Compositions for the prevention and the treatment of hair loss

Abstract

The present invention concerns a composition comprising shikimic acid and ursolic acid, wherein shikimic acid can be preferably in the form of a Illicium verum extract comprising 5 to 40% by weight, preferably from 9.8 to 30% by weight, more preferably 9.8-20% by weight of shikimic acid, independently ursolic acid can be in the form of Ocimum sactum extract comprising from 0.1 to 5% by weight, preferably from 1 to 3% by weight, more preferably 1-2% by weight of ursolic acid. It is also disclosed a cosmetic use of the composition according to the invention for stimulating the physiological growth of hair and the composition for use in the prevention and treatment of hair loss.

Description

[0001] “COMPOSITIONS FOR THE PREVENTION AND THE TREATMENT OF HAIR LOSS”

[0002] ***** ***** *****

[0003] FIELD OF THE INVENTION

[0004] The present invention relates to a composition comprising shikimic acid and ursolic acid for preferably stimulating the physiological growth of hair and specifically for use in the prevention and treatment of hair loss.

[0005] In a preferred embodiments the two active principles are present in the form of extract. Therefore, the present invention relates also the composition lllicium verum extract comprising 5 to 40% by weight, preferably from 9.8 to 30% by weight, more preferably 9.8-20% by weight of shikimic acid and Ocimum sactum extract comprising 0.1 to 5% by weight, preferably from 1 to 3% by weight, more preferably 1 -2% by weight of ursolic acid.

[0006] STATE OF THE ART

[0007] Recently, hair loss prevention using natural products, or their extracts has gained interest. Plants and their extracts contain multiple components, such as polyphenols, flavonoids, terpenoids, carotenoids, and fatty acids, which support the maintenance of hair follicle health.

[0008] In this view, many attempts have been made to apply the regenerative ability of plant stem cells to hair loss treatment, but the precise mechanism has not yet been elucidated.

[0009] Several factors affect hair health, including genetics, chemical exposure, lifestyle, smoking, drugs, stress, infection, and menopause.

[0010] Recently, Shikimic acid (SA, 3,4,5-trihydroxy-1-cyclohexene-1 -carboxylic acid, C7H10O5) has been found as a major component of plant stem cells or callus that induces tissue regeneration when a plant is injured (Morus M, Baran M, Rost- Roszkowska M, Skotnicka-Graca U. Plant stem cells as innovation in cosmetics. Acta. Pol. Pharm. 2014;71 :701-707).

[0011] SA was originally known as an extract obtained from lllicium anisatum (Japanese star anise) and lllicium verum Hook. f. (Chinese star anise) (Bochkov DV, Sysolyatin SV, Kalashnikov Al, Surmacheva IA. Shikimic acid: review of its analytical, isolation, and purification techniques from plant and microbial sources. J. Chem. Biol. 2012;5:5- 17. doi: 10.1007 / sl 2154-011-0064-8 and Wang GW, Hu WT, Huang BK, Qin LP. Illicium verum: a review on its botany, traditional use, chemistry and pharmacology. J. Ethnopharmacol. 2011 ;136:10-20. doi: 10.1016 / j.jep.2011.04.051 ). I. verum is a species commonly used in traditional Chinese medicine as a therapeutic agent (Wang GW, Hu WT, Huang BK, Qin LP. Illicium verum: a review on its botany, traditional use, chemistry and pharmacology. J. Ethnopharmacol. 2011 ;136:10-20. doi: 10.1016 / j.jep.2011.04.051 ).

[0012] Recent scientific studies have proven the biological activities of both the fruit and essential oil of Illicium verum as antibacterial, antifungal, anti-inflammatory, and antioxidant effects.

[0013] The plant has been principally employed as the main source of (-)-shikimic acid (SA) or 3R,4S,5R-trihydroxycyclohexo-1 -encarboxylic acid, a natural organic compound (Eykman, J. F. (1881). "The botanical relations of Illicium religiosum Sieb., Illicium anisatum Lour". American Journal of Pharmacy. 53 (8) and Bochkov, D.V., Sysolyatin, S.V., Kalashnikov, A. I. et al. A search for raw materials for the isolation of shikimic acid. Russ J Bioorg Chem 39, 750-754 (2013)). It is an important intermediate in the biosynthesis of lignin, aromatic amino acids phenylalanine, tyrosine and tryptophan, and most plant alkaloids and microorganisms. The name comes from the plant from which the Illicium anisatum or Japanese Anise was extracted for the first time.

[0014] Numerous medicinal effects have been indicated for shikimic acid and its derivatives, namely anticancer, anticoagulant, anti-inflammatory, antioxidant, analgesic, antithrombotic, and antiseptic effects, demonstrated via in vitro and in vivo studies. In a study of Choi and collaborators (Choi M, Choi SJ, Jang S, Choi HI, Kang BM, Hwang ST, Kwon O. Shikimic acid, a mannose bioisostere, promotes hair growth with the induction of anagen hair cycle. Sci Rep. 2019 Nov 18;9(1): 17008. doi: 10.1038 / S41598-019-53612-5. PMID: 740717; PMCID: PMC6861222) the effects of SA on hair growth was examined. The effect of SA on a mouse model of C57BL / 6 has been studied. In particular, the expression of mannose receptor (MR), which is a known SA receptor, in human follicles and the effect of SA on human dermal papilla cells (hDPC), on external root sheath cells (hORSC) were examined. SA has been shown to significantly prolong anagen hair growth, hDPC and hORSC proliferation. The results in the analyzed model show that SA promotes hair growth and can act as a new therapeutic agent in the treatment of alopecia (Choi M, Choi SJ, Jang S, Choi HI, Kang BM, Hwang ST, Kwon 0. Shikimic acid, a mannose bioisostere, promotes hair growth with the induction of anagen hair cycle. Sci Rep. 2019 Nov 18;9(1):17008. doi: 10.1038 / S41598-019-53612-5. PMID: 740717; PMCID: PMC6861222).

[0015] Another biologically active metabolite derived from plant with proven benefit on hair growth is ursolic acid.

[0016] Ursolic acid is a pentacyclic triterpenoid compound that has anti-inflammatory and antimicrobial properties. It promotes hair growth by stimulating peripheral blood flow in the scalp and helps prevent alopecia and dandruff (Wozniak L, Skqpska S, Marszatek K. Ursolic Acid--A Pentacyclic Triterpenoid with a Wide Spectrum of Pharmacological Activities. Molecules. 2015 Nov 19;20(11):20614-41. doi: 10.3390 / molecules201119721. PMID: 26610440; PMCID: PMC6332387).

[0017] A botanical extract particularly rich in ursolic acid is the Ocimum sanctum L., also named Ocimum tenuiflorum L., that possesses well-known activity in preventing hair loss by strengthening the hair follicles. It also treats dandruff and itching and prevents premature graying of hair.

[0018] One of the mechanisms of action of ursolic acid on hair growth is related to the stimulation of the so-called "clock genes", genes responsible for controlling hair growth through the mechanism of circadian rhythms (Geyfman M, Andersen B. Clock genes, hair growth and aging. Aging (Albany NY). 2010 Mar 31 ;2(3): 122-8. doi: 10.18632 / aging.100130. PMID: 20375466; PMCID: PMC2871241).

[0019] Hair follicles undergo recurrent cycles of controlled growth (anagen), regression (catagen) and relative quiescence (telogen) with a defined periodicity. By adopting a genomic approach to study gene expression during the synchronized cycle of the hair follicle of the mouse, it was discovered that, in addition to circadian fluctuation, genes regulated by CLOCK are also modulated in phase with the hair growth cycle. During telogen and early anagen, circadian clock genes are expressed prominently in the secondary hair germ, which contains precursor cells for the growing follicle. Some studies have shown that a modification of genes involved in the circadian clock can change the hair growth phase suggesting that circadian clock genes regulate anagen progression through their effect on the cell cycle.

[0020] Therefore, many studies have linked the production of the stress hormone, cortisol, on the function and cyclic regulation of the hair follicle (Thom E. Stress and the Hair Growth Cycle: Cortisol-Induced Hair Growth Disruption. J Drugs Dermatol. 2016 Aug 1 ;15(8):1001 -4. PMID: 27538002; Arck PC, Handjiski B, Hagen E, Joachim R, Klapp BF, Paus R. Indications for a 'brain-hair follicle axis (BHA)': inhibition of keratinocyte proliferation and up-regulation of keratinocyte apoptosis in telogen hair follicles by stress and substance P. FASEB J. 2001 Nov;15(13):2536-8. doi: 10.1096 / fj.00- 0699fje. Epub 2001 Sep 17. PMID: 11641256; Ito N, Ito T, Kromminga A, Bettermann A, Takigawa M, Kees F, Straub RH, Paus R: Human hair follicles display a functional equivalent of the hypothalamic-pituitary-adrenal (HPA) axis and synthesize cortisol. FASEB. 2005; 19: 332-4 and Foitzik K, Krause K, Nixon AJ, et al: Prolactin and its receptor are expressed in murine hair follicle epithelium, show hair cycle-dependent expression, and induce catagen. Am. J. Pathol. 2003; 162:1611-21).

[0021] When cortisol is present at high levels it reduces the synthesis and accelerate the degradation of important skin elements, namely hyaluronan and proteoglycans by approximately 40%.

[0022] Cortisol is the primary stress hormone also at the follicular level. Chronic stress is deeply harmful to the health of the scalp and hair because it triggers the excessive production of the hormone cortisol. This causes negative reactions such as accelerated hair loss and thinning.

[0023] O. sanctum is able to block the release of cortisol acting at the level of the enzyme 11 beta-HSD1 : 11 p-hydroxysteroidal dehydrogenase type 1 which is a key activator of the stress hormone cortisol. The sacred basil blocks the action of 11 beta-HSD1 , thereby reducing the activation of cortisol (Richard EJ, llluri R, Bethapudi B, Anandhakumar S, Bhaskar A, Chinampudur Velusami C, et al. Anti-stress Activity of Ocimum sanctum: Possible Effects on Hypothalamic-Pituitary-Adrenal Axis. Phytother Res. 2016;30(5): 805-814. pmid:26899341).

[0024] It is also known that Melandrium firmum (MF) extract contributes to hair growthpromoting effect and regulates the expression of growth factors and inhibited 5a- reductase activity.

[0025] Other more commons other mechanisms through which botanical extract and their metabolites can stimulate hair growth include the 5-alpha reductase activity and the stimulation of growth factors (Park S, Lee J. Modulation of Hair Growth Promoting Effect by Natural Products. Pharmaceutics. 2021 Dec 15; 13(12):2163. Doi: 10.3390 / pharmaceutics13122163. PMID: 34959442; PMCID: PMC8706577).

[0026] Among this, Insulin-growth factor 1 (IGF-1) enhances hair follicle production and prolongs the anagen phase. The IGF stimulates the proliferation of cells in the matrix of hair follicles, as well as down-regulating TGFpl . The TGFpl also increases androgen receptor activation (Harada, N.; Okajima, K.; Arai, M.; Kurihara, H.; Nakagata, N. Administration of capsaicin and isoflavone promotes hair growth by increasing insulin-like growth factor-l production in mice and in humans with alopecia. Growth Horm. IGF Res. 2007, 17, 408-415. [Google Scholar] [CrossRef] and An, S.M.R.; El, M.; Philpott, P.; Thonlas, A.; Ealey, T.K. The Role of IGF-1 in Human Skin and its Appendages: Morphogen as Well as Mitogen? J. Investig. Dermatol. 1997, 109, 770-777).

[0027] The products currently used in the trichological field and based on products of a natural origin like the extracts have the drawback of being made through complex preparation processes and, therefore, are particularly expensive.

[0028] Consequently, there is currently a need to provide products containing substances that are active in stimulating the physiological process of hair growth as above indicated and the use of which does not cause significant side effects.

[0029] One of the objects of the invention is to provide a composition or preparation containing active agents of a non-synthetic origin which are suitable for stimulating the hair growth in subjects suffering from hair loss or thinning and the use of which is nearly free from significant side effects.

[0030] Another object of the invention is to provide a composition for trichological use which may be applied locally or administered orally, the active agents of which are of a vegetal origin obtainable via simple methods.

[0031] SUMMARY OF THE INVENTION

[0032] The inventors found out that a composition comprising shikimic acid and ursolic acid was capable to synergistically stimulate the physiological process of hair growth, preferably when these two active principles were in the form of extract, specifically as lllicium verum extract comprising from 5 to 40% by weight, preferably from 9.8 to 30% by weight, more preferably 9.8-20% by weight of shikimic acid and Ocimum sactum extract comprising from 0.1 to 5% by weight, preferably from 1 to 3% by weight, more preferably 1 -2% by weight of ursolic acid.

[0033] The Applicant has unexpectedly found out that shikimic acid and ursolic acid, when preferably and independently to each other in the form of extract of lllicium verum and Ocimum sactum extract, respectively, together synergistically stimulate the activity of the hair bulb and contribute to restoring the physiological conditions of hair growth. Their effects are hence not additive, but the composition showed a synergistic effect deriving from putting together the two active principles as it will clear from the experimental part.

[0034] In accordance with a first aspect, therefore, the invention relates to a composition comprising shikimic acid and ursolic acid.

[0035] In a preferred embodiment the one of the two active principles, shikimic acid or ursolic acid, can be present in the form of extract, independently to each other.

[0036] In another aspect, the present invention relates also a composition comprising lllicium verum extract comprising from 5 to 40% by weight, preferably from 9.8 to 30% by weight, more preferably 9.8-20% by weight of shikimic acid and Ocimum sactum extract comprising from 0.1 to 5% by weight, preferably from 1 to 3% by weight, more preferably 1-2% by weight of ursolic acid .

[0037] In a still further aspect, the invention relates to the use of the composition comprising shikimic acid and ursolic acid or, preferably, the composition comprising lllicium verum extract comprising from 5 to 40% by weight, preferably from 9.8 to 30% by weight, more preferably 9.8-20% by weight of shikimic acid and Ocimum sactum extract comprising from 0.1 to 5% by weight, preferably from 1 to 3% by weight, more preferably 1-2% by weight of ursolic acid for stimulating the physiological growth of hair.

[0038] In a still further aspect, the invention relates to a composition comprising shikimic acid and ursolic acid or, preferably, a composition comprising lllicium verum extract comprising from 5 to 40% by weight, preferably from 9.8 to 30% by weight, more preferably 9.8-20% by weight of shikimic acid and Ocimum sactum extract comprising from 0.1 to 5% by weight, preferably from 1 to 3% by weight, more preferably 1 -2% by weight of ursolic acid for the prevention and treatment of hair loss.

[0039] Preferably the composition of the invention comprises lllicium verum extract is in amount from 0.005% w / w to 50% w / w with respect to the total amount of the composition. and Ocimum sactum extract is in amount from 0.005% to w / w to 50% w / w with respect to the total amount of the composition.

[0040] Surprisingly it has been observed that the compositions of the invention administered topically or orally in a subject suffering from hair thinning, determines a progressive thickening of thinner areas of the scalp.

[0041] When the administration is topical, the composition will comprise preferably lllicium verum extract in an amount from 0.005% w / w to 5% w / w with respect to the total amount of the composition and Ocimum sactum extract in an amount from 0.005% to w / w to 5% w / w with respect to the total amount of the composition.

[0042] When the administration is oral, the composition will comprise preferably lllicium verum extract in an amount from 5% w / w to 50% w / w with respect to the total amount of the composition and Ocimum sactum extract in an amount from 5% to w / w to 50% w / w with respect to the total amount of the composition.

[0043] The compositions of the invention can advantageously comprise a physiologically acceptable carrier.

[0044] Preferably, the composition of the invention contains a trichologically active amount of shikimic acid and ursolic acid as substances per se or in the form of extracts, preferably lllicium verum extract comprising from 5 to 40% by weight, preferably from 9.8 to 30% by weight, more preferably 9.8-20% by weight of shikimic acid and Ocimum sactum extract comprising from 0.1 to 5% by weight, preferably from 1 to 3% by weight, more preferably 1-2% by weight of ursolic acid .

[0045] The Applicant has found that the compositions of the invention comprising shikimic acid and ursolic acid, or preferably extracts containing them, when administered locally, topically or cutaneously, performs a stimulating action on the follicle cells, reactivating the life cycle of the hair follicles.

[0046] Furthermore, it has surprisingly been found that the trichological activity of the compositions of the invention is also exerted on the quiescent hair bulbs of the scalp, typically present in those areas of the scalp wherein a thinning of the hair is found.

[0047] In another preferred aspect, the cosmetic compositions of the invention, specifically in their preferred aspect as detailed below are hence used for improving an aesthetic aspect of the hair or of the skin.

[0048] BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The invention is described in detail below with reference to the Figures, specifically: Figure 1 : Expression of the Insuline-like growth factor 1 (IGF-1) gene in Human Follicle Dermal Papilla Cells (HFDPC) as determined by RT-PCR. HFDPC cells were treated at 37°C for 24h, under 5% CO2, with: basal medium (Control); Shikimic acid 3.2 pg / mL; ursolic acid 0.32 pg / mL; shikimic acid 3.2 pg / mL + Ursolic acid 0.32 pg / mL; lllicium verum 16 pg / mL; Ocimum sanctum 16 pg / mL; lllicium verum 16 pg / mL (containing 20% shikimic acid)+ Ocimum sanctum 16 pg / mL (containing 2% ursolic acid) in complete culture medium. Values are means of 2 experiments in duplicate. *p<0.05; **p<0.01.

[0050] Figure 2. Expression of the tumor necorsi growth factor (TGF-p1) gene in Human Follicle Dermal Papilla Cells (HFDPC) as determined by RT-PCR. HFDPC cells were treated at 37°C for 24h, under 5% CO2, with: basal medium (Control); lllicium verum 16 pg / mL; Ocimum sanctum 16 pg / mL; lllicium verum 16 pg / mL (containing 20% shikimic acid)+ Ocimum sanctum 16 pg / mL (containing 2% ursolic acid) in complete culture medium. Values were means of 2 experiments in duplicate. *p<0.05; **p<0.01 .

[0051] DETAILED DESCRIPTION OF THE INVENTION

[0052] The invention hence relates to a composition comprising shikimic acid and ursolic acid, preferably and independently to each other in the form of extract of lllicium verum and Ocimum sactum extract, respectively, that unexpectedly, together synergistically, stimulate the activity of the hair bulb and contribute to restoring the physiological conditions of hair growth.

[0053] In accordance with a first aspect, therefore, the invention relates to a composition comprising shikimic acid and ursolic acid.

[0054] In a preferred embodiment one of the two active principles is, independently to each other, present in the form of extract.

[0055] When these two active principles were in the form of extract, shikimic acid is present as lllicium verum extract comprising from 5 to 40% by weight, preferably from 9.8 to 30% by weight, more preferably 9.8-20% by weight of shikimic acid and ursolic acid as Ocimum sactum extract comprising from 0.1 to 5% by weight, preferably from 1 to 3% by weight, more preferably 1-2% by weight of ursolic acid.

[0056] In another aspect, the present invention relates also the composition comprising lllicium verum extract comprising from 5 to 40% by weight, preferably from 9.8 to 30% by weight, more preferably 9.8-20% by weight of shikimic acid and Ocimum sactum extract comprising from 0.1 to 5% by weight, preferably from 1 to 3% by weight, more preferably 1-2% by weight of ursolic acid.

[0057] In the present invention when the followings terms are used:

[0058] - “shikimic acid” it is intended 3,4,5-trihydroxy-1-cyclohexene-1 -carboxylic acid of formula

[0059] - “ursolic acid” it is intended a pentacyclic triterpenoid compound that has anti- inflammatory and antimicrobial properties of formula

[0060] - “Hair”, it is intended to comprise hair, as skin annexes, like eyebrows, eye-lashes and nails, hairs, mustache and beard.

[0061] The shikimic acid can be present as substance per se or in the form of an extract.

[0062] In a preferred aspect the composition of the invention comprises lllicium verum, also known as lllicium verum Hook. f. extract comprising from 5 to 40% by weight, preferably from 9.8 to 30% by weight, more preferably 9.8-20% by weight of shikimic acid .

[0063] The ursolic acid can be present in the composition as a substance per se or in the form of Ocimum sactum extract, also known as Ocimum tenuiflorum L, comprising 0.1 to 5% by weight, preferably from 1 to 3% by weight, more preferably 1 -2% by weight of ursolic acid.

[0064] In some embodiments, the preferred extract of the invention comprising shikimic acid and ursolic acid, respectively, are preferably derived from the cortex, fructus or any other appropriate part of the plant of lllicium verum or from summitas cum floribus, Folium-or any other appropriate part of the plant Ocimum sactum or from two or more of these parts of the plant.

[0065] According to some embodiments, the extracts of the invention are obtained by extraction from a part of the plant, in particular the aerial parts or its vegetal tissue, using as an extraction means a physiologically acceptable or edible solvent. The term edible means a physiologically acceptable solvent that may be absorbed by the human body.

[0066] A suitable solvent to obtain the extract is a physiologically acceptable and / or edible liquid wherein the biologically active components are soluble and wherein they do not undergo alteration that deprives them of their activity.

[0067] In some embodiments the solvent is hydrophilic and is selected from water, ethyl acetate, ethanol or mixtures thereof.

[0068] Preferably, the composition of the invention contains a trichologically active amount of shikimic acid and ursolic acid as substances per se or in the form of extracts, preferably lllicium verum extract comprising 9.8-20% by weight of shikimic acid as active principle shikimic acid and Ocimum sactum extract comprising 1-2% by weight of ursolic acid as active principle ursolic acid.

[0069] The shikimic acid is preferably present in an amount from 0.001 mg to 150 mg; preferably 0.01 to 90 mg, more preferably 10-30 mg or 0.01-1 mg per dosage unit.

[0070] Preferably, the lllicium verum extract comprising from 5 to 40% by weight, preferably from 9.8 to 30% by weight, more preferably 9.8-20% by weight of shikimic acid is preferably present in an amount from 0.001 to 150mg, 0.01 to 100 mg, preferably 90 to 110 mg or 0.01 to 1 .5 per dosage unit.

[0071] The ursolic acid is preferably present in an amount from 0.001 to 10 mg, preferably 1 to 3 mg or 0.01 to 0.03 per dosage unit.

[0072] Preferably, the Ocimum sactum extract, also known as Ocimum tenuiflorum L, comprising from 0.1 to 5% by weight, preferably from 1 to 3% by weight, more preferably 1 -2% by weight of ursolic acid is preferably present in an amount from 0.001 to 150mg, 0.01 to 100 mg, preferably 90 to 110 mg or 0.01 to 1.5 mg per dosage unit.

[0073] Preferably the composition of the invention comprises lllicium verum extract is in amount from 0.005% w / w to 50% w / w with respect to the total amount of the composition.

[0074] Preferably the composition of the invention comprises Ocimum sactum extract is in amount from 0.005% to w / w to 50% w / w with respect to the total amount of the composition.

[0075] The compositions of the invention can preferably comprise further ingredients.

[0076] In a preferred aspect the compositions of the invention comprise rutin, a flavonic

[0077] glycoside of formula for example contained in the extracts of Carpobrotus edulis, Capers, Olives,

[0078] Asparagus, Raspberry, Buckwheat, Green Currants, Plums, Black Currants,

[0079] Blackberries, Tomatoes, Plums, Fenugreek, Marjoram, Tea, Grapes, Pumpkin,

[0080] Apricots.

[0081] Rutin, when present, is preferably in an amount of 0.001 to 50 mg, more preferably 5 to 15 mg, still more preferably 9 to 11 mg per dosage unit.

[0082] In a preferred aspect the compositions of the invention comprise Reishi (Ganoderma lucidum), containing ganodermic acids of formula more preferably in an amount 0.001 to 150 mg, preferably 50 to 90 mg, more preferably 60 to 80 mg per dosage unit.

[0083] Reishi (Ganoderma lucidum) when present is in amount of XX % by weight with respect to the total amount of the composition.

[0084] In accordance with certain embodiments, the compositions of the invention further comprise one or more active substances, such as vitamins, minerals, micronutrients and other substances active in stimulating the activity of the hair follicle.

[0085] The compositions of the invention can advantageously comprise a physiologically acceptable carrier.

[0086] In a still further aspect, the invention relates to the use of the composition comprising shikimic acid and ursolic acid for stimulating the physiological growth of hair.

[0087] In a preferred embodiment one of the two active principles can be present as an extract, preferably, lllicium verum extract comprising from 5 to 40% by weight, preferably from 9.8 to 30% by weight, more preferably 9.8-20% by weight of shikimic acid and Ocimum sactum extract comprising from 0.1 to 5% by weight, preferably from 1 to 3% by weight, more preferably 1 -2% by weight of ursolic acid .

[0088] In another aspect the invention relates to a use of the composition comprising lllicium verum extract comprising from 5 to 40% by weight, preferably from 9.8 to 30% by weight, more preferably 9.8-20% by weight of shikimic acid and Ocimum sactum extract comprising from 0.1 to 5% by weight, preferably from 1 to 3% by weight, more preferably 1 -2% by weight of ursolic acid for stimulating the physiological growth of hair.

[0089] In a still further aspect, the invention relates to a composition comprising shikimic acid and ursolic acid or, preferably, the composition comprising lllicium verum extract comprising from 5 to 40% by weight, preferably from 9.8 to 30% by weight, more preferably 9.8-20% by weight of shikimic acid and Ocimum sactum extract comprising from 0.1 to 5% by weight, preferably from 1 to 3% by weight, more preferably 1-2% by weight of ursolic acid for use in the prevention and treatment of hair loss.

[0090] Preferably the composition of the composition comprises lllicium verum extract comprising from 5 to 40% by weight, preferably from 9.8 to 30% by weight, more preferably 9.8-20% by weight of shikimic acid and Ocimum sactum extract comprising from 0.1 to 5% by weight, preferably from 1 to 3% by weight, more preferably 1-2% by weight of ursolic acid, is for use in the prevention and treatment of hair loss due to stress. Surprisingly it has been observed that the compositions of the invention administered topically or orally in a subject suffering from hair thinning, determines a progressive thickening of thinner areas of the scalp. When the compositions of the invention are administered topically and orally perform a stimulating action on the follicle cells, reactivating the life cycle of the hair follicles.

[0091] Furthermore, it has surprisingly been found that the trichological activity of compositions of the invention is also exerted on the quiescent hair bulbs of the scalp, typically present in those areas of the scalp wherein a thinning of the hair is found.

[0092] In certain aspects, therefore, there is provided the cosmetic use of the compositions of the invention for stimulating the physiological growth of hair.

[0093] In accordance with a still further aspect, there is provided a composition according to the for use in stimulating the physiological growth of hair.

[0094] Extracts can be used in place of shikimic acid and ursolic acid, independently each other.

[0095] A vegetal extract for the uses of the invention may be derived from the cortex, fructus, Aetheroleum of the plant of lllicium verum or from summitas cum floribus, Folium of the plant Ocimum sactum or from two or more of these parts of the plant.

[0096] The vegetal extracts of the invention can be those available on the market or obtained according to well known extraction techniques.

[0097] For example, in order to obtain the vegetal extracts extraction techniques may be employed using supercritical CO2 or solid-liquid techniques suitable for separating / extracting from the vegetal tissues of the plant one or more biologically active components to restore physiological conditions of activity of the hair bulb.

[0098] In certain embodiments the vegetal extract of the plant lllicium verum or of the plant Ocimum sactum is administered at a concentration from 0.01 to 700 mg / ml.

[0099] Without being bound to any theory the compositions of the invention perform an inhibitory activity towards the 5-alpha-reductase enzyme, which plays an important role in androgenetic alopecia and is a target of finasteride, a known compound having antiandrogenetic activity, effective in stimulating hair growth and in reactivating the physiological cycle of hair follicles. Further methods for obtaining the vegetal extract of the invention include extraction techniques by digestion, infusion, squeezing, decoction, leaching, counter-current extraction, soxhlet, extraction with supercritical gases or ultrasounds.

[0100] According to the invention, the biologically active component contained in the extract of lllicium verum is shikimic acid and of Ocimum sactum is ursolic acid.

[0101] The biologically active components contained in the vegetal extract of the invention reactivate the life cycle of hair follicles, even when quiescent, of the scalp in thinned areas and / or areas affected by baldness. This action translates into the re-growth of hair also in areas of the scalp where the hair is thinned out.

[0102] The present invention hence provides the use of a composition according to the invention in the treatment and / or prevention of hair loss or for stimulating the physiological growth of hair.

[0103] Preferably the treatment and / or prevention of hair loss is the treatment and / or prevention of hair loss due to stress.

[0104] In particular, the composition of the invention comprises an active amount of shikimic acid and ursolic acid, preferably each present as an extract, independently to each other.

[0105] The compositions of the invention can be a medical device, a pharmaceutical formulation, a dietary or nutritional supplement, or a cosmetic composition.

[0106] The compositions of the invention, when administered locally or systemically, increase the viability of follicle cells, both in the anagen phase and in the miniaturization phase and reactivates quiescent cells of the scalp by regenerating the follicles and stimulating the growth of new hair.

[0107] In some embodiments the compositions of the invention comprise a physiologically and / or pharmaceutically acceptable carrier, diluent or excipient.

[0108] Typically, the physiologically acceptable carrier of the compositions of the invention is an excipient, carrier or diluent suitable for topical application and / or oral administration.

[0109] Within the scope of the present document, the term "carrier" relates to an excipient, carrier, diluent or adjuvant that may be present in the composition of the invention. Any carrier and / or excipient suitable for the form of preparation desired for administration is contemplated in the uses herein described active ingredients present therein.

[0110] Typically, the compositions of the invention comprise components of a vegetal origin which are biologically active and substantially free from side effects, when administered orally or locally.

[0111] In some embodiments, the compositions of the invention are administered orally. In other embodiments, the composition are topically applied to the skin, and in particular to the scalp.

[0112] The shikimic acid in the oral administration is preferably present in an amount from 0.01 mg to 150mg, more preferably 10 mg to 50 mg; still more preferably 10-30 mg for unitary dosage.

[0113] Preferably in the oral administration, the lllicium verum extract comprising from 5 to 40% by weight, preferably from 9.8 to 30% by weight, more preferably 9.8-20% by weight of shikimic acid is preferably present in an amount from 0.001 mg to 150mg, more preferably 10 to 100 mg, still more preferably 90 to 110 mg per dosage unit.

[0114] In the oral administration the lllicium verum extract comprising from 5 to 40% by weight, preferably from 9.8 to 30% by weight, more preferably 9.8-20% by weight of shikimic acid is preferably present in the composition in an amount in the range of 5 % to 50% by weight with respect to the total amount of the invention.

[0115] The ursolic acid in the oral administration is preferably present in an amount from 0.001 to 50 mg, more preferably 0.1 to 10 mg, still more preferably 1 to 3 per dosage form.

[0116] Preferably, the Ocimum sactum extract, also known as Ocimum tenuiflorum L, comprising 0.1 to 5% by weight, preferably from 1 to 3% by weight, more preferably 1-2% by weight of ursolic acid is preferably present in the oral administration in an amount from 0.001 mg to 150mg, more preferably 10 to 100 mg, still more preferably 90 to 110 mg per dosage unit.

[0117] In the oral administration the Ocimum sactum extract, also known as Ocimum tenuiflorum L, comprising 0.1 to 5% by weight, preferably from 1 to 3% by weight, more preferably 1 -2% by weight of ursolic acid is preferably present in the composition in an amount in the range of 5 to 50% by weight with respect to the total amount of the invention. The physiologically and / or pharmaceutically acceptable carrier, diluent or excipient may be selected based on the route of administration for which the resulting pharmaceutical composition is intended.

[0118] In some embodiments, the route of administration of the compositions of the invention is topical. In these cases, the composition of the invention may be applied, in an effective amount, directly to the scalp.

[0119] The shikimic acid in the topical administration is preferably present in an amount from 0.001 mg to 150mg, more preferably 0.01 mg to 10 mg; still more preferably 0.01 -1 mg for unitary dosage.

[0120] Preferably in the topical administration, the lllicium verum extract comprising 5 to 40% by weight, preferably from 9.8 to 30% by weight, more preferably 9.8-20% by weight of shikimic acid is preferably present in an amount from 0.001 mg to 150mg, more preferably 0.01 to 10 mg, still more preferably 0.01 to 1.5 mg per dosage unit.

[0121] In the topical administration the lllicium verum extract comprising 5 to 40% by weight, preferably from 9.8 to 30% by weight, more preferably 9.8-20% by weight of shikimic acid is preferably present in the composition in an amount in the range of 0.005 to 5% by weight with respect to the total amount of the invention.

[0122] The ursolic acid in the topical administration is preferably present in an amount from 0.001 to 50 mg, more preferably 0.001 to 10 mg, still more preferably 0.01 to 0.03 mg per dosage form.

[0123] Preferably, the Ocimum sactum extract, also known as Ocimum tenuiflorum L, comprising from 0.1 to 5% by weight, preferably from 1 to 3% by weight, more preferably 1-2% by weight of ursolic acid is preferably present in the topical administration in an amount from 0.001 to 50 mg, more preferably 0.01 to 10 mg, still more preferably 0.01 to 1.5 mg per dosage unit.

[0124] In the topical administration the Ocimum sactum extract, also known as Ocimum tenuiflorum L, comprising from 0.1 to 5% by weight, preferably from 1 to 3% by weight, more preferably 1 -2% by weight of ursolic acid is preferably present in the composition in an amount in the range of 0.005 to 5% by weight with respect to the total amount of the invention.

[0125] For example, in the treatment of androgenetic alopecia a cosmetically active amount of compositions of the invention may be applied directly to the scalp once or multiple times a day conveniently for cycles lasting 2-3 months, alternating with rest periods. In a preferred embodiment the cosmetically active amount of compositions of the invention can be applied in a pulsed manner, for example twice a week for a determined period of time, once every three days and once a week.

[0126] According to another aspect of the invention, there is provided a method of cosmetic treatment which comprises the application to the scalp of an effective amount of a composition of the type described previously.

[0127] The composition for topical application may be in the solid, semi-solid or fluid form. Suitable formulations in the solid form include creams, gels, pastes, ointments.

[0128] In other embodiments the formulation for local administration is in the fluid form, for example in the form of lotions, gels, shampoos, suspensions, emulsions.

[0129] In the case of formulations in the fluid or semi-fluid form, the two active principles also in the form of extracts, independently to each other, may be diluted in a carrier in the physiologically acceptable liquid form such as water, alcohol, hydroalcoholic or glyceryl solution or mixed with other liquids suitable for local application.

[0130] By way of example, the compositions of the invention in the liquid form may be prepared dissolving the active components, also in the form of the extract in water and / or alcohol. The liquid composition may be buffered to achieve a pH range conveniently selected between 5 and 7 so as to be compatible with the pH of the scalp and then be filtered and packaged in suitable containers such as vials or ampoules.

[0131] In some embodiments, the compositions of the invention may comprise excipients commonly used in the formulation of cosmetic or pharmaceutical preparations, for local use, such as preservatives, bactericidal agents, stabilizing agents, emulsifying agents, buffers, wetting agents, coloring agents and other excipients commonly used in the cosmetic / pharmaceutical preparation techniques. In one embodiment, the formulation for local application is in the form of an emulsion containing the extract carried in a suitable excipient.

[0132] By way of example, suitable excipients are cellulose derivatives such as microcrystalline cellulose, hydroxymethyl cellulose, hydroxypropyl methylcellulose, methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, carboxyethyl cellulose, ethylhydroxyethyl cellulose, cellulose acetate butyrate, cellulose acetate phthalate, and mixtures thereof.

[0133] Further examples of suitable excipients comprise the polymers belonging to the family of lactams, such as pyrrolidone and derivatives thereof, for example polyvinylpyrrolidone, polyvinylpolypyrrolidone and mixtures thereof, inorganic salts such as calcium or dicalcium phosphate, lubricants such as for example magnesium stearate, triacylglycerols and mixtures thereof.

[0134] In some embodiments, the composition for topical application comprises an excipient of the type hydroxymethyl cellulose and / or gelling agents with HLB suitable for the formulation and substances.

[0135] According to other embodiments, the administration of the compositions of the invention takes place orally.

[0136] The compositions for oral administration may be in the solid or liquid form. Typical compositions in the solid form comprise tablets, capsules, powders, granules, pills. Typical compositions in the liquid form comprise solutions, emulsions, suspensions, syrups. All the compositions also comprise controlled-release forms of the same.

[0137] Tablets generally comprise a suitable carrier or excipient wherein the vegetal extract is dispersed, typically in the dry form.

[0138] The active components of the composition of the invention, ursolic acid and shikimic aid, may be present in variable amounts, for example in the range from 0.0001 % by weight to 10% by weight, more preferably from 0.01 to 5% by weight.

[0139] In accordance with certain embodiments as above indicated, the compositions of the invention further comprise one or more active substances, such as vitamins, minerals, micronutrients and other substances active in stimulating the activity of the hair follicle. In accordance with some embodiments, the compositions for oral administration are a functional food, a nutraceutical composition, a dietary product, a supplement or a nutritional product or a medical device.

[0140] Functional food means any modified food or food ingredient which may provide a benefit or protection against a drawback or a physiological condition, besides the traditional nutrients it contains.

[0141] Nutraceutical product means a product isolated or purified from edible substances. A nutraceutical product is such when it shows to have a physiological advantage or to provide protection against a drawback or physiological disorder.

[0142] Dietary or nutritional supplement means a product containing a vitamin, mineral, vegetal extract, amino acid, metabolite, extract, concentrate or mixtures of these ingredients.

[0143] The amount administered and the frequency of administration of the composition will depend on the type and severity of the trichological condition to be treated.

[0144] The compositions of the invention are effective in preventing and / or treating the nonscarring alopecia (such as for example androgenetic alopecia, alopecia areata, telogen effluvium, anagen effluvium) and cicatricial alopecia.

[0145] The present invention shall now be described with reference to the following examples which are provided for illustration purposes and shall not be intended as limiting of the scope of the present invention.

[0146] EXPERIMENTAL PART

[0147] EXAMPLE 1

[0148] Composition for the oral administration having the following formulation:

[0149] EXAMPLE 2

[0150] Composition for the oral administration having the following formulation: EXAMPLE 3

[0151] Composition for the oral administration having the following formulation:

[0152] EXAMPLE 4

[0153] Composition for the oral administration having the following formulation: EXAMPLE 5

[0154] Composition for the topical administration having the following formulation:

[0155]

[0156] EXAMPLE 6

[0157] Composition for the topical administration having the following formulation: EXAMPLE 7

[0158] Composition for the topical administration having the following formulation:

[0159] EXEMPLE 8 Composition for the topical administration having the following formulation Cremophor RH40 was PEG-40 HYDROGENATED CASTOR OIL.

[0160] Prunizen was a mixture of GLYCERIN, AQUA and PRUNELLA VULGARIS EXTRACT.

[0161] Aristoflex TAC was AMMONIUM ACRYLOYLDIMETHYL- TAURATE / CARBOXYETHYL ACRYLATE CROSSPOLYMER.

[0162] EXPERIMENTAL PART

[0163] PromoCell® Human Follicle Dermal Papilla Cells (HFDPC) were used in order to test the hair growth promoting activity of Ocimum sanctum extract (2% of ursolic acid) and lllicium verum Hook. f. (20% shikimic acid). Shikimic acid (Sigma Aldrich Sri) and ursolic acid (Sigma Aldrich Sri) were also tested.

[0164] Human follicle dermal papilla cells are found in the dermal papillae at the base of hair follicles. These cells are essential for induction and maintenance of hair growth. Since these cells express androgen receptors, they represent a well-suited model system for androgen-related research.

[0165] PromoCell® Human Follicle Dermal Papilla Cells (HFDPC) were purchased by PromoCell. The cell line was cultured in a specific medium (follicle dermal papilla cell growth medium-ready to use with its supplement mix, PromoCell) in conditions of complete sterility and maintained at 37 °C with 5% carbon dioxide (CO2) atmosphere. HFDPC are isolated principally from normal human dermis of the lateral scalp. Shortly after isolation, all Human Follicle Dermal Papilla Cells are cryopreserved at passage 2 (P2) using proprietary serum-free freezing medium, Cryo-SFM.

[0166] Every two days confluent cultures were splitted 1 :3-1 :6, after washing with phosphate buffered saline (PBS) (without Ca2+ and Mg2+), using trypsin / EDTA, and seeded at a density of 1 *106 cells per well into 12-well plates for qRT-PCR.

[0167] All the compounds were tested in single and in combination.

[0168] Following concentrations were used:

[0169] 1.*Negative control: HFDPC in complete culture medium

[0170] 2. Shikimic acid 3.2 pg / mL in complete culture medium

[0171] 3. Ursolic acid 0.32 pg / mL in complete culture medium

[0172] 4. Shikimic acid 3.2 pg / mL + Ursolic acid 0.32 pg / mL in complete culture medium 5.lllicium verum 16 pg / mL in complete culture medium

[0173] Q.Ocimum sanctum 16 pg / mL in complete culture medium

[0174] 7.lllicium verum 16 pg / mL (containing 20% shikimic acid)+ Ocimum sanctum 16 pg / mL (containing 2% ursolic acid) in complete culture medium

[0175] Twenty-four hours after seeding on 12-well plates, HFDPC 80% confluent cells were exposed to compounds tacking into account the ratio at which the single compounds in the botanical extracts, respectively.

[0176] RNA for qRT-PCR analysis was extracted 24h after the treatment. Tri Reagent (Sigma Aldrich) methods as described by Chomczynski and Mackey (Chomczynski P, Mackey K. Modification of the TRI reagent procedure for isolation of RNA from polysaccharide- and proteoglycan-rich sources. Biotechniques 1995;19:942-5 )was used and the cDNA was then synthesized from 2pg RNA template in a 20pl reaction volume, using the PrimeScript RT-PCR Kit (Takara, Japan). The cDNA was amplified and detected by the Stratagene Mx3000P Real-Time PCR System (Agilent Technologies Italia S.p.A., Milan, Italy). The amplification of cDNA from HFDPC cells was conducted using the following Taqman gene expression assays: Hs01547656_m1 (IGF-1), Hs00155410_m1 (TGF- p1), and Hs999999 ml (human glyceraldehyde-3-phosphate dehydrogenase, GAPDH). GAPDH was used as housekeeping genes, respectively. PCR amplifications were carried out in a 20pl of total volume. The mixture of reaction contained 10pl of 2X Premix Ex Taq (Takara, Japan), 1 l of 20 TaqMan gene expression assay, 0.4 pl of RoX Reference Dye II (Takara, Japan), 4.6 pl of water and 4pl of DNA. PCR conditions were the following: 95°C for 30 sec followed by 40 cycles of 95°C for 5 sec, 60°C for 20 sec. PCR reactions were performed in duplicate using an MX3000p PCR machine (Stratagene, La Jolla, CA). A cycle threshold (Vigetti D, Viola M, Karousou E, Rizzi M, Moretto P, Genasetti A, et al. Hyaluronan-CD44-ERK1 / 2 regulate human aortic smooth muscle cell motility during aging. J Biol Chem 2008;283:4448-58.) was used for the calculation of the relative abundance in the expression of each gene.).

[0177] RESULTS

[0178] In order to assess the synergic activity of the two active principles components (ursolic acid and shikimic acid) and of the two botanical extracts were also assayed as single compound or in different combination.

[0179] Surprisingly, the combination of the two active principles showed a synergistic effect as it is evident from Figure 1 .

[0180] The same occurs with the combination of lllicium verum 16 pg / mL (containing 20% shikimic acid)+ Ocimum sanctum 16 pg / mL (containing 2% ursolic acid), that showed an higher significant stimulatory effect on IGF-1 gene than the botanical extracts tested alone (same concentration) and their main component (Figure 1 ).

[0181] Contextually, the same results were achieved evaluating the inhibitory effects on TGF-p1 gene of the combination of lllicium verum 16 pg / mL (containing 20% shikimic acid)+ Ocimum sanctum 16 pg / mL (containing 2% ursolic acid). The combination of lllicium verum 16 pg / mL (containing 20% shikimic acid)+ Ocimum sanctum 16 pg / mL (containing 2% ursolic acid), showed an higher inhibitory effect on TGF-p1 gene than the botanical extracts tested alone (same concentration) and their main component (Figure 2).

[0182] CONCLUSION

[0183] Taking together, the data on the expression of IGF-1 and TGF-p1 genes suggested a strong hair growth promoting effect of the combination of lllicium verum 16 pg / mL (containing 20% shikimic acid)+ Ocimum sanctum 16 pg / mL (containing 2% ursolic acid) and these effects are synergic.

[0184] Surprisingly and preferably the composition comprising the two extracts was the most effective among all the tested compositions.

Claims

CLAIMS1. A composition comprising shikimic acid and ursolic acid.

2. The composition according to claim 1 wherein shikimic acid is in the form of a lllicium verum extract comprising from 5 to 40% by weight, preferably from 9.8 to 30% by weight, more preferably 9.8-20% by weight of shikimic acid.

3. The composition according to claim 2 wherein the lllicium verum extract is in amount from 0.005% w / w to 50% w / w with respect to the total amount of the composition.

4. The composition according to anyone of claims 1 -3 wherein ursolic acid is in the form of Ocimum sactum extract comprising from 0.1 to 5% by weight, preferably from 1 to 3% by weight, more preferably 1-2% by weight of ursolic acid.

5. The composition according to claim 4 wherein the Ocimum sactum extract is in amount from 0.005% w / w to 50% w / w with respect to the total amount of the composition.

6. The composition according to anyone of claims 1 to 5, wherein the composition comprises rutin.

7. A composition comprising lllicium verum extract comprising from 5 to 40% by weight, preferably from 9.8 to 30% by weight, more preferably 9.8-20% by weight of shikimic acid and Ocimum sactum extract comprising from 0.1 to 5% by weight, preferably from 1 to 3% by weight, more preferably 1 -2% by weight of ursolic acid.

8. A cosmetic use of the composition according to anyone of claims 1 to 6 or to claim 7 for stimulating the physiological growth of hair.

9. A composition according to anyone of claims 1 to 6 or to claim 7 for use in the prevention and treatment of hair loss.

10. The cosmetic use according to claim 8 or the composition for use according to claim 9, wherein the composition is orally administered.

11. The cosmetic use or the composition for use according to claim 10 wherein the composition comprises lllicium verum extract is in amount from 5% w / w to 50% w / w with respect to the total amount of the composition and the Ocimum sactum extract is in amount from 5% w / w to 50% w / w with respect to the total amount of thecomposition.

12. The cosmetic use according to claim 8 or the composition for use according to claim 9, wherein the composition is topically administered.

13. The cosmetic use or the composition for use according to claim 12 wherein the composition comprises lllicium verum extract is in amount from 0.005% w / w to 5% w / w with respect to the total amount of the composition and the Ocimum sactum extract is in amount from 0.005% w / w to 5% w / w with respect to the total amount of the composition.

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