Formulation for promoting hair growth and improving quality and method for preparing same

Abstract

The present invention relates to a formulation for stimulating hair growth and improving its quality, the active ingredients of which are derived from plant sources, in particular a 10-20% nitrate-enriched extract of amaranth. Furthermore, the formulation has one or more ingredients selected from liquid paraffin, mango butter, beeswax, cetyl alcohol, glycerol, sodium benzoate, and potassium sorbate. The present invention also discloses a method for preparing the formulation, its application, and its use for stimulating hair growth and improving its quality. The formulation has been found to have high wound healing potential at low concentrations by increasing the cell proliferation rate. It not only increases the hair growth rate, but also improves the total number and density (n / cm) of terminal, vellus, and anagen hairs. 2 Applying the formulation to the scalp helps stimulate hair growth and improve hair quality.

Description

[Technical Field]

[0001] The present invention relates to a formulation for stimulating hair growth and improving hair quality. The formulation of the present invention comprises an active ingredient, particularly an extract of amaranth, containing nitrates, from plant sources such as amaranth, spinach, lettuce, fennel, arugula, radish, Chinese cabbage, parsley, and other green leafy vegetables. The present invention also relates to a method for preparing the formulation, its application, and its use for stimulating hair growth and improving hair quality. [Background technology]

[0002] Hair is essential to our body image and can have a major impact on self-esteem and confidence. While hair growth and shedding may seem like a simple process, the hair growth cycle actually consists of four distinct phases. The first three phases (anagen, catagen, and telogen) involve hair growth and maturation and the activity of the hair follicle that produces each individual hair. During the final, or catagen, phase, the "old" hair falls out, usually preparing for a new hair to take its place. Each phase has its own timeline and can be affected by age, nutrition, and overall health.

[0003] The growth phase of hair begins with the anagen phase, which is the longest phase and lasts approximately 3-5 years. During the anagen phase, hair is pushed out of the follicle and continues to grow until it is either cut off or falls out at the end of its lifespan.

[0004] Catagen begins at the end of anagen and lasts for about 10 days. During this period, the hair follicle shrinks and hair growth slows. The hair detaches from the base of the follicle but remains in place for the last few days of growth.

[0005] The telogen phase usually lasts about three months. It is estimated that about 10-15% of scalp hairs are in this phase. During the telogen phase, hairs do not grow, but they do not usually fall out. This is also the time when new hairs begin to form in the hair follicles that lost hair during the catagen phase.

[0006] The exogenous phase is essentially an extension or part of the telogen phase of hair growth. During the exogenous phase, hair is shed from the scalp with washing or brushing. It is normal to lose 50 to 100 hairs per day during the exogenous phase.

[0007] Hair loss is a common problem experienced by many humans as well as many animals. There are many types of hair loss, also known as alopecia. Alopecia is a chronic skin condition that causes partial or total hair loss on the scalp and sometimes body hair as well. The main factors that affect hair loss are hormonal changes, genes, stress, illness, childbirth, drugs, burns, injuries, autoimmune diseases, cosmetic procedures, and diet. Hereditary male pattern baldness is the most common form of hair loss. It manifests as reduced hair volume or baldness and affects approximately 70% of men. Acute hair loss can be associated with chemotherapy, stress, severe malnutrition, iron deficiency, hormonal disorders, AIDS, or acute radiation therapy treatment.

[0008] Androgenetic alopecia (AGA), also known as androgenetic alopecia (MPHL), is a genetically determined, progressive disease in which terminal hair is gradually replaced by vellus hair. Its prevalence in men increases with age, but the age at which symptoms appear and the rate of progression vary. The temples, vertex, and central frontal area are the three most affected areas of the scalp. The process is tightly structured within these areas. In women, androgenetic alopecia (FPHL) is distinguished by thinning hair on the vertex and a thinning hairline at the frontal area.

[0009] Alopecia areata often develops suddenly and causes patchy hair loss in children and young adults. This condition can lead to complete baldness (alopecia totalis). Alopecia universalis is the loss of all body hair, including eyebrows, eyelashes, and pubic hair. Trichotillomania, most commonly seen in children, is a psychiatric disorder in which people pull out their own hair. Telogen effluvium is a temporary thinning of scalp hair due to an alteration in the hair growth cycle. Many hairs simultaneously enter the resting phase, causing hair to fall out and subsequently thin. Cicatricial alopecia causes permanent hair loss. Inflammatory skin diseases (such as cellulitis, folliculitis, and acne) and other skin diseases (such as certain types of lupus and lichen planus) often cause scarring that destroys hair's ability to regrow.

[0010] Literature has shown that the interaction between the dermal papilla and hair follicle stem cells involves autocrine and paracrine factors, along with signaling pathways. The binding of dihydrotestosterone (DHT) to AR is the primary driver of androgen-dependent processes. Significant progress has been made in understanding the key components of androgen metabolism involved. Various studies in patients with androgen insensitivity syndrome and 5α-reductase type 2 deficiency suggest that activation of the hair follicle androgen receptor by dihydrotestosterone induces male pattern baldness. The availability of weak androgens, their conversion to more potent androgens by 5α-reductase, low enzymatic activity of androgen-inactivating enzymes, and a high number of functionally active ARs are all required for DHT-dependent cellular function. Therefore, scalps prone to hair loss have high DHT levels and enhanced AR expression. The conversion of testosterone to DHT in the dermal papilla is important, and androgen regulators derived from dermal papilla cells are thought to influence the proliferation of other hair follicle components. The contribution of locally produced dihydrotestosterone and systemically produced dihydrotestosterone to the hair loss process is unclear. Therefore, a presumed component of the complex pathogenesis of AGA is prolonged microfollicle inflammation accompanied by connective tissue remodeling, ultimately leading to irreversible hair loss (Figure 1).

[0011] One known treatment for alopecia is hair transplantation. Another is medication. The only products approved by the U.S. FDA for the treatment of alopecia are oral finasteride, a competitive inhibitor of type 2, 5-alpha-reductase, and topical minoxidil, an adenosine triphosphate-sensitive potassium channel opener shown to stimulate vascular endothelial growth factor production in cultured dermal papilla cells. Minoxidil is available in topical formulations for use in both men and women. Minoxidil is a piperidinopyrimidine derivative (2,4-diamino-6-piperidinopyrimidine-3-oxide) that functions as a prodrug and requires conversion to the active metabolite, minoxidil sulfate (MS), to exert its pharmacological effect. The active metabolite is responsible for minoxidil's effects on blood vessels and hair follicles. It typically requires several months of use before it becomes effective. Discontinuing minoxidil treatment usually results in a resumption of the hair loss process. Regrowing hair may fall out 3 to 4 months after treatment is discontinued. One serious side effect associated with minoxidil is hirsutism, especially on the face in women. Many patients are reluctant to use minoxidil because it can cause hair to become dry, dull, and coarse. Patients have also reported experiencing palpitations, eye irritation and burning, and weight gain. Topical minoxidil has also been reported to have adverse effects on the cardiovascular system.

[0012] Finasteride has also demonstrated some effectiveness in treating hair loss. Finasteride is a 5α-reductase inhibitor, which works by blocking the conversion of testosterone to its active form, 5α-dihydrotestosterone (DHT). High levels of DHT are associated with hair loss. Studies have shown efficacy in approximately 50% of patients, although side effects such as erectile dysfunction and gynecomastia have been reported. Furthermore, finasteride cannot be used by women of childbearing age due to the potential for birth defects in the fetus.

[0013] Pharmacological approaches have their own drawbacks. As a result, recent medical interest in AGA has focused on discovering new, safer treatments, often provided by natural therapies. The literature indicates the use of herbal medicines to treat conditions such as alopecia. However, all of them lack explanations of their mechanisms of action. The literature documents the effects of seven common plants (Panax ginseng CA Mey., Malus pumila Mill. cultivar Annurca, Coffea arabica, Allium sativum L., Camellia sinensis (L.) Kuntze, Rosmarinum officinalis L., and Capsicum annum L.). They are believed to slow hair loss or stimulate new hair growth. While their positive effects have been widely discussed in the literature, they also have limitations, such as known gaps between various experimental strategies—in vitro, ex vivo, in vivo, and clinical trials—making it difficult to explain their integrated mechanisms. As a result, alternative treatments, such as physical or cosmetic treatments, supplements, and the use of herbal extracts, are needed.

[0014] Traditional edible plants are consumed by a large proportion of the population in developing countries such as India. It is estimated that there are approximately 30,000 edible plant species worldwide, of which only 7,000 are used for human consumption. Natural compounds extracted from plants, such as storage lipids, flavors, essential oils, flavonoids, and polyphenols, have been extensively studied for their edible value and are used as ingredients in the cosmetic and pharmaceutical industries.

[0015] In this invention, a topical preparation containing a nitrate-enriched extract of amaranth is used to stimulate hair growth and improve hair quality. Amaranth is an excellent source of protein, dietary fiber, nitrates, and minerals. Nitrates are converted to nitrites and then to nitric oxide in the body. Nitric oxide is known to be a powerful vasodilator. Increased blood flow to hair cells is hypothesized to promote hair follicle growth and regeneration. Amaranth also contains the important amino acid lysine, which the body needs to create healthy, strong hair with strong roots.

[0016] The present invention provides the in vitro and in vivo effects of a nitrate-enriched extract of amaranth on DHT-induced hair loss and elucidates its possible mechanism of action. Summary of the Invention

[0017] The present invention discloses a formulation for stimulating hair growth and improving hair quality, the active ingredient of which is obtained from a plant source, particularly a nitrate-rich extract. The plant source used in the formulation is selected from amaranth, spinach, lettuce, fennel, arugula, radish, Chinese cabbage, and parsley. Other sources include beetroot, radish, turnip, watercress, and celery. In a preferred embodiment, a nitrate-rich extract from amaranth is used in the formulation. In addition to the nitrate-rich extract of amaranth in the formulation, the formulation may contain water and one or more ingredients selected from liquid paraffin, mango butter, beeswax, cetyl alcohol, glycerol, sodium benzoate, and potassium sorbate.

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

[0019] According to another embodiment of the present invention, the formulation of the present invention comprises 10-20% nitrate-enriched 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, and preferably 15% nitrate-enriched amaranth extract, 30% liquid paraffin, 23% beeswax, 6% cetyl alcohol, 20% glycerol, 0.5% sodium benzoate, 0.5% potassium sorbate and 5% water.

[0020] The formulation was found to have a high wound healing ability at a low concentration by increasing the cell proliferation rate. It not only increased the hair growth rate but also increased the total hair number and density (n / cm 2 As part of the efficacy evaluation, a 2g dose was applied once daily at night to the temples, scalp vertex, or mid-frontal scalp in patients with clinically diagnosed male pattern baldness (MPHL). The primary endpoint was the mean change in density, diameter, and number of terminal hairs. Secondary endpoints included the mean change in density, diameter, and number of vellus hairs, anagen, telogen, follicle index, terminal-to-vellus ratio, and anagen-to-telogen ratio.

[0021] Upon application, the formulation is found to increase VEGF mRNA expression, which indicates the formation of new blood vessels at the site of action / wound, and also increases the hair index, which is the product of the percentage of anagen hairs and their diameter.

[0022] 10. A method for producing a formulation for stimulating hair growth and improving hair quality according to claim 1, comprising: -Mix beeswax and liquid paraffin in a ratio of 20-30% each and melt them in a water bath to form an oil phase. Mix at a ratio of 5-10% and melt to form an oil phase. -Sodium benzoate and potassium sorbate are separately dissolved in water in the range of 0.1-3% each to form an aqueous phase. - Amaranth nitrate concentrated extract and glycerol are mixed with stirring in the range of 10-20% and 15-25%, respectively, to form an aqueous phase. The resulting aqueous and oil phases are added together with stirring to produce a formulation that stimulates hair growth and improves hair quality. [Brief explanation of the drawings]

[0023] These features, aspects, and advantages of the present invention will be better understood from a reading of the detailed description when taken in conjunction with the accompanying drawings.

[0024] [Figure 1] Figures 1(a) and (b) are representative examples of MTT assays that revealed that nitrate-enriched extracts of amaranth had no adverse effects on human skin fibroblasts.

[0025] Figure 1(c), (d) and (e) show the increase in VEGF expression in A1 and A2 for both minoxidil and nitrate-enriched extract of amaranth.

[0026] [Figure 2] FIG. 2 depicts the total hair counts after application of the topical formulation and placebo.

[0027] [Figure 3] FIG. 3 depicts total hair density after application of the topical formulation and placebo.

[0028] [Figure 4] FIG. 4 depicts the total hair length after application of the topical formulation and placebo.

[0029] [Figure 5] FIG. 5 depicts the terminal hair counts after application of the topical formulation and placebo.

[0030] [Figure 6] FIG. 6 depicts terminal hair density after application of the topical formulation and placebo.

[0031] [Figure 7] FIG. 7 depicts the vellus hair count after application of the topical formulation and placebo.

[0032] [Figure 8] FIG. 8 depicts vellus hair density after application of the topical formulation and placebo.

[0033] [Figure 9] FIG. 9 depicts the anagen hair counts after application of the topical formulation and placebo.

[0034] [Figure 10] FIG. 10 depicts anagen hair density after application of the topical formulation and placebo.

[0035] [Figure 11] FIG. 11 depicts the hair follicle index after application of the topical formulation and placebo.

[0036] [Figure 12] FIG. 12 depicts the anagen / telogen ratio after application of the topical formulation and placebo. DETAILED DESCRIPTION OF THE INVENTION

[0037] The present invention discloses a formulation for stimulating hair growth and improving hair quality. Each novel feature is disclosed through embodiments. Through these embodiments, the formulation, a method for preparing said formulation, and a method of use for stimulating hair growth and improving hair quality are disclosed herein. The embodiments should be understood in their broadest sense. The figures also disclose the efficacy of said formulation relative to known equivalents through various in vivo and in vitro studies. The figures of the present invention are intended to enable those skilled in the art to better understand the invention and are not intended to narrow the scope of the claimed subject matter.

[0038] As used herein, the term "hair" refers to protein filaments that grow from hair follicles in the dermis, and includes scalp, head, facial, and / or body hair, eyelashes, eyebrows, mustache, beard, ear hair, nose hair, chest hair, pubic hair, secondary hair, fur, etc.

[0039] "Enhancing hair growth" or "stimulating hair growth" or "inducing hair growth" or "promoting hair growth" means the earlier induction of new hair growth and / or the prolongation of the active growth phase (anagen) of the hair cycle and / or an increase in the rate of hair growth and / or an increase in the width of the hair shaft, including, but not limited to, inducing and making visible hair growth.

[0040] As used herein, "improving hair quality" means increasing the diameter of the hair shaft and / or enhancing the visual properties of the hair, such as hair volume, hair shine, hair thickness, and / or affecting the properties of the hair shaft and / or hair cuticle, including, but not limited to, creating a smoother appearance and feel and / or increasing shine.

[0041] Improved vasodilation and blood flow to the scalp provide a greater supply of oxygen and nutrients to the dermal papilla, stimulating the hair follicle to grow a new hair shaft and prolonging the anagen phase. While the duration of anagen is genetically determined, adequate nutrition from the circulation supports mitosis and hair follicle cell proliferation. Although other factors besides nutrition control the transition from anagen to telogen, a reduced blood supply is detrimental by activating endothelial production of vasoconstrictor factors, particularly androgen-mediated vasoconstriction.

[0042] The hair cycle is regulated by the pilosebaceous 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, which acts on the androgen receptor. This vasoconstriction may be responsible for male pattern baldness by limiting nutrients to the dermal papilla and causing miniaturization of the hair follicles.

[0043] Nitric oxide (NO) is a potent vasodilator and signaling molecule in the body. Its production in endothelial cells triggers a cascade of cell signaling pathways that stimulate hair growth. This signaling activates anti-inflammatory pathways, creating an optimal microenvironment for hair regrowth, whereas chronic inflammation damages hair follicles and leads to progressive hair loss. NO promotes hair follicle cellular metabolism and energy production. This metabolic stimulation provides a greater supply of biological fuels and building blocks required for hair follicle cell proliferation and hair shaft production. Therefore, nitric oxide constantly balances the vasoconstrictor effects of ET-1 in the blood vessels surrounding the dermal papilla.

[0044] In one embodiment, the present invention relates to a method of promoting hair growth in a subject, the method comprising applying to the subject a formulation enriched in nitrates.

[0045] In one embodiment, the present invention relates to a method of improving the quality of hair in a subject, the method comprising applying a nitrate-enriched formulation to the subject.

[0046] In one embodiment, the present invention relates to a method of preventing and / or treating hair loss in a subject, the method comprising applying to the subject a formulation enriched in nitrates.

[0047] One embodiment of the present invention provides a formulation, such as a topical formulation, which comprises a nitrate-rich extract and an excipient and is effective for preventing hair loss and / or promoting hair growth and / or strengthening hair growth. "Topical formulation" means that the formulation can be applied externally to the dermis of mammals.For example, the formulation can be useful for administration to the skin of male subjects who are susceptible to or at risk of hair loss, such as subjects who suffer from or are at risk of developing male pattern baldness.In another example, the formulation of the present invention is useful for administration to the skin of subjects who suffer from hair loss-related diseases or conditions, such as alopecia, particularly acute alopecia or male pattern baldness.

[0048] In one embodiment, the topical formulations of the present invention are useful for stimulating hair growth in subjects suffering from or predisposed to developing androgenetic alopecia or male pattern baldness.

[0049] In one embodiment, the topical formulations of the present invention are useful for improving the quality of a subject's hair.

[0050] One embodiment of the present invention provides a method for treating or preventing alopecia, comprising administering to a subject in need thereof, e.g., a subject suffering from or prone to developing alopecia, a formulation, e.g., a topical formulation, comprising a nitrate-rich extract and excipients effective to prevent hair loss and / or promote hair growth and / or enhance hair growth in the subject. The subject is typically a mammal, such as a human.

[0051] One embodiment of the present invention provides a method of improving hair quality, comprising administering to a subject in need thereof a formulation, e.g., a topical formulation, comprising a nitrate-rich extract and excipients effective to improve the quality of the subject's hair, the subject being typically a mammal such as a human.

[0052] The nitrate-rich extracts used to prepare the formulations are obtained from plant sources such as amaranth, spinach, green leafy vegetables such as lettuce, fennel, arugula, radish, Chinese cabbage, parsley, etc. Other sources include beetroot, radish, turnip, watercress, celery, etc. In a preferred embodiment, the plant source of nitrates is amaranth.

[0053] Excipients are commonly included in dosage forms, for example, to improve solubility and / or bioadhesion. Suitable excipients include solvents, cosolvents, emulsifiers, plasticizers, surfactants, thickeners, pH adjusters, emollients, antioxidants, and chelating agents, humectants, and water-absorbing agents. The formulation may also include one or more additives such as dyes, color pigments, pearlescent agents, deodorizing agents, and odor-masking agents.

[0054] Suitable excipients include, but are not limited to, liquid paraffin, petrolatum, cera microcrystalline, microcrystalline wax, ozokerite, ceresin isoparaffin, paraffin, synthetic wax, mango butter, shea butter, cocoa butter, avocado butter, rice bran wax, soy wax, lauric acid, olive oil, cetyl alcohol, glycerol, triethylene glycol, tripropylene glycol, and propylene glycol.

[0055] The formulation may be adapted for administration by any suitable route, for example oral, topical, or parenteral.

[0056] The formulations may be applied topically and may be in the form of a cream, lotion, ointment, gel, liquid, or other topical form.

[0057] The pharmaceutical formulation may be presented in the form of a unit dose containing a predetermined amount of the active agent.

[0058] In one embodiment, the formulation of the present invention comprises 10-20% nitrate-enriched extract of amaranth, 15-25% liquid paraffin, 20-30% mango butter, 10-15% beeswax, 5-10% cetyl alcohol and 15-25% glycerol.

[0059] In one preferred embodiment, the formulation of the present invention comprises 15% nitrate-enriched extract of amaranth, 20% liquid paraffin, 25% mango butter, 14% beeswax, 6% cetyl alcohol and 20% glycerol.

[0060] In another embodiment, the formulation of the present invention comprises 10-20% nitrate-enriched 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.

[0061] In a preferred embodiment, the formulation of the present invention comprises 15% nitrate-enriched extract of amaranth, 30% liquid paraffin, 23% beeswax, 6% cetyl alcohol, 20% glycerol, 0.5% sodium benzoate, 0.5% potassium sorbate and 5% water.

[0062] In one embodiment, a method for preparing a topical formulation is disclosed. Equal amounts of beeswax and liquid paraffin are mixed and melted in a water bath to form an oil phase. Cetyl alcohol and liquid paraffin are collected, mixed, and melted to form an oil phase. Sodium benzoate and potassium sorbate are separately dissolved in water to form an aqueous phase. A nitrate-enriched extract of amaranth and glycerol are mixed with stirring to form an aqueous phase. All aqueous phase components are added to the oil phase with continuous stirring.

[0063] The efficacy of nitrate-rich amaranth extract has been studied in vitro using human dermal fibroblast (HDFa) cells. MTT assays revealed that nitrate-rich amaranth extract had no adverse effects on human dermal fibroblast (HDF) cells. In fact, under the influence of nitrate-rich amaranth extract for 48 hours, HDF cells showed approximately 120% cell proliferation. Cytotoxicity, however, was observed at 72 hours.

[0064] Furthermore, in contrast to the nitrate-enriched extract of amaranth, minoxidil increased cell proliferation at lower concentrations, although it took 48 hours for minoxidil to increase cell proliferation, peaking at 72 hours, compared to the nitrate-enriched extract of amaranth, which showed an effect within 24 hours.

[0065] The MTT results also demonstrate that the nitrate-enriched amaranth extract is more rapid acting than minoxidil, likely due to the water-soluble nature of the nitrate-enriched amaranth extract, whereas minoxidil is an oil-based formulation.

[0066] Vascular endothelial growth factor (VEGF) is a potent angiogenic factor and was first described as an essential growth factor for vascular endothelial cells. VEGF mRNA expression was examined by quantitative real-time PCR. Nitrate-rich amaranth extracts showed greater relative mRNA expression, with a maximum at A2 over the 48-hour interval. This increase in relative mRNA expression also indicates the formation of new blood vessels at the site of action / wound. It is also important to note the mRNA expression of minoxidil at M1 and M2.

[0067] To investigate the effectiveness of a topical preparation for male pattern hair loss. Men with clinically diagnosed male pattern hair loss are selected for this study. A topical preparation of nitrate-rich amaranth extract is applied to the vertex at a dose of 2 g once daily at night for 90 days. The upper part of the occipital region is often referred to as the parietal region, and represents the location of the occipital fontanelle or the natural midline of the infantile soft tissue. Between these boundaries lies the occipital convexity. Terminal (non-vellus) hair density (n / cm 2 The mean change from baseline in hair shaft diameter (mm), hair growth rate (mm / day) will be measured from day 3 to day 93. The mean change from baseline in total hair count, thickness (mm), and length (mm) in the target area, mean change from baseline in hair index, mean change from baseline in vellus hair count, density, and percentage, mean change from baseline in anagen hair percentage and density, mean change from baseline in telogen hair percentage and density, mean change from baseline in terminal to vellus hair ratio, mean change from baseline in anagen to telogen hair ratio, mean change from baseline in Dermatology Life Quality Index (DLQI), and change from baseline in PGIC will be measured from day 3 to day 93.

[0068] 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 thicker than 40 μm and this value is used by the software 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 thinner than 40 μm. Anagen hair density (n / cm 2 ) is the number of anagen hairs per square centimeter. Anagen hairs grow at a rate of 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.

[0069] At 90 days, total hair count and hair density significantly increased from baseline by 23% compared to an 11% change in the placebo group. Hair growth rate (μm / day) averaged 380 μm / day in the test group and 310 μm / day in the placebo group over the 90-day study period. Terminal hair count and hair density significantly increased by 28% from baseline compared to a 15% change in the placebo group. Vellus hair count and hair density increased by 12% in the test group compared to only a 3% change in the placebo group.

[0070] In this study, anagen hair density increased by 54% compared to placebo. The anagen to telogen ratio showed a significant increase of 155%, while the placebo showed only a 21% change.

[0071] The hair index (PI) is a measure of the hair growth cycle in male pattern baldness. A lower hair index indicates a more advanced stage of male pattern baldness (MPHL).

[0072] In this study, a significant increase in PI (23%) was observed as early as 45 days after starting test product use, compared with a small change of 0.11% with placebo. At the end of the 90-day study, a significant increase of 25% was observed with test product compared with a 7% change with placebo.

[0073] The Dermatology Life Quality Index (DLQI) is a simple, self-administered, user-friendly, validated questionnaire with 10 questions about symptoms, emotions, daily activities, leisure time, work and school, personal relationships, and medical treatment.

[0074] Each response is scored as follows: The DLQI is calculated by adding the scores for each question. The maximum score is 30 and the minimum score is 0. A higher score indicates a poorer quality of life.

[0075] What does the DLQI score mean? 0-1-No impact on patient's life 2-5-Minimal impact on patient's life 6-10-Moderate impact on patient's life 11-20 - Extremely significant impact on patients' lives 21-30 - Extremely impactful on patients' lives [Change in overall improvement of subject]

[0076] The PGIC is a 7-point scale that assesses a patient's overall improvement. Patients rate their change as "very improved," "much improved," "slightly improved," "no change," "slightly worse," "much worse," or "very worse." [Example]

[0077] Methods for preparing topical formulations 230g of beeswax and 230g of liquid paraffin were mixed and melted in a water bath to form the oil phase. 60g of cetyl alcohol and 70g of liquid paraffin were mixed and melted to form the oil phase. 5g of sodium benzoate and 5g of potassium sorbate were separately dissolved in water to form the aqueous phase. 150g of nitrate-enriched extract of amaranth and 200g of glycerol were mixed with stirring to form the aqueous phase. All aqueous phase components were added to the oil phase with continuous stirring. In vitro study of the efficacy of nitrate-enriched extract of amaranth using human dermal fibroblasts (HDFa). cell culture

[0078] Human dermal fibroblasts (HDFa) were maintained in DMEM high glucose medium (Himedia) containing 10% FBS, 40 units / ml penicillin, and 40 μg / ml streptomycin (Thermo Fisher) in a humidified chamber at 37°C with 5% CO. Cells were passaged every 2–3 days using trypsin-EDTA. MTT assay

[0079] The cells were trypsinized, centrifuged, and resuspended in culture medium. They were then counted using the trypan blue method using a hemocytometer and seeded at 5,000 cells per 50 μl into wells of a 96-well microtiter plate. The cells were then incubated overnight under conditions suitable for cell line adhesion. The cells were then treated with different concentrations of nitrate-enriched extracts of amaranth or minoxidil for different periods of time (24, 48, and 72 hours). After each treatment period, 10 μl of MTT reagent was added to each well and the plate was incubated for 4 hours. After 4 hours, when a purple precipitate was clearly visible under a microscope, the supernatant was carefully removed with a pipette, and 100 μl of solubilization solution (DMSO) was added to all wells. The plate was then gently shaken in the dark at room temperature for 20 minutes. The absorbance of each well, including the blank, was then measured at 570 nm using a microtiter plate reader, with the reference wavelength set to 630 nm. The mean value was determined from triplicate measurements and the mean blank value was subtracted. The absorbance of the test wells was normalized to the control wells to determine cell viability. Cell viability was plotted against drug concentration to examine the increase in cell proliferation. Real-time PCR RNA extraction

[0080] HDFa cells were treated with the desired concentrations of nitrate-enriched extracts of amaranth and minoxidil for 24 and 48 hours. At each time point, cells were isolated and resuspended in 1 ml of an RNA extraction solution called Tri Reagent (Takara). Samples were stored overnight at -20°C before further analysis. Tri Reagent was added at 200 μl chloroform / ml. The mixture was then vortexed and incubated at room temperature for 15 minutes, followed by centrifugation at 12,000 g at 2-8°C. The aqueous phase containing the RNA was transferred to a new tube and mixed with Tri Reagent at 0.5 ml isopropyl alcohol / ml. The mixture was incubated at room temperature for 10 minutes to allow complete precipitation of the RNA. The RNA was then precipitated by centrifugation at 12,000 g for 10 minutes at 2-8°C. After removing the supernatant, the RNA pellet was washed once with 1 ml of 75% ethanol per ml of Tri Reagent. The RNA pellet was then 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. cDNA preparation

[0081] The isolated RNA was treated with DNase to remove any DNA present in the sample. To do this, 1 U of DNase and 1X DNase buffer were mixed with 1.5 μg of RNA to create a total reaction mixture of 10.0 ml. The reaction mixture was then incubated at 37°C for 30 minutes, followed by the addition of 1 μl of EDTA and heating at 65°C for 10 minutes to terminate the reaction. The reverse transcriptase reaction for cDNA preparation was performed 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 ribonuclease inhibitor rRNA and 200 U of Moloney murine leukemia virus ribonuclease reverse transcriptase were added to the final 20 μl reaction mixture containing 10 mmol deoxy NTPs and 5 μl of Moloney murine leukemia virus reaction buffer. The mixture was then incubated at 42°C for 1 hour and heated at 70°C for 10 minutes. Primer design and standardization

[0082] 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 to genome sequences in the database and check the sequence specificity. Primers were ordered from Eurofins. The primer annealing temperature was standardized by performing gradient PCR at temperatures 5°C above and below the melting temperature (Tm) of the primers. 20 μl reactions were set up.

[0083] The contents were mixed gently to avoid bubbles and stored in the PCR machine. Afterwards, 2% agarose gel electrophoresis was performed to confirm product formation, and the annealing temperature that gave the maximum intensity of the desired product size was selected. Agarose gel electrophoresis

[0084] PCR products were analyzed by electrophoresis on a 1.5% agarose gel in 1X TAE buffer. A 1.5% equivalent volume of agarose was weighed and added to an Erlenmeyer flask containing 1X TAE. The mixture was boiled in a microwave until a clear solution was obtained. After cooling, ethidium bromide (EtBr) was added, and the gel was carefully poured into a casting plate using an appropriate comb. The gel was allowed to set and then submerged in 1X TAE. Samples were loaded into each well and run until the bromophenol blue reached the bottom of the gel. Gel bands were analyzed by gel documentation. For real-time quantification, a temperature showing significant intensity of the desired PCR product was selected. Real-time PCR

[0085] After determining the annealing temperature by gradient PCR, real-time PCR for VEGF and GAPDH was performed in all study subjects. Real-time detection of amplified PCR products is based on the detection of fluorescent signals generated by SYBR Green binding to double-stranded DNA. The fluorescent signal from each PCR reaction was collected as a peak-normalized value plotted against cycle number. Reactions were characterized by comparing threshold cycle (Ct) values. Ct is a unitless value defined as the fractional cycle number at which the normalized sample's fluorescent signal passes a fixed threshold above baseline, always within the linear phase of amplification. Samples with a higher starting copy number of cDNA will have lower Ct numbers due to increased fluorescence early in the PCR process. β-actin was used as an internal control.

[0086] Aliquots containing total cDNA (100-200 ng) obtained from equal amounts of total RNA were subjected to PCR using primers specific for VEGF and GAPDH. PCR reactions were performed in a total volume of 10 μl containing 0.3 μM of each primer and 1X SyBr Green Mix (GBiosciences). PCR conditions consisted of 30 cycles of denaturation at 94°C for 25 seconds, annealing at 60°C for 25 seconds, extension at 72°C for 25 seconds, and fluorescence recording after the extension step. GAPDH was used as an internal control for normalization. Western blotting

[0087] Western blotting was performed to examine changes in VEGF protein expression after each treatment, with β-actin used as an endogenous control for normalization. Preparation of cell lysates

[0088] Cells were harvested after each incubation period and centrifuged at 200 g for 10 minutes to pellet the cells. They were then resuspended in approximately 50 μl of working RIPA lysis buffer containing 49.5 μl of stock RIPA lysis buffer and 0.5 μl of protease inhibitor cocktail. The cells were kept on ice for 30 minutes with gentle pipetting every 15 minutes. They were then centrifuged at high speed (15,000 g) for 20 minutes at 4°C. The supernatant containing cellular proteins was collected and stored in aliquots at -80°C until further use. Protein concentration estimation

[0089] Protein concentrations in cell lysates were determined using the Bradford assay, using 1X Bradford reagent as an estimate and BSA (5 mg / ml) as a standard. BSA stock was made in distilled water and diluted to various concentrations. Endpoint absorbance was read at 595 nm after a 5-minute incubation, and a standard curve was plotted. Protein concentrations in unknown samples were determined using the BSA standard curve. Preparing samples for loading onto the gel

[0090] Twenty micrograms of cell lysate was mixed with 3X protein loading dye containing β-mercaptoethanol and SDS to denature the sample and conjugate the proteins to negatively charged amino acids. The mixture was boiled at 95°C for 10 minutes and then loaded into the gel wells. SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis)

[0091] 10–12% polyacrylamide gels (for high and low molecular weight proteins, respectively) were formed from the polymerization of acrylamide and N,N-methylenebisacrylamide (or bisacrylamide). The latter served as a cross-linking agent for gel formation. Polymerization was initiated by the addition of ammonium persulfate along with TEMED. The gel was a neutral, hydrophilic, three-dimensional network of long hydrocarbon chains cross-linked by methylene groups.

[0092] The prepared samples were loaded onto a gel and electrophoresed at 60 V and 100 V on a 10-12% SDS-PAGE gel using 1X Tris-glycine electrophoresis buffer in a BioRad Western apparatus. A protein ladder (GBiosciences) was used to confirm the molecular weight of the protein bands of interest. The movement of bromophenol blue within the loading dye marked the electrophoresis front, and the gel was stopped when the dye migrated out of the gel. Protein transfer and staining (Western blotting)

[0093] The gel contents were transferred to a nitrocellulose membrane, which is more rigid and convenient for further processing. The gel and membrane were sandwiched between a sponge and a filter pad (sponge / filter pad / gel / membrane / filter pad / sponge), and after ensuring there were no air bubbles between the gel and membrane, everything was firmly secured. The sandwich was then immersed 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. An 80V electric field was applied for 2 hours. Negatively charged proteins migrate toward the positive electrode and bind to the nitrocellulose membrane. The transferred proteins were visualized by Ponceau's Red staining to assess whether the protein transfer was successful, after which the next step in Western blotting could be performed. blocking

[0094] To prevent nonspecific binding of the antibody to other parts of the membrane, the nitrocellulose membrane was blocked with 5% BSA for 1 hour at room temperature. After blocking, the membrane was washed once with 1X Tris-buffered saline Tween (TBST). Primary antibody incubation

[0095] The membrane was then incubated overnight at 4°C with primary antibodies against VEGF and β-actin (Santa Cruz Biotechnology). The antibodies were diluted in 1% BSA / 1X TBST. After incubation, the membrane was washed three times with 1X TBST (10 min each wash). Secondary antibody incubation

[0096] The membrane was then incubated with a secondary antibody anti-mouse IgG HRP conjugate (1:7000, Santa Cruz Biotechnology) for 2 hours at room temperature. The secondary antibody was diluted in 1% BSA-1X TBST. After incubation, the membrane was washed three times with 1X TBST, as in the case of the primary antibody incubation. Enhanced chemiluminescence reaction

[0097] The membrane was developed using the enhanced chemiluminescence method, which involves mixing peroxide and luminol in a 1:1 ratio in the dark, as provided in the chemiluminescence detection kit. The solution was then poured onto the membrane and incubated for approximately 30 seconds. Band images were captured and subsequently quantified using Azure Biosystems. immunohistochemistry

[0098] Paraffin blocks were cut into 5 μm-thick tissue sections and mounted on poly-L-lysine-coated slides. The slides were then deparaffinized in xylene and rehydrated with decreasing concentrations of alcohol. The slides were then placed in citrate buffer (pH 6.0) and endogenous peroxides were neutralized using 3% H2O2. Primary antibodies were applied to the tissue sections at the indicated dilutions and incubated overnight at 4°C. The following day, after washing, HRP-tagged secondary antibodies were applied for 1 hour. Color development was achieved using the substrate Impact Novared and counterstained with hematoxylin. The tissue sections were then dehydrated and mounted with DPX mounting medium. result

[0099] MTT assays revealed that the nitrate-enriched extract of amaranth had no adverse effects on human dermal fibroblasts (HDFs). In fact, HDFs exposed to the nitrate-enriched extract of amaranth for 48 hours showed approximately 120% cell proliferation. However, cytotoxicity was observed at 72 hours (Figures 1a and 1b). Furthermore, in contrast to the nitrate-enriched extract of amaranth, minoxidil increased cell proliferation at lower concentrations. However, compared to the nitrate-enriched extract of amaranth, which showed an effect within 24 hours, minoxidil required 48 hours to increase cell proliferation, peaking at 72 hours. The MTT results also revealed that the nitrate-enriched extract of amaranth has a faster action than minoxidil. This faster action is likely due to the water-soluble nature of the nitrate-rich extract of amaranth, whereas minoxidil is an oil-based formulation.

[0100] The in vitro cell scratch assay is one of the most suitable and economical procedures for preliminary testing of the wound-healing ability of any medicinal extract. This study aimed to evaluate the wound-healing ability of nitrate-rich amaranth extract. HDFs were treated with different concentrations of nitrate-rich amaranth extract (0.2 mg / ml, 0.4 mg / ml, 1.6 mg / ml) and minoxidil (20 mg / ml, 60 mg / ml, 100 mg / ml) for 48 hours. Cell migration was monitored at 0, 24, and 48 hours, and the wound closure distance was calculated using ImageJ software. Images from the scratch assay clearly show the superior proliferation and migration behavior of A1 (0.2 mg / ml, 48-hour duration). A1 closed 92.142% of the scratch-induced gap at 48 hours. On the other hand, minoxidil was close to the negative control (89.798%) in its performance, narrowing the gap by 89.358% at 120 mg / ml (SI1).

[0101] VEGF mRNA expression was examined by quantitative real-time PCR. The results clearly show that relative mRNA expression was greater in the case of nitrate-rich amaranth extract, with A2 being the highest after 48 hours. This increase in relative mRNA expression also indicates the formation of new blood vessels at the treatment / wound site. It is also noteworthy that minoxidil mRNA expression decreased sharply in M1 and M2 (Figure 1c, d, and e).

[0102] However, the main aim of this study was to investigate the modulation of VEGF protein expression in HDFs by nitrate-enriched extract of amaranth and minoxidil.The results revealed that nitrate-enriched extract of amaranth upregulated VEGF expression in human dermal fibroblasts, among which A1 significantly showed the presence of VEGF.

[0103] VEGF is one of the most important pro-angiogenic molecules in the skin. VEGF has been shown to be involved not only in hair growth but also in the development of skin diseases such as psoriasis and skin cancer. VEGF family members act on endothelial cells by binding to and activating tyrosine kinase receptors on the cell surface. VEGF can bind to multiple receptors, including VEGF receptor 1 (VEGFR-1) and VEGF receptor 2 (VEGFR-2). VEGFR-2 is considered 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. [Effectiveness of topical preparations for male pattern baldness]

[0104] Subjects aged 25 to 45 years with clinically diagnosed male pattern hair loss (MPHL) and Norwood classification III vertex, IV, or V were enrolled in this study. They were divided into two groups of 25 subjects each. A topical formulation of nitrate-rich amaranth extract was applied to the vertex at a dose of 2 g once daily at night for 90 days. The primary outcome measure was the mean change in terminal hair density, diameter, and number. Secondary outcomes included mean change in vellus hair, anagen, telogen, follicle index, terminal-to-vellus hair ratio, and anagen-to-telogen ratio.

[0105] Hair density (n / cm 2 )-Hair density was calculated as the number of hairs per square centimeter.

[0106] Terminal hair density (n / cm 2 ): The number of terminal hairs per square centimeter. Terminal hairs are thicker than 40 μm, and this value is used by the software to identify terminal hairs in the image.

[0107] Vellus density (n / cm 2 ): The number of vellus hairs per square centimeter. By definition, vellus hairs are hairs thinner than 40 μm. The number of vellus hairs relative to terminal hairs is also calculated and presented in the analysis.

[0108] Growth phase hair density (n / cm 2 ): The number of anagen hairs per square centimeter. Anagen hairs grow at a rate of approximately 0.3 mm per day.

[0109] Telogen hair density (n / cm 2 ): The number of telogen hairs per square centimeter. Telogen hairs do not grow.

[0110] Hair index: The percentage of hair in the anagen phase multiplied by the diameter of the hair.

[0111] At 90 days, total hair count and hair density significantly increased from baseline by 23% compared with an 11% change for placebo (Figures 2 and 3). The mean difference at 90 days was 31 and 50, respectively. Hair growth rate (μm / day) averaged over the 90-day study period was 380 μm / day in the study group and 311 μm / day in the placebo group (Figure 4).

[0112] Terminal hair count and density significantly increased by 28% from baseline compared to a 15% change with placebo (Figures 5 and 6). The mean difference from placebo was 30.88 and 48.95, respectively, at the end of the 90-day study. Vellus hair count and density increased by 12% in the test group compared to only a 3% change with placebo (Figures 7 and 8).

[0113] Anagen hair count and density significantly increased by 54% from baseline, compared to a 17.5% change with placebo (Figures 9 and 10). The mean difference from placebo at the end of the study was 47.08 and 73.18, respectively. Hair index significantly increased by 25% from baseline, compared to a 7.3% change with placebo (Figure 11). The mean difference from placebo at the end of the study was 530.83. Anagen to telogen ratio showed a significant increase of 155%, compared to only a 21% change with placebo (Figure 12).

[0114] In this study, hair density in the anagen phase increased by 54% compared to placebo. This effect can be explained by the fact that the test formulation contains natural nitrates, which, when absorbed by the scalp, are converted into nitric oxide, increasing blood circulation and thereby stimulating hair follicle cell proliferation.

[0115] The hair index (PI) is a measure of the hair growth cycle in male pattern baldness. A lower hair index indicates a more advanced stage of male pattern baldness (MPHL).

[0116] In this study, significant increases in PI (23%) were observed as early as 45 days after the start of use of the test product, compared with only a 0.11% change with placebo. At the end of the study at 90 days, a significant increase in PI was observed, with a 25% change compared with a 7% change with placebo.

[0117] The present invention has described some of the preferred embodiments of the present invention, thereby clarifying the novel features of the present invention and enabling those skilled in the art to understand and visualize the present invention. It should also be understood that the present invention is not limited in its application to the details set forth in the above description or shown in the drawings. Although the present invention has been described in considerable detail with reference to its preferred embodiments, various modifications can be made thereto without departing from the scope of the present invention as set forth above and defined in the following claims.

Claims

1. A formulation for stimulating hair growth and improving quality, comprising a nitrate-enriched extract of amaranth.

2. 2. The hair growth stimulating and quality improving formulation of claim 1, comprising 10-20% of a nitrate-enriched extract of amaranth.

3. 2. A hair growth stimulating and quality improving formulation according to claim 1, comprising 15% of a nitrate-enriched extract of amaranth.

4. 10. The hair growth stimulating and quality improving formulation of claim 1, comprising one or more ingredients selected from liquid paraffin, mango butter, beeswax, cetyl alcohol, glycerol, sodium benzoate, and potassium sorbate.

5. 2. The hair growth stimulating and quality improving formulation of claim 1, which is a composition of amaranth extract containing amaranth nitrate and one or more ingredients selected from liquid paraffin, mango butter, beeswax, cetyl alcohol, glycerol, sodium benzoate, and potassium sorbate.

6. The hair growth stimulating and quality improving preparation according to claim 1, which has high wound healing ability at a lower concentration by increasing cell proliferation rate.

7. 2. The hair growth stimulating and quality improving preparation according to claim 1, which is used to increase the rate of hair growth.

8. Total hair count and density (n / cm ２ 2. The hair growth stimulating and quality improving preparation according to claim 1, which is used to increase hair growth.

9. Number and density of terminal hairs (n / cm ２ 9. The hair growth stimulating and quality improving formulation according to claim 8, which is used to increase hair growth.

10. Number and density of vellus hairs (n / cm ２ 9. The hair growth stimulating and quality improving formulation according to claim 8, which is used to increase hair growth.

11. 9. A hair growth stimulating and quality improving formulation according to claim 8, which is used to increase the anagen to telogen ratio.

12. Number and density of anagen hair (n / cm ２ 9. The hair growth stimulating and quality improving formulation according to claim 8, which is used to increase hair growth.

13. The hair growth stimulating and quality improving formulation according to claim 8, which is used to increase the ratio of terminal hair to vellus hair.

14. The hair growth stimulating and quality improving formulation according to claim 8, which is used to increase vascular endothelial growth factor (VEGF) mRNA expression.

15. 9. A hair growth stimulating and quality improving formulation according to claim 8, used to increase the hair index, which is the percentage of hair in the anagen phase multiplied by its diameter.

16. 10. A method for preparing the hair growth stimulating and quality improving formulation of claim 1, comprising: - combining beeswax and liquid paraffin in amounts ranging from 20-30% each and melting them in a water bath to form an oil phase; - combining cetyl alcohol and liquid paraffin in amounts ranging from 5-10% each and melting to form an oil phase; - dissolving sodium benzoate and potassium sorbate separately in water in an amount ranging from 0.1 to 3% each to form an aqueous phase; - A nitrate-enriched extract of amaranth and glycerol are mixed under stirring in amounts ranging from 10 to 20% and from 15 to 25%, respectively, to form an aqueous phase; and - adding the obtained aqueous phase and oil phase together under stirring to form a hair growth stimulating and quality improving formulation; A method comprising:

17. 10. A method of using the hair growth stimulating and quality improving formulation of claim 1, comprising applying the formulation at a dose of 2 g once daily at night to the temples, vertex scalp, and / or central frontal scalp in patients with clinically diagnosed male pattern baldness (MPHL).

18. 6. A method of using the formulation of claims 1 to 5, wherein the formulation is applied for a period of 45 days or more.

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