EXTRACTS OF COPRINUS COMATUS AND THEIR USE FOR REGULATING THE PILOSEBACEOUS UNIT IN A HUMAN

Abstract

Suggested is an extract of Coprinus comatus obtained or obtainable according to the following protocol: (a) providing a source of dry biomass of Coprinus comatus; (b) extracting said source of biomass with water, C.sub.1-C.sub.4 aliphatic alcohols or mixtures thereof; (c) separating the extract thus obtained from the remaining cell material; and optionally (d) repeating steps (b) and (c) once or twice, which is particularly useful for regulating the pilosebaceous unit of a human.

Claims

1-18. (canceled)

19. A method for obtaining an extract of Coprinus comatus, comprising the steps of: (a) providing a source of dry biomass of Coprinus comatus; (b) extracting said source of biomass with water, C.sub.1-C.sub.4 aliphatic alcohols or mixtures thereof; (c) separating the extract thus obtained from the remaining cell material; and optionally (d) repeating steps (b) and (c) once or twice.

20. The method according to claim 19, comprising the additional step of using the extract of Coprinus comatus for regulating the pilosebaceous unit of a human in need thereof.

21. The method according to claim 19, comprising the additional step of using the extract of Coprinus comatus for (a) stimulating hair growth, (b) modulating hair cycle, (c) preventing hair loss, and/or (d) down-regulating sebogenesis by sebaceous glands, in a human in need thereof.

22. The method according to claim 19, comprising the additional step of using the extract of Coprinus comatus for treating or preventing greasy hair or oily skin in a human in need thereof.

23. A method for regulating the pilosebaceous unit in a human in need thereof, comprising the steps of: (i) providing the extract of Coprinus comatus obtained according to claim 19, and (ii) applying said extract either topically to human skin or scalp, or (iii) applying said extract by oral administration.

24. A method for (a) stimulating hair growth, (b) modulating hair cycle, (c) preventing hair loss, and/or (d) down-regulating sebogenesis by sebaceous glands, in a human in need thereof, comprising the steps of: (i) providing the extract of Coprinus comatus obtained according to claim 19, and (ii) applying said extract either topically to human skin or scalp, or (iii) applying said extract by oral administration.

25. A method for treating or preventing greasy hair or oily skin in a human in need thereof, comprising the steps of: (i) providing the extract of Coprinus comatus obtained according to claim 19, and (ii) applying said extract either topically to human skin or scalp, or (iii) applying said extract by oral administration.

Description

EXAMPLES

Preparation of the Extract

[0254] The extract of Coprinus comatus has been obtained by the following protocol: [0255] Each gram of dry biomass of Coprinus comatus was grinded in a mortar and extracted by treatment with 25 ml of solvent, stirring the suspension at room temperature for 16 hours in the dark; [0256] the residual cell material was separated from the extract by centrifugation at 2000G for 15 minutes; [0257] the residual biomass was washed by suspending it in 12.5 ml of solvent; [0258] the cell material was separated from the washing solvent by centrifugation at 2000G for 15 minutes; [0259] the residual biomass was washed again by suspending it in 12.5 ml of solvent; [0260] the cell material was separated from the washing solvent by centrifugation at 2000G for 15 minutes; [0261] the firstly collected extract and the washing solvent volumes were mixed.

[0262] According to the present invention, cell material of the Coprinus comatus biomass was extracted with a liquid extractant selected from the group consisting of ethanol and water. The extractant can also comprise a mixture of the two aforementioned solvents. Quantity and quality of compounds which are present in the extracts may vary with respect to both solvent properties and preparation protocol.

[0263] Coprinus comatus (Class: Agaricomycetes; Order: Agaricales) is a common mushroom which occurs widely in Europe, UK and North America. It has been introduced to Australia, New Zealand and Iceland.

[0264] The Coprinus comatus biomass used in these experiments has been produced by a German company and bought from an Italian company.

Activity of the Extracts on Hair Follicle Growth

Examples 1 to 4

Activity on the Growth of Hair Follicles of Ethanolic (EtOH) and Aqueous (Water) Extracts Obtained from Coprinus comatus

[0265] The following experiment was conducted to demonstrate the activity on hair follicle growth of the ethanolic extract (EtOH) and aqueous extract (water) obtained from Coprinus comatus. Hair follicles were micro-dissected from a single donor's scalp sample and plated into sterile 48-well plates at a density of 1 hair follicle/well, with 200 μl/well of a modified Williams' Medium E.

[0266] After 18 h of cultivation, the selection of the hair follicles suitable to be maintained in culture occurred. Only those follicles showing a good vital stage and a growth of not less than 0.2 mm were selected for the experimental plan. The experimental treatment of the follicles started after the follicle selection and lasted for eight days. All experimental groups and the control were prepared comprising 12-18 follicles. The hair follicles showing evident signs of suffering during the culture for reasons not depending on the experimental treatment were excluded from the final analysis.

[0267] The growth performances observed in the treated hair follicles were compared to a control group cultured in the same culture medium without extract supplement. At day 9 of culture (day 8 of treatment) the growth of the hair follicles was studied by image analysis.

[0268] The activity of the treatment is demonstrated by the increase of hair follicles growth expressed as a variation of the average elongation of the experimental groups in comparison to the control group (Table 1) and is expressed as % ratio of the control group performance.

TABLE-US-00001 TABLE 1 Growth of hair follicles at day 9 of culture (8 days of treatment) Total no. Example Sample Amount Average Std. error of HFs 0 Control 0 100.0 3.1 15 1 EtOH 0.1 μg/ml 107.2 4.8 12 2 EtOH 1 μg/ml 123.7 5.3 11 3 Water 0.2 μg/ml 109.1 4.0 12 4 Water 2 μg/ml 104.4 6.2 12

[0269] The treatment performed with 1 μg/ml of EtOH extract increased the follicle elongation by 23.7%, while 0.2 μg/ml of water extract stimulated the hair growth by 9.1%, in comparison to the control group. These results show that the addition of these extracts leads to an increase in growth of the hair follicles.

[0270] The increase of hair growth, in culture conditions, can be achieved by improving the general health of the organ and/or by delaying the catagen, which physiologically occurs when the follicle is explanted from the scalp. Both these effects are strongly desirable and make the extracts very interesting for cosmetic and therapeutic applications, in particular as an ingredient for preparations aimed at combatting hair loss.

Examples 5 to 12

Activity on the Growth of Hair Follicles of Ethanolic (EtOH) and Aqueous (Water) Extracts Obtained from Coprinus comatus

[0271] The experimental procedure reported above was also adopted for testing the same extracts of Coprinus comatus on hair follicles taken from another donor. The average elongation of the experimental groups in comparison to the control group is reported in Table 2 and is expressed as % ratio of the control group performance.

TABLE-US-00002 TABLE 2 Growth of hair follicles at day 9 of culture (8 days of treatment) Total no. Example Sample Amount Average Std. error of HFs 0 Control 0 100.0 4.0 16 5 EtOH 0.01 μg/ml 111.6 5.3 12 6 EtOH 0.1 μg/ml 122.7 6.9 9 7 EtOH 1 μg/ml 115.6 5.7 11 8 EtOH 10 μg/ml 104.0 6.1 9 9 Water 0.01 μg/ml 110.2 4.4 12 10 Water 0.1 μg/ml 119.7 5.5 12 11 Water 1 μg/ml 113.2 5.9 12 12 Water 10 μg/ml 98.8 4.6 11

[0272] The results confirmed the stimulant activity exerted by the extracts. The EtOH extract increased the follicle elongation up to 22.7%, while the water extract stimulated the hair growth up to 19.7%, in comparison to the control group. These results show that the addition of these extracts leads to an increase in growth of the hair follicles and can promote their metabolic activities typically expressed in anagen phase.

Examples 13 to 15

Activity on the Growth of Hair Follicles of Ethanolic (EtOH) and Aqueous (Water) Extracts Obtained from Coprinus comatus

[0273] The experimental procedure reported above was also adopted for testing the same extracts of Coprinus comatus on hair follicles taken from a third donor. The average elongation of the experimental groups in comparison to the control group is reported in Table 3 and is expressed as % ratio of the control group performance.

TABLE-US-00003 TABLE 3 Growth of hair follicles at day 9 of culture (8 days of treatment) Total no. Example Sample Amount Average Std. error of HFs 0 Control 0 100.0 6.3 14 13 EtOH 0.01 μg/ml 114.8 5.6 10 14 EtOH 0.1 μg/ml 102.1 4.6 11 15 Water 0.1 μg/ml 114.1 4.4 10

[0274] The results confirmed the stimulant activity exerted by the extracts in comparison to the control group. Both the ethanolic and aqueous extracts increased the hair growth performance of about 14-15%, at the concentrations of 0.01 and 0.1 μg/ml, respectively.

Examples 16 to 17

Activity on the Growth of Hair Follicles of Aqueous (Water) Extract Obtained from Coprinus comatus

[0275] The experimental procedure reported above was adopted for testing the water extract of Coprinus comatus on hair follicles taken from another donor. The average elongation of the experimental groups in comparison to the control group is reported in Table 4 and expressed as % ratio of the control group performance.

TABLE-US-00004 TABLE 4 Growth of hair follicles at day 9 of culture (8 days of treatment) Total no. Example Sample Amount Average Std. error of HFs 0 Control 0 100.0 3.7 17 16 Water 0.1 μg/ml 118.2 5.3 12 17 Water 1 μg/ml 120.1 5.8 11

[0276] The water extract of Coprinus produced a relevant stimulation of the hair growth at both the concentrations of treatment tested.

Examples 18 to 21

Activity of Ethanolic (EtOH) and Aqueous (Water) Extracts Obtained from Coprinus comatus on the Hair Cycle Staging of Cultivated Human Follicles

[0277] The examples previously reported show that the C. comatus extracts allow to increase the hair follicle growth in culture. These results are due to a prolonged persistence of the growing phase, i.e. anagen, throughout the culture time. Indeed, as each skilled technician knows, in culture conditions the hair follicles switch from anagen to catagen in a few days, whereas their growth accordingly slows and finally stops. Therefore, it can be assumed that an increase of growth of the treated follicles, in culture condition, is due to a prolonged stay in anagen phase.

[0278] In order further to prove this conclusion, the cycle stage of cultured hair follicles treated with C. comatus extracts was evaluated at the day 5 of culture (day 4 of treatment). Day 5 is the pivotal time for the catagen onset among the cultured follicles. The culture method was the same adopted for the previous examples, but at day 5 the hair follicles were subjected to histological analysis in order to verify the morphological state of the dermal papilla. The cycle stage of each hair follicle was classified on the basis of the dermal papilla morphology. The frequency of each cycle stage was evaluated group by group and expressed as intra-group percentage. All the experimental groups comprised 12 follicles at the beginning of the culture, but some follicles were not analyzed as they appeared dystrophic at day 5 of culture. The results are shown in Table 5. Hair Cycle Staging expressed as % frequency of each stage within the group.

TABLE-US-00005 TABLE 5 Hair follicle staging at day 5 of culture (4 days of treatment) obtained by histological analysis of the dermal papilla morphology Early Late Total no. Example Sample Amount Anagen catagen catagen of HFs 0 Control 0 60% 30% 10% 10 18 EtOH 0.1 μg/ml 70% 30% 10 19 EtOH   1 μg/ml 81.8%   18.2%   11 20 Water 0.1 μg/ml 66.7%   33.3%   9 21 Water   1 μg/ml 75% 25% 12

[0279] The results indicate that the experimental extracts have significantly delayed the onset of the catagen phase among the hair follicles. Indeed, the treated groups had a higher frequency of hair follicles in anagen than the control group.

Activity of the Extracts on Sebaceous Glands

[0280] Description of the Experimental Model Based on Ex-Vivo Culture of Human Sebaceous Glands (hSGs) and Subsequent Quantification of their Sebum Content

[0281] All the reported examples are intended to show the modulation of sebum production exerted by the experimental preparations, evaluated on human hSGs microdissected and cultivated up to day 6. At the end of the culture time, the sebum was extracted and quantified from each experimental group of hSGs and then normalized by the proteins extracted from the residual hSG material (mg lipids/mg proteins). As a result, the biological activity of the tested compounds is inferred by comparing the lipids/proteins ratio of the treated glands with that of the control group.

Organ Culture Technique

[0282] Using micro-scissors and tweezers, hSGs were isolated from the pilosebaceous units of a scalp skin sample. They were seeded in 24-well plates at the density of 8 hSGs/well and then cultivated in 500 μl/well of Williams' medium E, appropriately modified, hereinafter referred to as standard medium. After 24 hours of culture the viability of the glands was assessed via resazurin assay and then, having attested their good viability, the culture medium was substituted with the same medium supplemented with experimental extracts, whereas the control group received again the standard medium. The culture medium was renewed every other day. At day six of the organ culture, after having preventively verified the good viability of the hSGs via resazurine assay, each group of hGSs was collected and analyzed for quantifying the sebum content.

Analysis of the Sebum Content

[0283] In order to make comparable the estimated productivity of the glands, which are variable in biomass, their total sebum content was estimated and divided by the proteins extracted from the gland tissue, obtaining the ratio between the produced sebum and the tissue proteins (i.e. mg of lipids/mg of proteins). Methods are described in detail in WO2016020339 A8.

[0284] The amounts of normalized lipids obtained from the treated groups, i.e. the sebum produced by each group of hSGs, were expressed in percentages with respect to the value obtained from the control group, in order to point out the regulatory effect performed by the experimental treatment.

Examples 22 to 23

Activity of Aqueous (Water) Extract of Coprinus comatus on Human Sebaceous Glands (hSGs)

[0285] hSGs were dissected and cultivated as previously described. Two experimental groups of hSGs were cultivated in culture media supplemented with 1 and 10 μg/ml of water extract obtained from Coprinus comatus, respectively. The control group was cultured in standard medium. As positive control, a 5 μM Capsaicin treatment was included in the experimental design. Capsaicin is an active component of chili peppers suitable to inhibit sebogenesis [T6th et al., J. Invest. Derm. (2009), 129: 329-339]. The results obtained from the experiment are reported in Table 6. Responses are expressed as % ratio of the control group performance. The capsaicin treatment was included as positive control.

TABLE-US-00006 TABLE 6 Sebum content in hSGs following treatment with water extract Coprinus comatus Example Sample Amount Average Std. error 0 Control 0 100.0 2.8 0 capsaicin 5 μM 73.5 1.8 22 Water 1.0 μg/ml 92.1 1.5 23 Water 10.0 μg/ml 70.4 2.4

[0286] The positive control treatment reduced the sebum content of the hSGs by 26.5% in comparison with the control group. However, surprisingly, also the Coprinus extract produced an intense inhibition of the sebogenesis, reaching −29.6% in comparison with the control group. These data show that the experimental extract has a biological activity comparable or higher than capsaicin.

Examples 24 to 26

Activity of Ethanolic (EtOH) Extract of Coprinus comatus on Human Sebaceous Glands (hSGs)

[0287] hSGs were dissected and cultivated as previously described and treated with EtOH extract of C. comatus. The control group was cultured in standard medium and the positive control was treated with 5 μM Capsaicin. The results obtained from the experiment are reported in Table 7. Responses are expressed as % ratio of the control group performance. The capsaicin treatment was included as positive control.

TABLE-US-00007 TABLE 7 Sebum content in hSGs following treatment with ethanol extract of Coprinus comatus Example Sample Amount Average Std. error 0 Control 0 100.0 1.2 0 capsaicin 5 μM 88.3 2.2 24 EtOH 0.1 μg/ml 72.7 2.2 25 EtOH 1.0 μg/ml 61.3 1.0 26 EtOH 10.0 μg/ml 46.2 0.8

[0288] The experimental results show that the EtOH extract produced a down-regulation of sebogenesis much more intense than capsaicin, increasing with the dosage of treatment.

CONCLUSIVE REMARKS

[0289] The reported examples attest that the fungus Coprinus comatus is a suitable source of natural extracts for modulating the hair growth and hair cycling and/or regulating the sebum production. The activity on the hair follicle indicates that these extracts can be exploited for improving the hair wellness, prolonging the anagen phase and delaying the hair loss.

[0290] Besides, the biological activity as sebum regulators was shown to be comparable to or higher than that of capsaicin, a well-known sebum-inhibitor. The described results support the proposed uses of the extracts to treat skin, hair and genitals, in order to prevent and/or treat the excessive secretion of sebum and the related aesthetic problems or skin disorders (greasy hair and skin, dandruff, acne, itch, discomfort of the vulvar region etc.).

[0291] A special comment is required in order to interpret correctly the effective concentrations exemplified for the C. comatus extracts. The optimal concentration of treatment can vary a little with the sensitivity of each particular donor from which the organs have been taken. This is perfectly normal and consistent with the cases observed in vivo. More critical is to estimate the order of magnitude of the treatment concentrations required for obtaining in vivo the same responses observed in the reported examples. These latter are based on experimental models consisting of ex-vivo cultivated organs, maintained at constant concentrations of treatment throughout the culture time. This experimental condition is not obtainable in vivo, since both topical and oral administration produce fluctuating concentrations, depending on the frequency of administration and product formulation. It can therefore be assumed that any effective concentration obtained from ex-vivo experiments needs to be opportunely increased in the final product formulations. This is especially true for topical preparations, in which only a limited part of the active ingredients reaches the target organ. It is assumed, as a very general indication, that a topical preparation should be formulated with 10 to 2000 folds the effective concentration detected by means of the experimental model adopted here. The magnitude of the multiplication factor required to obtain an effective formulation mainly depends on the effectiveness of the cosmetic vehicle and the frequency of application suggested. On the basis of these comments and taking into account the tested concentrations, it is assumable that the minimal effective topical dosage of fungal extract can vary between 0.0001 wt.-percent and 2 wt.-percent, preferably between 0.001 wt.-percent and 1 wt.-percent, more preferably between 0.01 wt.-percent and 0.1 wt.-percent and most preferably between 0.02 wt.-percent and 0.5 wt.-percent.