Formulations for stimulating hair growth and improving quality and methods of making same

Abstract

The present invention relates to a formulation for stimulating hair growth and improving quality, the active ingredient of which is derived from a plant source, in particular comprising 10-20% of an amaranth nitrate-rich extract. In addition, the formulation has one or more ingredients selected from the group consisting of liquid paraffin, mango butter, beeswax, cetyl alcohol, glycerol, sodium benzoate, potassium sorbate. Also disclosed are methods of preparing the formulations, their use and their use for stimulating hair growth and improving hair quality. The formulations have been found to have high wound healing potential at lower concentrations by increasing the cell proliferation rate. The hair growth rate is increased, and the total number and density (n / cm < 2 >) of final hair, vellus hair and hair in the growing period are increased. Application of the formulation on the scalp helps stimulate hair growth and improve hair quality.

Description

Technical Field

[0001] The present invention relates to a preparation for stimulating hair growth and improving hair quality. The preparation of the present invention contains active ingredients from plant sources such as nitrate-containing green leafy vegetables (such as amaranth, spinach, lettuce) and fennel, arugula, radish, Chinese cabbage and parsley, and more particularly amaranth extract. The present invention also relates to a method for preparing the preparation, its application and its use for stimulating hair growth and improving hair quality. Background Art

[0002] Hair is an integral part of our body image and can have a profound impact on our self-esteem and confidence. The growth and shedding of hair may seem like a simple process, but the hair growth cycle actually consists of four different stages. The first three stages - anagen, catagen and telogen - cover the growth and maturation of hair and the activity of hair follicles that produce single hairs. In the last or shedding stage (exogen), the "old" hair falls out, but usually new hair is ready to take its place. Each stage has its own schedule, which can be affected by age, nutrition and overall health.

[0003] The hair growth phase begins with the anagen phase. This is the longest phase, lasting about 3 to 5 years. In the anagen phase, the hair follicles grow hair, and these hairs continue to grow until they are cut off or until they reach the end of their lifespan and fall out.

[0004] The catagen phase begins at the end of the anagen phase and usually lasts about 10 days or so. In this phase, the hair follicles contract and hair growth slows down. The hair also detaches from the bottom of the hair follicle but remains in place for the last few days of its growth.

[0005] The telogen phase usually lasts about 3 months. It is estimated that 10% to 15% of the hair on our scalp is in this phase. Hair does not grow in the telogen phase, but usually does not fall out either. The telogen phase is also when new hair begins to form in the hair follicles that have just released hair in the catagen phase.

[0006] The shedding phase is essentially an extension or part of the telogen phase of hair growth. In the shedding phase, hair falls out from the scalp, usually facilitated by washing and combing. It is normal to lose 50 to 100 hairs per day in the shedding phase.

[0007] Hair loss is a common problem that affects many people and many animals. There are many types of hair loss, also known as alopecia. Alopecia is a chronic skin disease in which a person loses some or all of the hair on their head and sometimes also the hair on their body. The main factors that can affect hair loss are hormonal changes, genes, stress, illness, childbirth, medications, burns, trauma, autoimmune diseases, cosmetic surgery, and diet. Inherited androgenetic alopecia is the most common form of hair loss: it is characterized by a reduction in hair volume or even baldness and affects up to about 70% of men. Acute hair loss can be associated with chemotherapy treatment, stress, severe malnutrition, iron deficiency, hormonal imbalances, AIDS, or acute radiation.

[0008] Androgenetic alopecia (AGA), also known as male-pattern hair loss (MPHL), is a genetically determined progressive condition in which terminal hair is gradually replaced by vellus hair. The prevalence in men increases with age, but the age of onset and the rate of progression vary. The temples, vertex scalp, and mid-frontal scalp are the three parts of the scalp most affected. This process has a precise structure within these areas. In women, female pattern hair loss (FPHL) is characterized by diffuse thinning on the top of the head while the frontal hairline remains intact.

[0009] Alopecia areata usually occurs suddenly, causing patchy hair loss in children and young people. This condition can lead to complete baldness (alopecia totalis). Alopecia universalis causes the loss of all body hair, including eyebrows, eyelashes, and pubic hair. Trichotillomania is most common in children and is a psychological disorder in which patients pull out their own hair. Telogen effluvium is a temporary thinning of the scalp hair due to a change in the hair growth cycle. A large number of hairs enter the telogen phase simultaneously, resulting in hair loss and subsequent thinning. Scarring alopecia leads to permanent hair loss. Inflammatory skin conditions (such as cellulitis, folliculitis, acne) and other skin diseases (such as certain forms of lupus and lichen planus) often cause scarring, which destroys the hair's regenerative capacity.

[0010] The literature indicates that autocrine and paracrine factors, together with signaling pathways, are involved in the crosstalk between dermal papilla and hair follicle stem cells. The binding of dihydrotestosterone (DHT) to the AR is the main cause of androgen-dependent processes. Significant progress has been made in understanding the key elements of androgen metabolism involved. Various studies conducted on patients with androgen insensitivity syndrome and type 2 5α-reductase deficiency have shown that the activation of hair follicle androgen receptors by DHT induces androgenetic alopecia. The availability of weak androgens, their conversion to stronger androgens through the action of 5α-reductase, the low enzymatic activity of androgen-inactivating enzymes, and a large number of functionally active ARs are all necessary for DHT-dependent cellular functions. Therefore, DHT levels are higher in the susceptible scalp, and AR expression is enhanced. The conversion of testosterone to DHT in the dermal papilla is very important, and androgen regulatory substances derived from dermal papilla cells are thought to affect the proliferation of other hair follicle components. The proportional contribution of locally produced DHT and systemically produced DHT to the balding process is not clear. Thus, it can be said that the putative components in the complex etiology of AGA are chronic microscopic follicular inflammation and connective tissue remodeling, ultimately leading to irreversible hair loss (Scheme 1).

[0011] A known treatment for hair loss is hair transplantation. Another treatment is medical treatment. The only products approved by the US FDA for the treatment of hair loss are oral finasteride (a competitive inhibitor of type 2 5-α reductase) and topical minoxidil (an ATP-sensitive potassium channel opener, which has been shown to stimulate the production of vascular endothelial growth factor in cultured dermal papilla cells). Minoxidil is available as a topical preparation for both men and women. Minoxidil is a piperidinylpyrimidine derivative (2,4-diamino-6-piperidinyl-pyrimidine-3-oxide), which acts as a prodrug and needs to be converted to its active metabolite, minoxidil sulfate (MS), to exert its pharmacological effects. The active metabolite is responsible for the vascular and follicular effects of minoxidil. It usually takes several months of use to see the effects. If minoxidil treatment is stopped, the balding process usually resumes. The regrown hair may fall out three to four months after stopping treatment. One of the serious effects associated with minoxidil is hirsutism, especially on the faces of women. Many patients are hesitant to use it because their hair sometimes looks dry, dull, and stiff. It has been reported that patients complain of palpitations, eye inflammation or burning, and weight gain. It has also been reported that topical minoxidil has an adverse effect on the cardiovascular system.

[0012] Finasteride also shows some efficacy in the treatment of hair loss. Finasteride is a 5α-reductase inhibitor, which functions to prevent the conversion of testosterone into the active 5α-dihydrotestosterone (DHT) form, and an increase in the latter's level has been associated with hair loss. Studies have shown it to be effective in approximately 50% of patients, and reported side effects include erectile dysfunction and gynecomastia. In addition, finasteride is not suitable for women of childbearing age as it can cause birth defects in unborn babies.

[0013] Pharmacological approaches have their own set of drawbacks. Therefore, the recent medical community's attention to AGA has focused on the discovery of new and safer treatments, which are often provided by natural therapies. The literature points to the use of herbs in treating diseases such as hair loss. However, they all lack an explanation of the mechanism of their action. There is literature introducing the activities of seven common phytomedicines (Panax ginseng C.A.Mey., Malus pumila Mill cultivar Annurca, Coffea arabica, Allium sativum L., Camellia sinensis (L.) Kuntze, Rosmarinum officinalis L., Capsicum annum L.), which are considered to be able to reduce the hair loss rate or stimulate new hair growth. Although their positive effects have been widely discussed in the literature, they also have limitations, such as the known gap between different experimental strategies (in vitro, ex vivo, in vivo, and clinical trials), which makes it difficult to provide a comprehensive mechanistic explanation. Therefore, alternative treatments are needed, such as physical or cosmetic treatments, supplements, and the use of herbal extracts.

[0014] In developing countries (such as India), a large part of the population consumes traditional edible plants. It is estimated that there are approximately 30,000 plant species edible worldwide, of which only 7,000 are used as food. Natural compounds extracted from plants, such as storage lipids, spices, essential oils, flavonoids, and polyphenols, have been widely studied for their edible value and are used as precursors by the cosmetics and pharmaceutical industries.

[0015] In the present invention, a topical preparation containing amaranth rich in nitrate is used to stimulate hair growth and improve hair quality. Amaranth is an excellent source of protein, dietary fiber, nitrate, and minerals. Nitrate is converted into nitrite in the body and then into nitric oxide. Nitric oxide is known to be an effective vasodilator. It is speculated that an increase in blood flow to hair cells promotes hair follicle growth and regeneration. It also contains a key amino acid, lysine, which the body needs to grow healthy and strong hair with strong hair roots.

[0016] The present invention provides the in vitro and in vivo effects of amaranth rich-nitrate extract on DHT-induced hair loss and elucidates its possible mechanism of action. SUMMARY OF THE INVENTION

[0017] We disclose a preparation for stimulating hair growth and improving quality, the active ingredient being from a plant source, especially a rich-nitrate extract. The plant sources used in the preparation are selected from amaranth, spinach, lettuce as well as fennel, arugula, radish, Chinese cabbage and parsley. Other sources are beetroot, radish, turnip, watercress, celery, etc. In a preferred embodiment, a rich-nitrate extract derived from amaranth is used in the preparation. In addition to the amaranth rich-nitrate extract in the preparation, it may also have one or more components selected from liquid paraffin, mango butter, beeswax, cetyl alcohol, glycerol, sodium benzoate, potassium sorbate and water.

[0018] According to a preferred embodiment of the present invention, the preparation contains 10-20% amaranth rich-nitrate extract. In another embodiment, the preparation contains 10-20% rich-nitrate extract, 20-30% liquid paraffin, 20-30% mango butter, 10-15% beeswax, 5-10% cetyl alcohol and 15-25% glycerol. Preferably, the preparation consists of 15% amaranth rich-nitrate extract, 20% liquid paraffin, 25% mango butter, 14% beeswax, 6% cetyl alcohol and 20% glycerol.

[0019] According to another embodiment of the present invention, the preparation consisting of the preparation of the present invention contains 10-20% rich-nitrate extract, 20-30% liquid paraffin, 20-30% beeswax, 5-10% cetyl alcohol, 15-25% glycerol, 0.1-3% sodium benzoate, 0.1-3% potassium sorbate and 1-10% water. Preferably, it consists of 15% amaranth rich-nitrate extract, 30% liquid paraffin, 23% beeswax, 6% cetyl alcohol, 20% glycerol, 0.5% sodium benzoate, 0.5% potassium sorbate and 5% water.

[0020] It was found that the preparation has a high wound healing potential by increasing the cell proliferation rate at a lower concentration. It can not only increase the hair growth rate, but also increase the total hair number and density (n / cm 2 ). As part of the efficacy evaluation, it was applied at a dose of 2 g every night on the temples, the top of the head scalp and / or the mid-frontal scalp clinically diagnosed with male pattern hair loss (MPHL) for 90 days. The main outcome was the average change in the density, diameter and number of terminal hairs. The secondary outcomes were the average change in the density, diameter and number of vellus hairs, the anagen phase, the telogen phase, the hair index, the ratio of terminal hairs to vellus hairs and the ratio of anagen phase to telogen phase.

[0021] When in use, it was found that the said preparation increased the expression of VEGF mRNA, indicating that new blood vessels were formed at the site of action / wound site. It also increased the hair index, which is the percentage of anagen hair multiplied by its diameter.

[0022] The method for preparing a preparation for stimulating hair growth and improving quality as claimed in claim 1, comprising:

[0023] - Combining beeswax and liquid paraffin each in an amount of 20 - 30% and melting them in a water bath to form an oil phase;

[0024] - Combining cetyl alcohol and liquid paraffin each in an amount of 5 - 10% and melting them to form an oil phase;

[0025] - Dissolving sodium benzoate and potassium sorbate each in an amount of 0.1 - 3% separately in water to form an aqueous phase;

[0026] - Stirring and mixing amaranth nitrate - rich extract and glycerol each in an amount of 10 - 20% and 15 - 25% respectively to form an aqueous phase; and

[0027] - Adding the obtained aqueous phase and oil phase together while stirring to obtain the said preparation for stimulating hair growth and improving quality. Description of the Drawings

[0028] These and other features, aspects, and advantages of the present invention will become more readily understood when the detailed description is read with reference to the accompanying drawings.

[0029] Figures 1(a) and (b) represent MTT assays, which show that the amaranth nitrate - rich extract has no side effects on human dermal fibroblasts.

[0030] Figures 1(c), (d), and (e) depict the elevated VEGF expression of both A1 and A2 for minoxidil and the amaranth nitrate - rich extract.

[0031] Figure 2 Represents the total hair count after applying the topical preparation and placebo.

[0032] Figure 3 Represents the total hair density after applying the topical preparation and placebo.

[0033] Figure 4 Represents the total hair length after applying the topical preparation and placebo.

[0034] Figure 5 Represents the terminal hair count after applying the topical preparation and placebo.

[0035] Figure 6 Represents the terminal hair density after applying the topical preparation and placebo.

[0036] Figure 7 Indicates the number of vellus hairs after administration of the topical preparation and placebo.

[0037] Figure 8 Indicates the vellus hair density after administration of the topical preparation and placebo.

[0038] Figure 9 Indicates the number of anagen hairs after administration of the topical preparation and placebo.

[0039] Figure 10 Indicates the anagen hair density after administration of the topical preparation and placebo.

[0040] Figure 11 Indicates the hair index after administration of the topical preparation and placebo.

[0041] Figure 12 Indicates the anagen / telogen ratio after administration of the topical preparation and placebo. Specific embodiments

[0042] The present invention discloses a preparation for stimulating hair growth and improving hair quality. Each novel feature is disclosed by way of embodiments. Through these embodiments, a preparation for stimulating hair growth and improving hair quality, a method for preparing the preparation, and a method of use are disclosed herein. These embodiments must be understood in the broadest sense. These illustrations also disclose the efficacy of the preparation relative to known counterparts through various in vivo and in vitro studies. The illustrations of the present invention are intended to enable those skilled in the art to better understand the present invention and are not intended to narrow the scope of any subject matter claimed.

[0043] As used herein, the term "hair" refers to protein filaments growing from hair follicles present in the dermis, including scalp, head, facial, and / or body hair, eyelashes, eyebrows, beards, sideburns, ear hair, nose hair, chest hair, pubic hair, accessory hair, fur, etc.

[0044] "Enhancing hair growth" or "stimulating hair growth" or "inducing hair growth" or "promoting hair growth" means inducing the growth of a new hair cycle earlier, and / or extending the active growth phase (anagen) of the hair cycle, and / or increasing the growth rate of the hair, and / or increasing the width of the hair shaft, including but not limited to inducing hair growth and making it more visible to the eye.

[0045] "Improving hair quality" as used herein means increasing the diameter of the hair shaft and / or enhancing the visual properties of the hair, such as hair volume, hair shine, and hair thickness, and / or affecting the properties of the hair shaft and / or hair cuticle, including but not limited to producing a smoother appearance or feel, and / or increasing shine.

[0046] Improved vasodilation and scalp blood flow provide more oxygen and nutrients to the dermal papilla, which can stimulate hair follicles to grow new hair shafts and extend the anagen growth phase. Although the duration of the anagen phase is genetically determined, sufficient nutrients from the circulation can support mitosis and follicular cell proliferation. While other factors besides nutrients also regulate the transition from anagen to telogen, reduced blood supply is detrimental, especially when androgens mediate vasoconstriction by activating endothelial cells to produce vasoconstrictors.

[0047] The regulation of the hair cycle occurs in the hair follicle sebaceous unit, which includes the sebaceous gland, hair follicle, and hair shaft. Endothelin-1 is a potent vasoconstrictor produced by vascular endothelial cells in response to high testosterone levels acting on androgen receptors. This vasoconstriction may contribute to the induction of male pattern hair loss by restricting nutrient supply to the dermal papilla and leading to follicular miniaturization in androgenetic alopecia.

[0048] Nitric oxide (NO) is a potent vasodilator and signaling molecule in the body. Nitric oxide produced by endothelial cells triggers a series of cell signaling pathways that stimulate hair growth. This signaling activates anti-inflammatory pathways, creating an optimal microenvironment for hair regeneration because chronic inflammation can damage hair follicles and lead to progressive hair loss. NO increases cell metabolism and energy production in hair follicles. This metabolic stimulation provides more biofuel and building blocks required for follicular cell proliferation and hair shaft production. Thus, nitric oxide always balances the vasoconstrictive action of ET-1 in the blood vessels surrounding the dermal papilla.

[0049] In one embodiment, the present disclosure relates to a method for enhancing hair growth in a subject. The method includes administering a formulation comprising a nitrate-rich extract to the subject.

[0050] In one embodiment, the present disclosure relates to a method for improving the hair quality of a subject. The method includes administering a formulation comprising a nitrate-rich extract to the subject.

[0051] In one embodiment, the present disclosure relates to a method for preventing and / or treating hair loss in a subject. The method includes administering a formulation comprising a nitrate-rich extract to the subject.

[0052] One embodiment of the present invention provides a preparation comprising a nitrate-rich extract and excipients, such as a topical preparation, which is effective in preventing hair loss and / or stimulating hair growth and / or enhancing hair growth. A "topical preparation" means that the preparation can be externally applied to the dermis of a mammal. For example, the preparation can be used to apply to the skin of male subjects prone to hair loss or at risk of hair loss (such as subjects suffering from male pattern baldness or at risk of male pattern baldness). In another example, the preparation of the present invention can be used to apply to the skin of subjects suffering from a disease or disorder associated with hair loss (such as alopecia, especially acute alopecia or androgenetic alopecia).

[0053] In one embodiment, the topical preparation of the present invention can be used to stimulate hair growth in subjects suffering from androgenetic alopecia or male pattern baldness or having a tendency to suffer from androgenetic alopecia or male pattern baldness.

[0054] In one embodiment, the topical preparation of the present invention can be used to improve hair quality in a subject.

[0055] One embodiment of the present invention provides a method for treating or preventing hair loss, the method comprising administering to a subject in need thereof (such as a subject suffering from hair loss or having a tendency to suffer from hair loss) a preparation (such as a topical preparation) comprising a nitrate-rich extract and excipients, which is effective in preventing hair loss and / or promoting hair growth and / or enhancing hair growth in the subject. The subject is usually a mammal, such as a human.

[0056] One embodiment of the present invention provides a method for improving hair quality, the method comprising administering to a subject in need thereof a preparation (such as a topical preparation) comprising a nitrate-rich extract and excipients, which is effective in improving hair quality in the subject. The subject is usually a mammal, such as a human.

[0057] The nitrate-rich extract for preparing the preparation is obtained from plant sources, such as green leafy vegetables, such as amaranth, spinach, lettuce, and fennel, arugula, radish, Chinese cabbage, and parsley. Other sources are beetroot, radish, turnip, watercress, celery, etc. In a preferred embodiment, the nitrate plant source is amaranth.

[0058] Excipients are usually included in the dosage form, for example, to improve solubility and / or bioadhesion. Suitable excipients include solvents, cosolvents, emulsifiers, plasticizers, surfactants, thickeners, pH regulators, emollients, antioxidants and chelating agents, wetting agents and water absorbents. The preparation can also contain one or more additives, such as dyes, colored pigments, pearlescent agents, deodorants, and odor masking agents.

[0059] Suitable excipients include, but are not limited to, liquid paraffin, vaseline, microcrystalline beeswax (ceramicrocristallina), microcrystalline wax, ozokerite, ceresine, isoparaffin, paraffin wax, synthetic wax, mango butter, shea butter, cocoa butter, avocado butter, rice bran wax, soybean wax, lauric acid, olive oil, cetyl alcohol, glycerol, triethylene glycol, tripropylene glycol, and propylene glycol.

[0060] The formulation may be suitable for administration by any appropriate route, such as by oral, topical, or parenteral routes.

[0061] The formulation may be administered topically and may be in the form of a cream, lotion, ointment, gel, liquid, or any other topical form.

[0062] The pharmaceutical formulation may be presented in unit dose form containing a predetermined amount of the active agent.

[0063] In one embodiment, the formulation of the present invention comprises 10 - 20% amaranth nitrate-rich extract, 15 - 25% liquid paraffin, 20 - 30% mango butter, 10 - 15% beeswax, 5 - 10% cetyl alcohol, and 15 - 25% glycerol.

[0064] In a preferred embodiment, the formulation of the present invention comprises 15% amaranth nitrate-rich extract, 20% liquid paraffin, 25% mango butter, 14% beeswax, 6% cetyl alcohol, and 20% glycerol.

[0065] In another embodiment, the formulation of the present invention comprises 10 - 20% nitrate-rich extract, 20 - 35% liquid paraffin, 20 - 30% beeswax, 5 - 10% cetyl alcohol, 15 - 25% glycerol, 0.1 - 3% sodium benzoate, 0.1 - 3% potassium sorbate, and 1 - 10% water.

[0066] In a preferred embodiment, the formulation of the present invention comprises 15% amaranth nitrate-rich extract, 30% liquid paraffin, 23% beeswax, 6% cetyl alcohol, 20% glycerol, 0.5% sodium benzoate, 0.5% potassium sorbate, and 5% water.

[0067] In one embodiment, a method for preparing a topical formulation is disclosed. Equal amounts of beeswax and liquid paraffin are combined and melted in a water bath to form an oil phase. Cetyl alcohol and liquid paraffin are taken, combined and melted to form an oil phase. Sodium benzoate and potassium sorbate are separately dissolved in water to form an aqueous phase. The amaranth nitrate-rich extract and glycerol are stirred and mixed to form an aqueous phase. All the aqueous phases are added to the oil phase with continuous stirring.

[0068] The efficacy of amaranth nitrate-rich extract was studied in vitro using human dermal fibroblasts (HDFa) cells. MTT assay revealed that amaranth nitrate-rich extract had no side effects on human dermal fibroblasts (HDF). In fact, under the influence of amaranth nitrate-rich extract for 48 hours, HDF showed a cell proliferation of approximately 120%. However, cytotoxicity was observed at the 72-hour time interval.

[0069] In addition, minoxidil showed an increase in cell proliferation at lower concentrations compared to amaranth nitrate-rich extract. Nevertheless, minoxidil took 48 hours to increase the cell proliferation rate and reached its peak at 72 hours, compared to amaranth nitrate-rich extract which showed its efficacy within 24 hours.

[0070] It was also evident from the MTT results that amaranth nitrate-rich extract had a faster mode of action compared to minoxidil. This faster mode of action can be attributed to the water solubility of amaranth nitrate-rich extract, while minoxidil is an oil-based preparation.

[0071] Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that was initially described as an essential growth factor for vascular endothelial cells. VEGF mRNA expression was examined by quantitative real-time PCR. In the case of amaranth nitrate-rich extract, the relative mRNA expression was greater, with A2 reaching its maximum at the 48-hour time interval. This increased relative mRNA expression also indicated the formation of new blood vessels at the site of action / wound site. It is also important to note the mRNA expression of minoxidil in M1 and M2.

[0072] The efficacy of topical formulations for male pattern hair loss was studied. Men clinically diagnosed with male pattern hair loss were selected for the study. A topical formulation of amaranth nitrate-rich extract was applied to the vertex of the head at a dose of 2 g every night for 90 days. The top of the back of the head is commonly referred to as the vertex and represents the original midline position of the posterior fontanelle or baby soft spot. Between these boundary lines is the occipital protuberance. The mean changes in terminal hair (non-vellus) density (n / cm 2 )), hair shaft diameter (mm), and hair growth rate (mm / day) relative to the baseline were measured from day 3 to day 93. The mean changes in the total number of hairs, thickness (mm), and length (mm) in the target area relative to the baseline were measured from day 3 to day 93, the mean change in the hair index relative to the baseline, the mean changes in the number, density, and percentage of vellus hairs relative to the baseline, the mean changes in the percentage and density of anagen hairs relative to the baseline, the mean changes in the percentage and density of telogen hairs relative to the baseline, the mean change in the terminal hair to vellus hair ratio relative to the baseline, the mean change in the anagen hair to telogen hair ratio relative to the baseline, the mean change in the dermatology quality of life index (DLQI) relative to the baseline, and the change in PGIC relative to the baseline.

[0073] Hair density is calculated as the number of hairs per square centimeter. Terminal hair density is the number of terminal hairs per square centimeter. Terminal hairs are coarser than 40 μm, and the software uses this value to identify terminal hairs in the image. Vellus hair density (n / cm 2 ) is the number of vellus hairs per square centimeter. By definition, vellus hairs are finer than 40 μm. Anagen hair density (n / cm 2 ) is the number of anagen hairs per square centimeter. Anagen hairs grow at approximately 0.3 mm per day. Telogen hair density (n / cm 2 ) is the number of telogen hairs per square centimeter. Telogen hairs do not grow. The hair index is the percentage of anagen hairs multiplied by their diameter.

[0074] At day 90, the total hair count and hair density increased significantly by 23% relative to baseline, while the placebo had a 11% change. During the 90-day study period, the hair growth rate (μm / day) of the test group averaged 380 μm / day, and 310 μm / day for the placebo. The terminal hair count and density increased significantly by 28% relative to baseline, while the placebo had a 15% change. The vellus hair count and density of the test group increased by 12%, while the placebo had only a 3% change.

[0075] In this study, the anagen hair density increased by 54% compared to the placebo. The anagen to telogen ratio showed a significant increase of 155%, while the placebo showed only a 21% change.

[0076] The hair index (PI) is a measure of the hair growth cycle in androgenetic alopecia. A lower hair index indicates a more advanced stage of male pattern hair loss (MPHL).

[0077] In this study, as early as 45 days after administration of the test product, the increase in PI (23%) was evident, while the placebo had only a negligible change of 0.11%. At the end of the 90-day study, there was a significant increase with a 25% change, while the placebo had a 7% change.

[0078] The Dermatology Life Quality Index (DLQI) is a simple self-administered and user-friendly validated questionnaire. Ten questions are asked regarding symptoms and feelings, daily activities, leisure, work and school, personal relationships, and treatment.

[0079] Each answer is scored as follows: The DLQI is calculated by adding up the scores for each question. The maximum score is 30 and the minimum score is 0. The higher the score, the more severely the quality of life is impaired.

[0080] Meaning of DLQI scores

[0081] 0 - 1 - No impact on the patient's life at all

[0082] 2 - 5 - Has little impact on the patient's life

[0083] 6 - 10 - Has a moderate impact on the patient's life

[0084] 11 - 20 - Has a very large impact on the patient's life

[0085] 21 - 30 - Has an extremely large impact on the patient's life

[0086] Overall improvement changes in the subjects

[0087] The PGIC is a 7 - component scale that describes the patient's assessment of overall improvement. Patients rate their changes as "very marked improvement", "marked improvement", "slight improvement", "no change", "slight worsening", "marked worsening", or "very marked worsening".

[0088] Examples

[0089] Preparation method of the topical preparation

[0090] Combine 230 g of beeswax and 230 g of liquid paraffin and melt them in a water bath to form an oil phase. Take 60 g of cetyl alcohol and 70 g of liquid paraffin, combine and melt them to form an oil phase. Dissolve 5 g of sodium benzoate and 5 g of potassium sorbate separately in water to form an aqueous phase. Stir - mix 150 g of amaranth nitrate - rich extract and 200 g of glycerol to form an aqueous phase. Add all the aqueous phases to the oil phase with continuous stirring.

[0091] An in - vitro study on the efficacy of amaranth nitrate - rich extract was conducted using human dermal fibroblast (HDFa) cells.

[0092] Cell culture

[0093] In a 37 °C humidified chamber with 5% CO 2 , maintain human dermal fibroblast (HDFa) cells in DMEM high - glucose medium (Himedia) containing 10% FBS and 40 units / ml penicillin and 40 μg / ml streptomycin (Thermo Fisher). Passage the cells every 2 - 3 days using trypsin - EDTA.

[0094] MTT assay

[0095] The cells were digested with trypsin, centrifuged and resuspended in the medium. Then the cells were counted by the trypan blue method using a hemocytometer and seeded at 5000 cells per 50 μl into the wells of a 96-well microtiter plate. Then the cells were incubated overnight under conditions suitable for cell line adhesion. Subsequently, the cells were treated with different concentrations of amaranth nitrate-rich extract or minoxidil for different time periods (24 hours, 48 hours and 72 hours). Then 10 μl of MTT reagent was added to each well after the corresponding time period and then incubated for 4 hours. After 4 hours, when the purple precipitate was clearly visible under the microscope, the supernatant was carefully removed with a pipette and 100 μl of solubilization solution (DMSO) was added to all wells. The plate was gently shaken in the dark for 20 minutes at room temperature. Then, the absorbance of each well (including the blank) was measured at 570 nm in a microtiter plate reader, with a reference wavelength of 630 nm. The mean value was determined from triplicate readings and the mean value of the blank was subtracted. The absorbance of the test groups was normalized to the control wells to determine cell viability and plotted against the drug concentration to study the increase in cell proliferation.

[0096] Real-time PCR

[0097] RNA extraction

[0098] HDFa cells were treated with the desired concentrations of amaranth nitrate-rich extract and minoxidil for 24 hours and 48 hours. The cells were separated after the corresponding time points and resuspended in 1 ml of RNA extraction solution (called Tri reagent (Takara)). The samples were kept at -20 °C overnight and then continued. 200 μl of chloroform / ml of Tri reagent was added. Then the mixture was vortexed and incubated at room temperature for 15 minutes and then centrifuged at 12000 g at 2 - 8 °C. The aqueous phase containing RNA was transferred to a new tube and mixed with 0.5 ml of isopropanol / ml of Tri reagent. The mixture was incubated at room temperature for 10 minutes to ensure complete precipitation of RNA. Then the RNA was precipitated by centrifuging at 12000 g for 10 minutes at 2 - 8 °C. After removing the supernatant, the RNA precipitate was washed once with 1 ml of 75% ethanol / ml of Tri reagent. Then the RNA precipitate was briefly air-dried at room temperature, dissolved in nuclease-free water and incubated at 56 °C for 10 minutes. Finally, the isolated RNA was quantified using a Nanodrop spectrophotometer.

[0099] cDNA preparation

[0100] The isolated RNA was treated with DNase to remove DNA content (if any) from the sample. For this, 1 U of DNase and 1X DNase buffer were mixed with 1.5 μg of RNA, with a total reaction volume of 10.0 ml. The reaction was then incubated at 37 °C for 30 minutes, and then 1 μl of EDTA was added to stop the reaction by heating at 65 °C for 10 minutes. The reverse transcriptase reaction for cDNA preparation was carried out by heating a 12.5 μl reaction mixture containing 1.0 μg of total RNA and 0.5 μg of random hexamers at 70 °C for 10 minutes. After cooling, 20 U of rRNA (as a ribonuclease inhibitor) and 200 U of Moloney murine leukemia virus ribonuclease reverse transcriptase were added to the final 20 μl reaction mixture containing 10 mmol of deoxy-NTP and 5 μl of Moloney murine leukemia virus reaction buffer, incubated at 42 °C for 1 hour, and heated at 70 °C for 10 minutes.

[0101] Primer Design and Standardization

[0102] For PCR analysis, primers for VEGF and GAPDH were designed using Primer3 software. To ensure the specificity of the selected primers, BLAST was performed to align the primers with genomic sequences in the database and check the sequence specificity. The primers were ordered from Eurofins. The annealing temperature of the primers was standardized by performing gradient PCR at temperatures five degrees higher and five degrees lower than the melting temperature (Tm) of the primers. A 20 μl reaction was set up.

[0103] The contents were gently mixed without forming bubbles and stored in a PCR machine. Then, 2% agarose gel electrophoresis was performed to check for product formation and select the annealing temperature to obtain the maximum intensity of the desired product size.

[0104] Agarose Gel Electrophoresis

[0105] The PCR products were analyzed by running them on a 1.5% agarose gel in 1X TAE buffer. Weigh an amount equivalent to 1.5% of agarose, add it to an Erlenmeyer flask containing 1X TAE. Boil the mixture in a microwave oven until a clear solution is obtained, cool for some time, then add ethidium bromide (EtBr), and carefully pour the gel onto a casting plate with a suitable comb. Let it solidify and then immerse it in 1X TAE. Load the samples into the corresponding wells and let it run until the bromophenol blue reaches the bottom of the gel. Analyze the gel bands in a gel documentation. Select the temperature that shows a significant intensity of the desired PCR product for real-time quantification.

[0106] Real-Time PCR

[0107] After determining the annealing temperature by gradient PCR, real-time PCR for VEGF and GAPDH was performed on all study subjects. Real-time detection of the PCR products after amplification was based on the detection of the fluorescence signal generated by the binding of SYBR Green to double-stranded DNA. Fluorescence signals from each PCR reaction were collected and plotted as peak-normalized values against the cycle number. The reactions were characterized by comparing the threshold cycle (Ct) values. Ct is a unitless value defined as the fractional cycle number at which the normalized sample fluorescence signal passes a fixed threshold above the baseline while always remaining within the linear phase of amplification. Samples with high starting cDNA copy numbers show an increase in fluorescence early in the PCR process and thus result in lower Ct numbers. β-actin was used as an internal control.

[0108] PCR was performed on aliquots containing total cDNA (100 - 200 ng) obtained from equal amounts of total RNA using specific primers for VEGF and GAPDH. The PCR reactions were carried out in a total volume of 10 μl containing 0.3 μM of each primer and 1X SyBr Green mixture (GBiosciences). The PCR conditions included 30 cycles: denaturation at 94 °C for 25 s, annealing at 60 °C for 25 s, extension at 72 °C for 25 s, and fluorescence was recorded after the extension step. GAPDH was used as an internal control for normalization.

[0109] Western blot

[0110] Western blot was performed to investigate the change in protein expression of VEGF after the corresponding treatment. β-actin was used as an endogenous control for normalization.

[0111] Cell lysate preparation

[0112] Cells were harvested after the corresponding incubation time and centrifuged at 200 g for 10 minutes to pellet the cells. The cells were then resuspended in approximately 50 μl of working RIPA lysis buffer, which contained 49.5 μl of stock RIPA lysis buffer and 0.5 μl of protease inhibitor mixture. The cells were kept on ice for 30 minutes and gently pipetted every 15 minutes. Then, they were centrifuged at 4 °C at high speed (15,000 g) for 20 minutes. The supernatant containing the cellular proteins was collected and stored in multiple aliquots at -80 °C for further use.

[0113] Protein concentration estimation

[0114] The protein concentration in the cell lysate was determined using the Bradford assay, where 1X Bradford reagent was used for the estimation and BSA (5 mg / ml) was used as the standard. The BSA stock solution was prepared in distilled water and diluted to different concentrations. After incubation for 5 minutes, the endpoint absorbance was read at 595 nm wavelength and a standard curve was plotted. The protein concentration in the unknown sample was determined using this BSA standard curve.

[0115] Prepare the sample for loading onto the gel

[0116] Mix 20 μg of cell lysate with 3X protein loading dye containing β-mercaptoethanol and SDS to denature the sample and make the proteins negatively charged by binding to the amino acids. Boil the mixture at 95 °C for 10 minutes, and then it can be loaded into the wells of the gel.

[0117] SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis)

[0118] 10 - 12% polyacrylamide gels (for high molecular weight and low molecular weight proteins respectively) were polymerized from acrylamide and N,N'-methylenebisacrylamide (or bisacrylamide). The latter acts as a cross-linking agent for gel formation. Polymerization was initiated by adding ammonium persulfate together with TEMED. The gel is a neutral hydrophilic three-dimensional network of long-chain hydrocarbons cross-linked by methylene groups.

[0119] Load the prepared sample onto the gel and perform electrophoresis on a 10 - 12% SDS-PAGE at 60 V and 100 V using 1X Tris-glycine electrophoresis buffer in a Biorad Western apparatus. A protein ladder molecular weight standard (GBiosciences) was used to confirm the molecular weight of the desired protein bands. The migration of bromophenol blue in the loading dye marked the electrophoresis front, and the gel was stopped when the dye migrated out of the gel.

[0120] Protein transfer and staining (Western blotting)

[0121] Transfer the contents of the gel to a more rigid nitrocellulose membrane that is convenient for further processing. Sandwich the gel and the membrane between sponges and filter pads (sponge / filter pad / gel / membrane / filter pad / sponge), and after ensuring that no air bubbles are formed between the gel and the membrane, clamp them all tightly together. Immerse the sandwich in 1X Tris-glycine transfer buffer in a Bio-Rad western transfer apparatus, with the gel facing the negative electrode and the membrane facing the positive electrode. Apply an 80 V electric field for 2 hours. Negatively charged proteins migrate towards the positively charged electrode and thus bind to the nitrocellulose membrane. The proteins transferred onto the membrane can be visualized by Ponceau S Red staining to evaluate the successful transfer of proteins, and then additional steps of western blotting can be carried out.

[0122] Blocking

[0123] Block the nitrocellulose membrane with 5% BSA for 1 hour at room temperature to prevent non-specific binding of antibodies to other parts of the membrane. After blocking, wash the membrane once with 1X tris-buffered saline Tween (TBST).

[0124] Primary antibody incubation

[0125] Then incubate the membrane with primary antibodies against VEGF and β-actin antibodies (Santa Cruz Biotechnology) overnight at 4°C. Dilute the antibodies in 1% BSA / 1X TBST. After incubation, wash the membrane three times with 1X TBST (10 minutes for each wash).

[0126] Secondary antibody incubation

[0127] Then incubate the membrane with a secondary antibody anti-mouse IgG HRP conjugate (1:7000, Santa Cruz Biotechnology) for 2 hours at room temperature. Dilute the secondary antibody with 1% BSA-1X TBST. After incubation, wash three times using 1XTBST as was done after incubation with the primary antibody.

[0128] Enhanced chemiluminescence reaction

[0129] Develop the membrane using enhanced chemiluminescence, where peroxide and luminol provided in the chemiluminescence detection kit are mixed at a 1:1 ratio in the dark. Then pour this solution onto the membrane and incubate for about 30 seconds. Obtain the band images in an Azure Biosystems and then perform quantification.

[0130] Immunohistochemistry

[0131] The paraffin blocks were cut into 5-μm thick tissue sections and mounted onto poly-L-lysine-coated glass slides. The slides were then dewaxed in xylene and gradually rehydrated in a decreasing gradient of alcohol. Thereafter, the slides were placed in citrate buffer (pH 6.0) and endogenous peroxidase was neutralized using 3% H 2 O 2 One primary antibody was used at the desired dilution on the tissue sections and incubated overnight at 4 °C. The next day, after washing, an HRP-conjugated secondary antibody was applied for 1 hour. Color development was carried out using SubstrateImpact Novared and counterstained with hematoxylin. The tissue sections were then dehydrated and mounted with DPX mounting medium.

[0132] Results

[0133] The MTT assay showed that the nitrate-rich amaranth extract had no side effects on human dermal fibroblasts (HDFs). In fact, under the influence of the nitrate-rich amaranth extract for 48 hours, the HDFs showed a cell proliferation of approximately 120%. However, cytotoxicity was observed at the 72-hour time interval ( Figure 1a and b). In addition, minoxidil showed an increase in cell proliferation at lower concentrations compared to the nitrate-rich amaranth extract. Nevertheless, compared to the nitrate-rich amaranth extract, which showed its efficacy within 24 hours, minoxidil required 48 hours to increase the cell proliferation rate and reached its peak at 72 hours. It was also evident from the MTT results that the nitrate-rich amaranth extract had a faster mode of action compared to minoxidil. This faster mode of action can be attributed to the water solubility of the nitrate-rich amaranth extract, while minoxidil is an oil-based preparation.

[0134] In vitro cell-based scratch assay is one of the most suitable and cost-effective methods for the preliminary examination of the wound healing potential of any medicinal extract. The present study aimed to evaluate the wound healing ability of the nitrate-rich amaranth extract, where HDFs were treated with different concentrations of the nitrate-rich amaranth extract (0.2 mg / ml, 0.4 mg / ml, and 1.6 mg / ml) and minoxidil (20 mg / ml, 60 mg / ml, and 100 mg / ml) for 48 hours. Cell migration was monitored at 0 hours, 24 hours, and 48 hours, and the wound closure distance was calculated using Image J software. The scratch assay images clearly showed that A1 (0.2 mg / ml; 48-hour time period) had excellent proliferative and migratory behavior. A1 closed 92.142% of the gap created by the scratch within 48 hours. On the other hand, minoxidil performed close to the negative control (89.798%), closing 89.358% of the gap at 120 mg / ml (SI 1).

[0135] VEGF mRNA expression was examined by quantitative real-time PCR. The results clearly showed that in the case of amaranth nitrate-rich extract, the relative mRNA expression was greater, with A2 reaching its maximum at a 48-hour time interval. This increased relative mRNA expression also indicated the formation of new blood vessels at the site of action / wound site. It is also important to note that the mRNA expression of minoxidil decreased sharply at M1 and M2 ( Figure 1c , d and e).

[0136] However, the main objective of this study was to investigate the regulation of VEGF protein expression in HDF by amaranth nitrate-rich extract and minoxidil. The results showed that amaranth nitrate-rich extract upregulated VEGF expression in human dermal fibroblasts; A1 significantly showed the presence of VEGF.

[0137] VEGF is one of the most important pro-angiogenic molecules in the skin. It has been shown to play a role in hair growth as well as in the development of skin diseases such as psoriasis and skin cancer. VEGF family members exert their effects on endothelial cells by binding and activating tyrosine kinase receptors located on the cell surface. VEGF is able to bind to multiple receptors, including VEGF receptor-1 (VEGFR-1) and VEGF receptor-2 (VEGFR-2). VEGFR-2 is considered to be the more important of the two receptors in controlling endothelial cell function and regulating angiogenesis based on its superior ability to stimulate downstream signaling cascades.

[0138] Efficacy of topical preparations in male pattern hair loss

[0139] Subjects aged 25 - 45 years, clinically diagnosed with male pattern hair loss (MPHL) and Norwood classified as III vertex, IV, V were selected for the study. The subjects were divided into two groups of 25 each. A topical preparation of amaranth nitrate-rich extract was applied to the vertex at a dose of 2 g every night for 90 days. The primary outcome was the mean change in the density, diameter, and number of terminal hairs. The secondary outcomes were the mean change in the density, diameter, and number of vellus hairs, anagen, telogen, hair index, terminal to vellus hair ratio, and anagen to telogen ratio.

[0140] Hair density (n / cm 2 ) - Hair density was calculated as the number of hairs per square centimeter.

[0141] Terminal hair density (n / cm 2 ) : The number of terminal hairs per square centimeter. Terminal hairs are coarser than 40 μm, and the software used this value to identify terminal hairs in the image.

[0142] Vellus hair density (n / cm 2):The number of vellus hairs per square centimeter. By definition, vellus hairs are finer than 40 μm. The number of vellus hairs relative to terminal hairs was also calculated and provided in the analysis results.

[0143] Anagen hair density (n / cm 2 ):The number of anagen hairs per square centimeter. Anagen hairs grow at approximately 0.3 mm per day.

[0144] Telogen hair density (n / cm 2 ):The number of telogen hairs per square centimeter. Telogen hairs do not grow.

[0145] Hair index: The percentage of anagen hairs multiplied by their diameter.

[0146] At day 90, the total hair count and hair density increased significantly by 23% relative to baseline, while the placebo had a 11% change ( Figure 2 and 3 ). The mean differences at day 90 were 31 and 50 respectively. During the 90-day study period, the hair growth rate (μm / day) of the test group averaged 380 μm / day, and 311 μm / day for the placebo ( Figure 4 ).

[0147] The number and density of terminal hairs increased significantly by 28% relative to baseline, while the placebo had a 15% change ( Figure 5 and 6 ). At the end of the 90-day study, the mean differences relative to the placebo were 30.88 and 48.95 respectively. The number and density of vellus hairs in the test group increased by 12%, while the placebo had only a 3% change ( Figure 7 and 8 ).

[0148] The number and density of anagen hairs increased significantly by 54% relative to baseline, while the placebo had a 17.5% change ( Figure 9 and 10 ). The mean differences relative to the placebo at the end of the study were 47.08 and 73.18 respectively. The hair index increased significantly by 25% relative to baseline, while the placebo had a 7.3% change ( Figure 11 ). The mean difference relative to the placebo at the end of the study was 530.83. The anagen-to-telogen ratio increased significantly by 155%, while the placebo showed only a 21% change ( Figure 12 )

[0149] In this study, the anagen hair density increased by 54% compared to the placebo. This effect may be explained by the fact that the test preparation contains naturally occurring nitrates, and the nitrates absorbed by the scalp are converted into nitric oxide, which increases blood circulation and thus stimulates the proliferation of hair follicle cells.

[0150] The hair index (PI) is a measure of the hair growth cycle in androgenetic alopecia. A lower hair index indicates a more advanced stage of male pattern hair loss (MPHL).

[0151] In this study, as early as 45 days after the administration of the test product, an increase in PI (23%) was evident, while the placebo had a negligible change of only 0.11%. At the end of the 90-day study, there was a significant increase with a 25% change, while the placebo had a 7% change.

[0152] We have presented the novel features of the present invention by explaining some preferred embodiments thereof, enabling those skilled in the art to understand and visualize our invention. It should also be understood that the application of the present invention is not limited to the details set forth in the above description or illustrated in the accompanying drawings. Although the present invention has been described in considerable detail with reference to some of its preferred embodiments, various modifications can be made without departing from the scope of the present invention as described above herein and defined by the following claims.

Claims

1. A preparation for stimulating hair growth and improving quality, said preparation comprising an amaranth nitrate-rich extract.

2. The preparation for stimulating hair growth and improving quality according to claim 1, said preparation comprising 10 - 20% of an amaranth nitrate-rich extract.

3. The preparation for stimulating hair growth and improving quality according to claim 1, said preparation comprising 15% of an amaranth nitrate-rich extract.

4. The preparation for stimulating hair growth and improving quality according to claim 1, said preparation comprising one or more components selected from liquid paraffin, mango butter, beeswax, cetyl alcohol, glycerol, sodium benzoate, and potassium sorbate.

5. The preparation for stimulating hair growth and improving quality according to claim 1, said preparation being a composition of amaranth extract, said composition of amaranth extract comprising amaranth nitrate and one or more components selected from liquid paraffin, mango butter, beeswax, cetyl alcohol, glycerol, sodium benzoate, and potassium sorbate.

6. The preparation for stimulating hair growth and improving quality according to claim 1, said preparation having a high wound healing potential by increasing the cell proliferation rate at a lower concentration.

7. The preparation for stimulating hair growth and improving quality according to claim 1, said preparation increasing the hair growth rate.

8. The preparation for stimulating hair growth and improving quality according to claim 1, wherein the preparation increases the total hair number and density (n / cm 2 ).

9. The preparation for stimulating hair growth and improving quality as claimed in claim 8, wherein the preparation increases the number and density of terminal hairs (n / cm 2 ).

10. The preparation for stimulating hair growth and improving quality according to claim 8, wherein the preparation increases the number and density of vellus hair (n / cm 2 ).

11. The preparation for stimulating hair growth and improving quality according to claim 8, said preparation increasing the anagen to telogen ratio.

12. The preparation for stimulating hair growth and improving quality according to claim 8, wherein the preparation increases the number and density of anagen hair (n / cm 2 ).

13. The preparation for stimulating hair growth and improving quality according to claim 8, said preparation increasing the terminal to vellus hair ratio.

14. The preparation for stimulating hair growth and improving quality according to claim 8, said preparation increasing the vascular endothelial growth factor (VEGF) mRNA expression.

15. The preparation for stimulating hair growth and improving quality according to claim 8, said preparation increasing the hair index, which is the percentage of anagen hairs multiplied by their diameter.

16. A method for preparing the preparation for stimulating hair growth and improving quality according to claim 1, said method comprises: - combining beeswax and liquid paraffin each in an amount of 20 - 30% and melting them in a water bath to form an oil phase; - combining cetyl alcohol and liquid paraffin each in an amount of 5 - 10% and melting them to form an oil phase; - dissolving sodium benzoate and potassium sorbate each in an amount of 0.1 - 3% separately in water to form an aqueous phase; - stirring and mixing the amaranth nitrate-rich extract and glycerol in amounts of 10 - 20% and 15 - 25% respectively to form an aqueous phase; and - adding the resulting aqueous phase and oil phase together while stirring to obtain the preparation for stimulating hair growth and improving quality.

17. A method of using the preparation for stimulating hair growth and improving quality according to claim 1, comprising applying the preparation at a dose of 2 g every night on the temples, vertex scalp, and / or mid-frontal scalp clinically diagnosed with male pattern hair loss (MPHL).

18. A method of using the preparation according to claims 1 to 5, wherein the preparation is applied for a period of 45 days or longer.

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