Composition for preventing hair loss and stimulating hair-growth

Abstract

The present disclosure relates to a composition including a Centipeda minima extract and the fraction thereof as an active ingredient. Since the composition of the present disclosure promotes the production and growth of hair, the composition not only presents excellent effects in the prevention, amelioration, and treatment of hair-loss, but may also be used for promoting hair-growth.

Claims

1. A method of treating hair-loss comprising administering a therapeutically effective amount of a Centipeda minima extract or a fraction of the Centipeda minima extract to a subject in need thereof, wherein the Centipeda minima is a Centipeda minima 30 to 100% by weight methanol aqueous solution extract or a Centipeda minima 30 to 100% by weight ethanol aqueous solution extract, or wherein the fraction is a fraction of a Centipeda minima 30 to 100% methanol or ethanol extract obtained using dichloromethane as the fractioning solvent.

2. The method of claim 1, wherein the hair-loss includes at least one selected from a group consisting of denutrition-based alopecia, endocrine disorder-based alopecia, vascular disorder-based alopecia, alopecia premature, traction alopecia, alopecia areata, alopecia neurotica, pityriasis alopecia, Trichotillomania, alopecia maligna, female pattern alopecia, male pattern alopecia, androgenetic alopecia, telogen effluvium, tinea capitis, alopecia totalis, hypotrichosis, genetic hypotrichosis simplex, systemic drug for alopecia-based hair-loss, mechanical hair-loss, traumatic alopecia, pressure alopecia, anagen effluvium, alopecia syphilltica, alopecia seborrheica, symptomatic alopecia, alopecia cicatrisata, and alopecia congenita.

3. The method of claim 1, wherein the Centipeda minima extract or the fraction of the Centipeda minima extract is applied to the scalp or hair.

4. The method of claim 1, wherein the administering of Centipeda minima extract or the fraction of the Centipeda minima extract is transdermal administration.

5. The method of claim 1, wherein the Centipeda minima extract or the fraction of the Centipeda minima extract is formulated as an aerosol, ointment, liquid, lotion, patch, powder for application, oil, cream or gel.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows a photograph of daily observation of a skin of each of a test substance-administered group, a positive control group 1 and a positive control group 2 in Example 1-1 until 7 days.

(2) FIG. 2 is a photograph of the normal group, negative control group, test substance-administered group, and positive control groups 1 (1, 2) to 2 (3) after 17 days in Example 1-1.

(3) FIG. 3 is a graph quantifying the results of the hair-growth efficacy evaluation score through visual inspection in Example 1-1.

(4) FIG. 4 is a graph quantifying the results of the hair-growth efficacy evaluation score through visual inspection after 7 days in Example 1-1.

(5) FIG. 5 shows the observation of the tissues of the normal group, negative control group, test substance-administered group, positive control groups 1 and 2 in Example 1-2 as stained and observed with an optical microscope.

(6) FIG. 6 shows a photograph of daily observation of a skin of each of the normal group, negative control group, test substance-administered group (Centipeda minima extract), positive control group 1 (Tofacitinib) and positive control group 2 (Minoxidil) in Example 2-1 until 12 days, and shows the result of the change in growth.

(7) FIG. 7 shows the results of the change in the weight of the mouse during the test period of Example 2-1.

(8) FIG. 8 shows the results obtained by quantifying the hair-growth efficacy evaluation score during the test period of Example 2-1. (A in FIG. 8: Daily hair-growth efficacy evaluation score, B in FIG. 8: Hair-growth efficacy evaluation score on Day 12.

(9) FIG. 9 shows the observation of the tissues of the normal group, negative control group, test substance-administered group (Centipeda minima extract), positive control group 1 and positive control group 2 in Example 2-2 as stained and observed with an optical microscope: Anagen_Early (A/e), Anagen_late (A/l), Catagen_early (C/e), telogen (t).

(10) FIG. 10 is the result of the change in the weight of the mouse during the test period of Example 3-1.

(11) FIG. 11 shows the results obtained by quantifying the hair-growth efficacy evaluation score during the test period of Example 3-1 (A in FIG. 11: Daily hair-growth efficacy evaluation score, B in FIG. 11: Hair-growth efficacy evaluation score on Day 12.

(12) FIG. 12 shows that the tissues of the normal group, negative control group, and test substance-administered group (Centipeda minima fraction (MC: dichloromethane)) in Example 3-2 as stained and observed under an optical microscope: Anagen_Early (A/e), Anagen_late (A/l), Catagen_early (C/e), telogen (t).

MODES OF THE INVENTION

(13) Hereinafter, the present disclosure will be described in more detail based on specific Preparation Examples and Examples. However, the following Examples are provided only to aid understanding according to the present disclosure, and the scope according to the present disclosure is not limited to Examples.

PREPARATION EXAMPLES

Preparation Example 1: Preparation of Centipeda minima Extract

(14) Centipeda minima (a dried aerial portion of Centipeda minima harvested in Goesan-gun, Chungcheongbuk-do in August 2017) was purchased in September 2017 and used as a sample (Purchased from: Goesan Herbal Organic Agricultural Products Cooperative).

(15) 1.0 kg of the purchased Centipeda minima was extracted twice (each: 15.0 L×3 days) with 80% methanol (MeOH) at room temperature and then filtered to obtain a Centipeda minima extract.

Preparation Examples 2 to 5: Preparation of Fraction of Centipeda minima Extract

(16) The Centipeda minima extract obtained in Preparation Example 1 was evaporated under reduced pressure to obtain 193.2 g of crude extract and then it was suspended in 800 mL distilled water.

(17) The suspension was subjected to systematic solvent fractionation in a sequential direction with increasing polarity using HX (800 mL×3), CH.sub.2C2 (800 mL×4), EtOAc (800 mL×3), and n-BuOH (800 mL×3). Each fraction was subjected to concentration under reduced pressure to prepare Preparation Example 2 (HX (23.0 g)) fraction, Preparation Example 3 (CH.sub.2C12 (9.0 g)) fraction, Preparation Example 4 (EtOAc (9.0 g)) fraction, and Preparation Example 5 (n-BuOH (32.7 g)) fraction.

Preparation Examples 6 to 11: Preparation of Centipeda minima Extract

(18) Centipeda minima (a dried aerial portion of Centipeda minima harvested in Goesan-gun, Chungcheongbuk-do in August 2018) was purchased in September 2018 and used as a sample (Purchased from: Goesan Herbal Organic Agricultural Products Cooperative).

(19) 1.0 kg of the purchased Centipeda minima was extracted twice (each: 15.0 L 3 days) using each of ethanol (EtOH) 30%, ethanol (EtOH) 70%, ethanol (EtOH) 100%, 30% methanol (MeOH), 70% methanol (MeOH) or 100% methanol (MeOH) at room temperature and then filtered to obtain a Centipeda minima extract.

Preparation Examples 12 to 17: Preparation of Fraction of Centipeda minima Extract

(20) Each extract of Preparation Examples 6 to 11 was subjected to systematic solvent fractionation in a sequential direction in the same manner as the fraction preparation in Preparation Examples 2 to 5. Each fraction was subjected to concentration under reduced pressure to preparea dichloromethane fraction.

PREPARATION EXAMPLE

Preparation Example 1: Preparation of Formulation Containing Centipeda minima Extract

(21) Tonic formulation-based test substance was prepared using a mixture solvent (65%:5%) of ethanol and glycerin so that the content of Centipeda minima extract of Preparation Example 3 was 5% in total.

Preparation Examples 2 to 13: Preparation of Test Substances Containing Centipeda minima Extract or Fraction

(22) A test substances were prepared using a mixture solvent of 5% Tween 80 and 5% glycerin in distilled water such that a content of each of Centipeda minima extracts or Dichloromethane fractions of Preparation Examples 6 to 17 were 20% in total.

<Example 1> Identification of Prevention, Treatment, Amelioration Effect of Hair-Loss, and Hair-Growth Stimulating Effect of Centipeda minima Extract Via Catagen Inhibition Animal Model Efficacy Test

(23) To proceed with the hair-growth efficacy test in the catagen inhibition animal model of Centipeda minima extract, male C57BL/6 mice (5 weeks of age) were purchased and subjected to acclimation for 1 week and selected animals weighing 16 to 21 g. In the case of an animal in which skin damage occurred during hair removal, or if the skin color was black, the growth period was already in progress, so it was inappropriate for this experiment and was excluded from the experiment.

(24) The test group was divided into a normal group, a negative control group, a test substance-administered group, and a positive control groups 1 and 2. Each of the normal group and the negative control group had 1 mouse, the test substance-administered group had 3 mice, and the positive control group 1 had two mice, and the positive control group 2 had one mouse. Specifically, the normal group is a group not administered with a solvent or a test substance, the negative control group is a group receiving only a solvent, and the test substance group is a group administered with Centipeda minima extract of Preparation Example 3, the positive control group 1 is a Minoxidil-administrated group, the positive control group 2 is a group administered with Litsea glutinosa extract.

(25) Tonic formulation (Preparation Example 1) of Preparation Example 3 was applied to the back skin for 7 days twice a day at 6 hour intervals at a dose of 100 μL. 0.1% 1 ml of dexamethasone (ferridex, green cross) as catagen-inducing substance was applied to the epilation site for 5 days. 5% 100 μl of dexamethasone was applied to the positive control groups 1 and 2.

(26) During the experiment period, after administration of the test substance, hair-growth level was observed via photography and visual inspection. After the end of the experiment, animals were sacrificed and a pathology test for skin tissue was conducted.

(27) Further, in order to quantify the results of hair-growth efficacy visual inspection, image analysis was performed on the entire epilation site (solvent or test substance, positive control substance application site) that is black (anagen) or a site where hair-growth begins. Image analysis was carried out with the image analysis program (ImagePro Plus 5.0, Cybernetics, USA). The result was quantified in %. The numerical data was converted to a numerical graph as produced using a graph production program (Prism 5.0, Graphpad, USA).

Example 1-1: Visual Inspection

(28) Based on a result of visual inspection of the mouse skin, as shown in FIG. 1, FIG. 3 and FIG. 4, the test substance-administered group showed a significantly higher tendency (18.5%, 20.4%, 32.8%) to migrate to anagen on 3 days of administration, compared to other groups and could not be observed hair growth in positive control group 1 (0.1%, 0.3%) and positive control group 2 (0.4%). On 7 days of administration, the best hair-growth efficacy could be identified in the test substance-administered group (84.9%, 88.2%, 97.0%).

(29) Further, as shown in FIG. 2, as a result of administering 0.1% of dexamethasone to maintain catagen, it may be visually identified that the hair-growth efficacy was excellent in the test substance-administered group (93.5%, 96.7%, 98.2%) finally on the Day 17 after administration. However, based on a result of more clearly identifying the hair follicle generation and hair-growth efficacy via the skin tissue pathology test, the positive control group 1 (6.1%, 21.2%) and positive control group 2 (3.3%) were unable to exhibit the excellent hair-growth efficacy like the test substance-administered group.

(30) Therefore, it may be seen that the Centipeda minima extract according to the present disclosure may effectively act on the prevention, treatment and amelioration of hair-loss, and the promotion of hair-growth.

Example 1-2: Skin Tissue Pathology Test

(31) The skin tissue pathology test was conducted by H&E staining using Hematoxylin and Eosin, which are generally used widely. A sample was collected from the skin tissue of the sacrificed animals and the generation of hair follicles and phenomena of hair-growth were identified through fixation, washing, clearing, paraffin infiltration, embedding, microtome cutting, staining and reading processes.

(32) In this study, Anagen_Early (A/e) indicates the state in which hair has just begun to develop and Anagen_late (A/l) and Catagen_early (C/e) indicate that hair-growth is in full swing. Anagen_late and Catagen_early have histologically similar positions of the hair bulb. When considering morphological changes according to tissue sections, it is necessary to consider that it is often unclear to distinguish there between. Nevertheless, the hair follicles in the Anagen_late and Catagen_early stages are generally considered as similar stages of development, and the results are interpreted.

(33) FIG. 5 shows the results of the analysis based on the above descriptions. As shown in FIG. 5, in the normal group and the negative control group, specific hair follicle generation and hair-growth efficacy could not be observed. Further, in the positive control group 1 and the positive control group 2, only minimal hair follicle generation and hair-growth efficacy degree could be observed. On the other hand, only the test substance-administered group to which Centipeda minima extract was administered was able to confirm the specifically excellent hair follicle generation and hair growth efficacy.

(34) Therefore, it may be seen that the Centipeda minima extract according to the present disclosure may effectively act on the prevention, treatment and amelioration of hair-loss, and the promotion of hair-growth.

<Example 2> Identification of Prevention, Treatment and Amelioration Effects of Hair-Loss, and Hair-Growth Stimulating Effect of Centipeda minima Extract

(35) To proceed with the hair-growth efficacy test in the catagen inhibition animal model of Centipeda minima extract, male C57BL/6 mice (5 weeks of age) were purchased and subjected to acclimation for 1 week and selected animals weighing 20 to 24 g. In the case of an animal in which skin damage occurred during hair removal, or if the skin color was black, the growth period was already in progress, so it was inappropriate for this experiment and was excluded from the experiment.

(36) The test group was divided into a normal group, a negative control group, a test substance-administered group, and a positive control groups 1 and 2. The normal group had three mice, the negative control group had 4 mice, the test substance-administered group had 4 mice, and the positive control group 1 (Tofacitinib) had four mice, and the positive control group 2 (Minoxidil) had four mice. Specifically, the normal group is a group not administered with a solvent or a test substance, the negative control group is a group receiving only a solvent, and the test substance group is a group administered with Centipeda minima extract of Preparation Examples 6, 8, 9 and 11 (30 or 100% ethanol, or 30 or 100% methanol), respectively. The positive control group 1 is a Tofacitinib-administrated group, and the positive control group 2 is a Minoxidil-administered group.

(37) Each of the test substance preparation solutions of Preparation Example 2, 4, 5, and 7 were applied to the dorsal skin twice a day at intervals of 6 hours for 7 days at a dose of 100 μL. 0.1% 1 ml of dexamethasone (ferridex, green cross) as catagen-inducing substance was applied to the epilation site two days before the administration of the test substance, and the application thereof was repeated for 12 days, the end of the test. 5% 100 μl of dexamethasone was applied to the positive control groups 1 and 2.

(38) During the experiment period, after administration of the test substance, hair-growth level was observed via photography and visual inspection. After the end of the experiment, animals were sacrificed and a pathology test for skin tissue was conducted.

(39) Further, to quantify the results of hair-growth efficacy visual inspection, a black portion (anagen) or a region where hair-growth begins in the entire hair removal site (solvent or test substance, positive control substance application site) was visually observed. When anagen symptom was observed in less than 10% of an area to which each of the test substance, the negative control substance, and the positive control substance was applied in the entire hair removal region, score 1 was assigned. When anagen symptom was observed in 10% to 30% thereof, score 2 was assigned. When anagen symptom was observed in 30% to 50% thereof, score 3 was assigned. When anagen symptom was observed in 50% to 80% thereof, score 4 was assigned. When anagen symptom was observed in 80% to 100% thereof, score 5 was assigned. In this way, a numerical graph of the hair-growth efficacy evaluation score was produced.

Example 2-1: Visual Inspection

(40) FIG. 6 shows the results of visual inspection of the mouse skin. As shown in FIG. 6, the test substance-administered group was found to have a significantly higher tendency to migrate to anagen on the Day 5 after 7 days of administration(+12 day), compared to the other groups.

(41) Specifically, the hair-growth efficacy in each administered group on the Day 5 after 7 days of administration was shown in Table 1 below.

(42) TABLE-US-00001 TABLE 1 Test group Hair-growth efficacy (%) Tofacitinib 25 Minoxidil 45 EtOH 30% 65 EtOH 100% 70 MeOH 30% 95 MeOH 100% 90

(43) As may be seen above, excellent hair-growth efficacy could be identified in the Centipeda minima extract-administered group according to the present disclosure.

(44) Further, the changes in body weight during the administration period and hair-growth efficacy evaluation scores during the administration period are shown in FIG. 7 and FIG. 8, respectively.

(45) As shown in FIG. 7, it may be identified that there was no significant change in the weight of the mouse during the test period.

(46) Further, as may be seen in FIG. 8, the Centipeda minima extract-administered group was able to exhibit a significant increase in the hair-growth efficacy during the test period. Specifically, as shown in A of FIG. 8, we may identify the excellent hair-growth efficacy in the test group administered with a Centipeda minima extract according to the present disclosure during the test period. As may be seen from B in FIG. 8, the test group administered with a Centipeda minima extract according to the present disclosure exhibits a much better effect than the positive control groups on the Day 12 of the final evaluation.

(47) From the above results, it may be seen that the Centipeda minima extract according to the present disclosure may be effective in preventing, treating and ameliorating hair-loss and stimulating hair-growth.

Example 2-2: Skin Tissue Pathology Test

(48) The skin tissue pathology test was conducted on day 12 by H&E staining using Hematoxylin and Eosin, which are widely used. A sample was collected from the skin tissue of the sacrificed animals and the generation of hair follicles and phenomena of hair-growth were identified through fixation, washing, clearing, paraffin infiltration, embedding, microtome cutting, staining and reading processes.

(49) FIG. 9 shows the results.

(50) As may be seen in FIG. 9, the formation of specific hair follicles and hair-growth efficacy could not be observed in the normal and negative control groups. Further, in the positive control group 1 and the positive control group 2, only minimal hair follicle generation and hair-growth efficacy degree could be observed.

(51) However, specific hair follicle production and hair-growth efficacy could be identified only in the test substance-administered group administered with Centipeda minima extract.

<Example 3> Identification of Prevention, Treatment and Amelioration Effect of Hair-Loss, and Hair-Growth Stimulation Effect of Centipeda minima Fraction

(52) In the same manner as in Example 2, the hair-loss prevention, treatment, amelioration effects, and hair-growth stimulating effects using Centipeda minima fraction were identified.

(53) Specifically, the normal group is a group not administered with a solvent or a test substance, and a negative control group is a group receiving only a solvent. The test substance group was a group administered with Centipeda minima fractions of Preparation Examples 12, 14, 15, and 17 (dichloromethane fraction of 30 or 100% ethanol, or dichloromethane fraction of 30 or 100% methanol), respectively.

(54) The test substance preparation solutions Preparation Examples 8, 10, 11 and 13 were applied to the dorsal skin twice a day at intervals of 6 hours for 7 days at a dose of 100 μL, respectively. 0.1% 1 mL of dexamethasone (ferridex, green cross) as catagen inducing substance was applied to the epilation site two days before the administration of the test substance, and the application thereof was repeated for 12 days, the end of test. 5% 100 μl of dexamethasone was applied to positive control groups 1 and 2. The rest of the experimental method was the same as in Example 2.

Example 3-1: Visual Inspection

(55) It may be specifically shown in Table 2 below that the test substance-administered group exhibited the significantly higher tendency to migrate to anagen on the Day 5 after 7 days of administration(+12 day), than those of the other groups. Table 2 shows hair-growth efficacy in each administered group on the Day 5 after 7 days of administration.

(56) TABLE-US-00002 TABLE 2 Test group Hair-growth efficacy (%) CH.sub.2Cl.sub.2 fraction of EtOH 30% extract 40 CH.sub.2Cl.sub.2 fraction of EtOH 100% extract 50 CH.sub.2Cl.sub.2 fraction of MeOH 30% extract 45 CH.sub.2Cl.sub.2 fraction of MeOH 100% extract 65

(57) As may be seen above, we may identify excellent hair-growth efficacy in the Centipeda minima fraction-administered group according to the present disclosure.

(58) Further, the changes in the mouse body weight during the administration period and hair-growth efficacy evaluation scores during the administration period are shown in FIG. 10 and FIG. 11, respectively.

(59) As may be seen in FIG. 10, we may identify that there was no significant change in the weight of the mouse during the test period.

(60) Further, as may be seen in FIG. 11, the Centipeda minima fraction-administered group was able to exhibit a significant increase in hair-growth efficacy during the test period. Specifically, as shown in A in FIG. 11, the excellent hair-growth effects were identified in test groups administered with Centipeda minima fraction during the test period. Further, as shown in B of FIG. 11, it was identified that hair-growth efficacy was remarkable on the Day 12 of the final evaluation.

(61) From the above results, it may be seen that the Centipeda minima fraction according to the present disclosure may be effective in preventing, treating and ameliorating hair-loss and stimulating hair-growth.

Example 3-2: Skin Tissue Pathology Test

(62) The skin tissue pathology test was conducted on day 12 by H&E staining using Hematoxylin and Eosin, which are widely used. A sample was collected from the skin tissue of the sacrificed animals and the generation of hair follicles and phenomena of hair-growth were identified through fixation, washing, clearing, paraffin infiltration, embedding, microtome cutting, staining and reading processes.

(63) The results are shown in FIG. 12.

(64) As may be seen from FIG. 12, specific hair generation and hair-growth efficacy could not be observed in the normal and negative control groups. However, specific hair follicle production and hair-growth efficacy could be identified only in the test substance-administered group administered with Centipeda minima extract.

(65) Based on the above results, it may be identified that the composition containing the Centipeda minima extract or the fraction thereof according to the present disclosure may promote hair generation and growth, and may quickly migrate the state of the hair follicle from the telogen to the anagen, thereby to achieve the excellent effect of stimulating hair-growth and its own health.

(66) From the above description, a person skilled in the art to which the present disclosure belongs may understand that the present disclosure may be implemented in other specific forms without changing the technical idea or essential characteristics. In this regard, the examples as described above should be understood as illustrative in all respects and not restrictive. The scope of the present disclosure should be construed to include the meaning and scope of the patent claims as described later rather than the detailed description and all changes or modified forms derived from the equivalent concept included in the scope according to the present disclosure.