary filter. The resulting second drained solution was added to the first and homogenized.

[0065] At this stage, 618 kg of total drained material were obtained after the two extractions of the plant drug without grinding.Concentration

[0066] The total drained material from the first and second extractions, for a total of 618 kg, were homogenized and concentrated in a Bernauer concentrator at a maximum temperature of 75°C. 30.2 kg of concentrated extract with solids content of 28.7% were obtained.Drying

[0067] Finally, the concentrate was dried in a spray dryer within the following parameters: inlet temperature = 165ºC; minimum outlet temperature = 90ºC; air flow pressure control = 2.0 bar. 6.15 kg of dry extract were obtained according to the present invention.

[0068] shows the chromatographic profile of the extract according to the present invention by HPLC-DAD.

[0069] Comparative tests showed significantly lower results.Testosterone-Induced Alopecia in Mice

[0070] Alopecia was induced in C57BL / 6 mice by topical application of 100 µL of testosterone solution (0.05% in 50% ethanol) to shaved back skin once daily for 30 days. The animals were treated topically daily with reference drugs and the extract according to the present invention at different doses. The vehicle (100 µL 50% ethanol) was applied to sham control animals that served as controls.Formulations

[0071] 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 comparison was propylene glycol:ethanol (20:80). The market comparison formulation was prepared using 0.25% carboxymethyl cellulose as the vehicle. All formulations were prepared daily immediately before treatment.Treatment

[0072] Each day, 30 minutes after testosterone application, groups of animals were treated with the test and reference formulation, once daily (groups 1 to 15) or twice a week (groups 16 to 18), as specified in the experimental design.Observations

[0073] Hair growth was assessed in each mouse on days 0, 7, 12, 15, 18, 21, 24, and 30 after alopecia induction, and classified according to the following scale:0 no hair growth observed1 less than 20% growth observed2 of 20% less than 40% observed growth3 of 40% less than 60% observed growth4 of 60% less than 80% observed growth5 of 80% to 100% growth observedHistopathology

[0074] On days 0, 7, 12, 15, 18, 21, 24, and 30, ten animals from each group per time point were euthanized using CO2. Samples from the treated area of their dorsal skin were collected and preserved in 10% neutral-buffered formalin. The samples were processed using the standardized paraffin fixation technique. The prepared blocks were sectioned into 3 to 5 µm thicknesses using a microtome and mounted on clean glass slides. The slides were stained with hematoxylin and 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 of each group are reported ().Results

[0075] The means ± SD of hair growth assessments were estimated in each group at different time points. 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.

[0076] The time course of hair growth is shown inshows a comparison of hair growth for the extract of the invention andshows representative animals from each group after 30 days of treatment.Discussion

[0077] Hair growth classification:

[0078] Finasteride (5%, o.d.) and Minoxidil (2%, o.d.) showed significant efficacy when compared to the testosterone control vehicle (p<0.05), after 30 days of treatment.

[0079] Alpha-estradiol (0.03%, o.d.) and the market comparator (30 mg / kg, p.o., o.d.) did not show significant efficacy when compared to the testosterone control vehicle (p>0.05), after 30 days of treatment.

[0080] The extract according to the present invention showed significant (p<0.05) dose-dependent efficacy at 2.5%, 5% and 10% (o.d.) when compared to the vehicle control on day 30. Efficacy was also observed on day 18 onwards for the highest dose and from day 21 onwards for the lower 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.

[0081] Carnosic acid demonstrated significant (p<0.05) dose-dependent efficacy at 1.25%, 2.5%, and 5% (o.d.) when compared to vehicle control on day 30. Efficacy was also observed on day 18 onward for the medium and high dose and from day 21 onward for the lower doses.

[0082] On day 30, the efficacy of the extract according to the present invention (5%, of ) and carnosic acid (5%, o.d.) was comparable to that of finasteride (5%, o.d.).

[0083] On day 30, the efficacy of the extract according to the present invention (5%, twice weekly) was not significantly different from the vehicle control (P<0.05) and was significantly lower (not higher) when compared to once-daily dosing.

[0084] Carnosic acid (2.5%, twice weekly) showed significant efficacy when compared to the control vehicle and was comparable to once-daily dosing.Histopathology

[0085] At the end of 30 days of treatment, finasteride and minoxidil showed significantly (p<0.05) high A / T ratios (P<0.05) when compared to the negative control.

[0086] The extract according to the present invention (once a day) 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.Vasodilation effect

[0087] The vasodilation effect of the extract according to the present invention was evaluated in an in vitro model of isolated rat aorta.

[0088] Eight naive male Wistar (Han) rats were euthanized by CO2inhalation and then exsanguinated. A segment of the thoracic aorta was carefully excised. The artery was cleaned 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% O2and 5% CO2.

[0089] The method evaluated the effect of the test substance on pre-contracted preparations.

[0090] Preparations were precontracted with 20 mM KCl (or 0.1 µM norepinephrine) until a stable tonic contraction was achieved. Six cumulative test concentrations (0.01, 0.1, 1, 10, 100, and 1,000 µg / mL) or 7 cumulative comparison concentrations (minoxidil: 0.1, 0.3, 1, 3, 10, 30, and 1,000 µM; gallic acid: 0.1, 0.3, 1, 3, 10, 30, and 3,000 µM) were then added to the bath, and relaxations were recorded to plot a concentration-response curve. Preparations were washed, and a second concentration-response curve was evaluated on the same preparation (with a new precontraction agent).

[0091] Relaxation / contraction responses to the test substance were expressed as the percentage change from agonist-induced pre-contraction.ResultsPre-contraction with norepinephrine

[0092] In isolated rat aortic rings pre -contracted with 0.1 µM norepinephrine, the vehicle (10-5% DMSO to 0.1%) had no substantial effect (-16 ± 4% to 0.1%). In contrast, at 1% DMSO induced a relaxation of aortic rings pre -contracted with 0.1 µM norepinephrine (-43 ± 6%).

[0093] The ACH38 extract according to the present invention (from 0.01 to 1 µg / mL) () had no relaxant 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 a relaxation in a concentration-dependent manner compared to the control group:- at 10 µg / mL: -23 ± 11% versus -11 ± 3% in the control group,- at 100 µg / mL: -43 ± 17% versus -16 ± 4% in the control group, and- at 1000 µg / mL: -80 ± 8% versus -43 ± 6% in the control group.

[0094] 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).

[0095] Minoxidil (0.1 to 10 µM) () had no relaxant effects on aortic rings precontracted with 0.1 µM norepinephrine. At 30 and 1000 µM, minoxidil relaxed the preparations in a concentration-dependent manner (-24 ± 5% and -77 ± 3%, respectively). Minoxidil was dissolved in distilled water. Therefore, time-matched control preparations were not included for comparison.

[0096] Gallic acid, one of the substances present in the extract according to the present invention, (from 0.1 to 3 µM) () had no relaxant effects on aortic rings pre -contracted with 0.1 µM norepinephrine. At 10, 30, and 3000 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 differed 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 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, time-matched control preparations were not included for comparison.Pre-contraction with KCl

[0097] In isolated rat aortic rings precontracted with 20 mM KCl, the vehicle (10-5% DMSO to 0.1%) had no substantial effect (-17 ± 2% to 0.1%). In contrast, at 1% DMSO induced a relaxation of aortic rings precontracted with 20 mM KCl (-42 ± 4%).

[0098] The ACH38 extract according to the present invention (from 0.01 to 100 µg / mL) () had no relaxant effects on aortic rings pre-contracted with 20 mM KCl compared with the vehicle (DMSO) control group. 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).

[0099] Minoxidil (0.1 to 30 µM) () had no relaxant effects on aortic rings precontracted with 20 mM KCl. At 1000 µM, minoxidil relaxed the preparations (-81 ± 10%). Minoxidil was dissolved in distilled water. Therefore, time-matched control preparations were not included for comparison.

[0100] Gallic acid (0.1 to 3 µM) () had no relaxant 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 three preparations (no. 5, 15, and 21), the tension was not modified. At the highest concentration (3000 µM), gallic acid relaxed 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, time-matched control preparations were not included for comparison.Conclusion

[0101] The data suggests that the extract according to the present invention relaxed the aortic rings from 100 µg / mL when pre-contracted with 0.1 µM noradrenaline and from 1000 µg / mL when pre -contracted 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 by prostaglandin modulation

[0102] Prostaglandins regulate a variety of physiological activities, such as inflammation, neurotransmitter release, and so on, but they are also involved in hair growth. Two subgroups of prostaglandins, PGE2and 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.

[0103] In this study, the effects of the extract of the invention were evaluated on prostaglandins E2, F2 and D2 (PGE2, PGF2and PGD2) released by human follicle dermal papilla cells (HFDPC) using a specific ELISA kit.

[0104] More specifically, the potential activity of the test compounds was evaluated on basal PGE2and PGF2release to verify the stimulatory effect, while their activity on PGD2release was evaluated under stimulation conditions, so as to show the inhibitory effects.

[0105] Prior to evaluating the extract according to the present invention for PGD2release analysis, a development assay was performed to determine the best experimental conditions for inducing PGD2release by HFDPC. For this purpose, two potential inducers were tested at three concentrations:- IL-1α at 0.1, 1 and 10 ng / ml, and- PMA at 0.1, 0.3 and 1 ng / ml.

[0106] It was then decided to evaluate the effects of the test compound under conditions stimulated by IL-1α at 1ng / ml.Cultures and treatmentsBasal conditions - release of PGE2 and PGF2

[0107] Human dermal papilla cells (HFDPC) were cultured in a 96-well plate and grown for 24 h in culture medium. The medium was replaced by the assay medium (2 mM L-glutamine, 50 U / mL penicillin - 50 µg / mL streptomycin, 10% FCS), and the cells were incubated for an additional 24 h. The medium was then replaced with the assay medium containing or not (control) the extract according to the present invention or the reference, and the cells were incubated for 48 h. All experimental conditions were performed with n = 3, except for the control n = 6.

[0108] Stimulated conditions - PGD2release - assay development

[0109] HFDPCs were grown in a 96-well plate and cultured for 24 h in culture medium. The medium was then replaced with an assay medium containing or not (stimulated control) the test or reference compound (indomethacin at 1 µM), and the cells were preincubated for 4 h. After preincubation, the inflammatory inducers, IL-1α (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 an inducer (unstimulated control) was prepared in parallel. All experimental conditions were performed with n = 3.

[0110] Stimulated conditions - PGD2release - effect of test compound

[0111] HFDPCs were grown in a 96-well plate and cultured for 24 h in culture medium. The medium was then replaced with an assay medium containing or not (stimulated control) the extract according to the present invention or a reference (indomethacin and dexamethasone at 1 µM), and the cells were preincubated for 4 h. After preincubation, the inflammatory inducer IL-1α at 1 ng / ml was added, and the cells were incubated for 48 h. A control without an inducer (unstimulated control) was prepared in parallel. All experimental conditions were performed with n=3, except for the stimulated control condition n=6.

[0112] For each test, at the end of the incubation period, culture supernatants were collected to measure the amount of PGE2, PGF2, and PGD2released.

[0113] Additionally, for the analysis of the effect of the test compound on the PGD2release assay, after collecting the supernatants, a cell viability assay was performed using a standard MTT reduction assay.Effect on basal PGE2 release

[0114] Under non-stimulating conditions, basal PGE2release by HFDPC was limited (~50 pg / ml), but treatment with 0.1 µg / ml reference PMA greatly stimulated PGE2release (~6800 pg / ml). These results were expected and validated the assay.

[0115] Under the experimental conditions of the test, the extract according to the present invention induced a significant increase in the release of PGE2, but only at the highest concentration tested (110% stimulation at 100µg / ml).

[0116] [Table 1] Effect of the extract according to the present invention on PGE2release by HFDPC under basal conditionsTreatmentBasic dataNormalized dataTested compoundConcentrationPGE2(pg / ml)adjusted dilution factorAverage(pg / ml)Without (pg / ml)% controlWithout (%)p% stimulationWithout (%)pControl-43<3940435074<48510011-011-PMA0.1 µg / ml637373066781682027014197562***14097562***Extract of the invention10 µg / ml42<39<39<401<842ns<-162ns30 µg / ml<396457<537<11115ns<1115ns100 µg / ml1031079310142108***1108***ns: >0.05, not significant***: <0.001, extremely significantEffect on basal PGF2 release

[0117] Under unstimulated conditions, basal PGF2release by HFDPC was substantial (571 pg / ml), but treatment with 0.1 µg / ml reference PMA resulted in an even stronger release (3172 pg / ml, 555% of control). These results were expected and validated the assay.

[0118] Under the experimental conditions of the assay, the extract according to the present invention did not significantly modulate the release of PGF2.

[0119] [Table 2] Effect of the extract according to the present invention on the release of PGF2by HFDPC under basal conditionsTreatmentBasic dataNormalized dataTested compoundConcentrationPGE2(pg / ml)adjusted dilution factorAverage(pg / ml)Without (pg / ml)% controlWithout (%)p% stimulationWithout (%)pControl-655564627595447539571301005-05-PMA0.1 µg / ml274533213450317221755538***45538***Extract of the invention10 µg / ml47055156552929935ns-75ns30 µg / ml47254161454241957ns-57ns100 µg / ml59746153753239937ns-77nsns: >0.05, not significant***: <0.001, extremely significantEffect on PGD2 release

[0120] No basal PGD2release was detected (<2 pg / ml), and IL-1α stimulation at 1 ng / ml in HFPDC resulted in a strong and significant increase in PGD2release. Indomethacin and dexamethasone, tested as alternative reference compounds, both completely inhibited IL-1α-induced PGD2release.

[0121] The extract according to the present invention showed a dual effect on IL-1α-induced PGD2release with an inhibitory effect at 10 and 30 µg / ml (24% and 48% inhibition) and then a stimulatory tendency at 100 µg / ml (37% stimulation). Depending on the individual ratio of the ingredients in the extract, the loss of inhibitory activity at high concentrations may be related to the stimulatory effect of apigenin in the extract.

[0122] The extract according to the present invention showed a stimulatory effect on PGE2release (at 100 µg / ml). It also significantly decreased IL-1α-induced PGD2release, but only when tested at moderate concentrations (10 and 30 µg / ml).

[0123] [Table 3] Effect of the extract according to the present invention on IL-1α-stimulated PGD2release by HFDPCTreatmentBasic dataNormalized dataFeasibilityMTTTested compoundConcentrationPGD2(pg / ml)Average(pg / ml)Without(pg / ml)% controlstimulatedwithout (%)p% Relative inhibitionwithout (%)p%stimulated controlUnstimulated control-< 2< 2< 2< 20< 70***1000***90Control-3025262934282911005-05-100Indomethacin1 µM< 2< 2< 2< 20< 70***>1000***108Dexamethasone1 µM< 2< 23< 20< 81***>991***91Extract of the invention10 µg / ml242518222788*248*10130 µg / ml141815161554***484***97100 µg / ml3539423921377**-398**18ns: >0.05, not significant*: 0.01 to 0.05. significant**: 0.001 to 0.01, very significant***: <0.001, extremely significant

Claims

1.PROCESS OF OBTAINING AN EXTRACT OFROSMARINUS OFFICINALIScharacterizedby the extraction being from its whole leaf and comprising the steps of:a) 1stextraction by maceration with agitation;b) Filtration of the product from a), obtaining drained I;c) 2ndextraction by maceration with agitation;d) Filtration of the product from c), obtaining drained II;e) Mixture of drained materials I and II;f) Concentration; andg) Drying.2.PROCESS, according to claim 1,characterizedin that the 1stand 2ndextractions are carried out with 50% ethanol, in a liquid:solid ratio of 10:1.3.PROCESS, according to claim 1 or 2,characterizedin that the 1stand 2ndextraction are carried out at a temperature of 70 ± 5ºC.4.EXTRACT OFROSMARINUS OFFICINALIS,characterizedin that it comprises hydroxynamic derivatives, rosmarinic acid, luteolin, neptin and phenolic diterpenes.5.EXTRACT, according to claim 4,characterizedin that said extract comprises 15 to 25% of hydroxynamic 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,characterizedin that said extract comprises 19% of hydroxynamic derivatives, 5% of rosmarinic acid, 5% of phenolic diterpenes, 0.5% of luteolin and 0.3% of neptin.7.EXTRACT OFROSMARINUS OFFICINALIS,characterizedin that it is for use in the treatment / prevention of alopecia and / or strengthening of hair strands, in which the extract comprises as major compounds hydroxynamic derivatives, rosmarinic acid, luteolin, neptin and phenolic diterpenes.8.USE OF AN EXTRACT OFROSMARINUS OFFICINALIS,as defined in claim 4 or 7,characterizedin that it is 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 OFROSMARINUS OFFICINALIS, as defined in claim 4 or 7,characterizedin that it is for the manufacture of a dermocosmetic or pharmaceutical product for strengthening keratin fibers.10.DERMOCOSMETIC OR PHARMACEUTICAL COMPOSITION,characterizedin that it comprises an extract ofRosmarinus 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,characterizedin that it comprises a concentration between 2.5% and 15% of the extract.12.COMPOSITION, according to claim 11,characterizedin that it comprises the extract in a concentration of 2.5%, 5% or 10%.13.COMPOSITION, according to any one of claims 9 to 12,characterizedin that it is for use in the treatment / prevention of alopecia and / or strengthening of hair strands.14.COMPOSITION, according to claim 13,characterizedin that it is used in the treatment / prevention of alopecia and / or strengthening of hair strands, by acting on the activation mechanisms of the WNT / β-catenin pathway, modulation of prostaglandins, inhibition of TGF-β, up -regulation of IGF-1 and VEGF genes and vasodilatory effect.15.METHOD OF TREATMENT AND / OR PREVENTION OF ALOPECIA,characterizedin that it comprises the administration of the composition, as defined in any one of claims 10 to 14, or of the extract ofRosmarinus officinalisas defined in any one of claims 4 to 7.16.METHOD, according to claim 15,characterizedin that the extract or composition acts on the activation of the WNT / β-catenin pathway, modulation of prostaglandins, inhibition of TGF-β, up-regulation of IGF-1 and VEGF genes and vasodilatory effect.17.METHOD, according to claim 15,characterizedin that the extract or composition does not act on the 5-alpha-reductase enzyme and does not produce androgenic effects.

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