Radix scrophulariae extract and oxoglutaric acid composition and application thereof in preventing hair loss and promoting hair growth

Abstract

The present application relates to A61K, more particularly, the present application relates to a gardenia extract and oxopentanedioic acid composition and application in the field of preventing hair loss and promoting hair growth, comprising: gardenia extract and oxopentanedioic acid. The present application provides a composition, by using the effect of gardenia extract and oxopentanedioic acid, the expression and inhibition of VEGF, TGF-beta, Wnt / beta-catenin factor can be affected, the balance promotes the differentiation and proliferation of hair papilla cells, promotes the proliferation and growth of hair follicles, reduces the atrophy or closure of hair follicles, thereby the effect of preventing hair loss and promoting hair growth can be effectively played, and the performance is stable, and is suitable for subsequent actual use. In addition, the composition of the present application is safe and non-irritating, can be used in different preparations for preventing and treating hair loss, and can achieve similar or even better effect than the current minoxidil and other drugs by using less amount.

Description

Technical Field

[0001] This invention relates to A61K, and more specifically, to a composition of gardenia extract and oxoglutaric acid and its application in the field of hair loss prevention and hair growth. Background Technology

[0002] Hair loss, an increasingly serious problem in dermatology, not only affects personal appearance but also poses numerous obstacles to life and work. The main types of hair loss include androgenetic alopecia (AGA), telogen effluvium, and alopecia areata (AA). Currently, the FDA has only approved two chemically synthesized drugs for the treatment of androgenetic alopecia: oral finasteride and topical minoxidil. Both drugs have some efficacy, but their side effects are also very significant.

[0003] Therefore, selecting safer plant extracts or their active ingredients is currently the focus of the hair loss prevention and hair growth field. However, the effectiveness of traditional Chinese medicine in treating hair loss is currently limited to clinical observation, and its effects are generally not ideal, making it difficult to replace finasteride and minoxidil.

[0004] CN111375018A discloses a plant extract solution for promoting hair growth, which is an alcoholic solution of gardenia extract and / or earthworm extract, exhibiting significant hair growth effects, especially the alcoholic solution. However, the use of alcohol is still not suitable for sensitive individuals. Summary of the Invention

[0005] To address the aforementioned problems, the first aspect of the present invention provides a composition of gardenia extract and oxoglutaric acid, comprising: gardenia extract and oxoglutaric acid.

[0006] As a preferred embodiment of the present invention, the weight ratio of gardenia extract to oxoglutaric acid (α-KG) is 0.5-210:1, and examples include 0.5:1, 1:1, 2:1, 3:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, 110:1, 120:1, 130:1, 140:1, 150:1, 160:1, 170:1, 180:1, 190:1, 200:1, and 210:1.

[0007] As a preferred embodiment of the present invention, the gardenia extract includes at least one of gardenia fruit extract, gardenia flower extract, gardenia extract, gardenia oil, gardenia fruit extract, and gardenia extract. Preferably, it is gardenia fruit extract (GFFE), which is an extract of the dried, ripe fruit of gardenia, and its main components are iridoid glycosides.

[0008] Hair growth is closely related to the growth of dermal papilla cells (DPCs), and the activity of DPCs is correlated with Wnt / β-catenin, TGF-β, and VEGF. Under normal circumstances, Wnt / β-catenin is beneficial for stimulating hair follicle proliferation, VEGF is beneficial for providing nutrients to hair follicles, while TGF-β reduces oxygen supply, causing hair follicle atrophy. The inventors discovered that by selecting gardenia extract and oxoglutarate as a combination and controlling their dosage, the expression of Wnt / β-catenin, TGF-β, and VEGF can be regulated to achieve a balance that promotes the growth of dermal papilla cells and hair growth. Thus, through the synergistic effect of the composition of this invention, a hair loss prevention and hair growth effect similar to minoxidil can be obtained.

[0009] As a preferred technical solution of the present invention, the carrier of the gardenia extract is at least one of water, alcohol, and sugar carrier, preferably water or sugar carrier.

[0010] Examples of alcohols include methanol, ethanol, and propanol. Examples of sugar carriers include dextrin, cyclodextrin, xylitol, and erythritol.

[0011] As a preferred embodiment of the present invention, the carrier accounts for less than 50 wt% of the weight of the gardenia extract, such as 40-50 wt% (excluding 50 wt%).

[0012] In addition, the inventors also discovered that when plant extracts are applied to hair or hair-related cells, such as dermal papilla cells, the effects are unstable. However, by using the gardenia extract and oxoglutaric acid provided by this invention, the inventors found that the effects are more stable, which is beneficial for subsequent applications in preventing hair loss and promoting hair growth.

[0013] A second aspect of this invention provides a hair loss prevention and hair growth preparation comprising the gardenia extract and oxoglutaric acid composition as described above, wherein the gardenia extract accounts for 0.001-2.1 wt% of the preparation, such as 0.005 wt%, 0.01 wt%, 0.02 wt%, 0.03 wt%, 0.05 wt%, 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.4 wt%, 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, etc. by weight. The oxoglutaric acid comprises 0.005-0.01 wt% of the formulation, such as 0.005 wt%, 0.006 wt%, 0.008 wt%, 0.009 wt%, or 0.01 wt%, in wt% of the amount of 0.9 wt%, 1 wt%, 1.1 wt%, 1.2 wt%, 1.3 wt%, 1.4 wt%, 1.5 wt%, 1.6 wt%, 1.7 wt%, 1.8 wt%, 1.9 wt%, 2 wt%, or 2.1 wt%, respectively. Compared to minoxidil, the composition used in this invention can achieve good hair growth effects with similar or even lower concentrations.

[0014] As a preferred embodiment of the present invention, the formulation is at least one of powder, tablet, granule, liquid formulation, and capsule, without specific limitation. When it is a solid formulation such as powder, tablet, capsule, or granule, the filler can be organic substances such as sugars or inorganic substances such as talc. When it is a liquid formulation, the filler can be water, alcohol, etc., without specific limitation, as long as it is acceptable for pharmaceutical use or can be used for external application.

[0015] In addition to the composition and fillers, the formulation may also include other pharmaceutically acceptable adjuvants, such as vitamins, binders, antioxidants, etc., without specific limitations.

[0016] A third aspect of the present invention provides the application of a composition of gardenia extract and oxoglutaric acid in the field of hair loss prevention and hair growth.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] This invention provides a composition that, through the action of gardenia extract and oxoglutaric acid, can influence the expression and inhibition of VEGF, TGF-β, and Wnt / β-catenin factors in dermal papilla cells within hair follicle tissue. This achieves a balanced promotion of dermal papilla cell differentiation and proliferation, promotes hair follicle proliferation and growth, and reduces hair follicle atrophy or closure, thereby effectively preventing hair loss and promoting hair growth. Furthermore, the composition is stable and suitable for subsequent practical use. In addition, this invention's composition is safe and non-irritating, and can be used in various hair loss prevention and treatment formulations, achieving similar or even better effects than current drugs such as minoxidil with a smaller dosage. Attached Figure Description

[0019] Figure 1 The effects of different concentrations of the composition and formulation on the proliferation of DPCs.

[0020] Figure 2 The effects of different concentrations of the composition and formulation on VEGF.

[0021] Figure 3 The effects of different concentrations of the composition and formulation on TGF-β.

[0022] Figure 4 The effect of different concentrations of the composition and formulation on Wnt / β-catenin.

[0023] Figure 5 These are photos from a mouse experiment.

[0024] Figure 6 This is a diagram showing the changes in mouse hair follicle tissue as determined by HE staining. Detailed Implementation

[0025] Example

[0026] In the examples, the gardenia extract was a commercially available product purchased from Dassef Biotechnology (including 51.26 wt% gardenia fruit extract and 48.74 wt% dextrin).

[0027] Examples 1-4 provide a composition and formulation for determining the proliferation rate of human dermal papilla cells and the proliferation rate of human dermal papilla cells. mRNA of VEGF, TGF-β, and Wnt / β-catenin factors

[0028] Examples 1-4 provide a composition comprising gardenia extract and oxoglutaric acid, in the weight ratios shown in Table 1.

[0029] Table 1

[0030]

[0031]

[0032] Examples 1-4 also provide formulations comprising the composition, wherein the raw materials for the preparation of the formulation are shown in Table 2 by weight percentage.

[0033] Table 2

[0034] 1 0.008 0.01 margin 2 0.016 0.01 margin 3 0.032 0.01 margin 4 0.064 0.01 margin

[0035] 1. Determination of human dermal papilla cell proliferation rate using the CCK-8 assay

[0036] Cell growth was detected using a colorimetric method according to CN111437299A. Cell proliferation was measured using a cell counting kit 8 (CCK-8) (Sigma, St. Louis, MO, USA). Specifically, 10 cells were seeded in each well of a 96-well microplate. 4Personal dermal papillary cells (purchased from Cell Applications, USA) were incubated for 24 h, then cultured for 24 h in 100 μL of serum-free Dulbecco modified Eagle medium (DMEM, Sigma). The formulations from Examples 1-4 were then added, with serum-free DMEM and polyethylene glycol octylphenyl ether (Sigma, St. Louis, Missouri, USA) medium used as negative (NC) and blank controls, respectively. 10 μM minoxidil was used as the in vitro positive control (PC). After 24 h of treatment, 10 μL of LCK-8 solution was added to each well, and the cells were incubated at 37°C for 3 h. Absorbance was measured at 450 nm (test wavelength) and 650 nm (reference wavelength) using a microplate spectrophotometer (Epoch, VT, USA). The measured absorbance was used to determine cell proliferation using the following equation:

[0037] Cell proliferation (%) =

[0038] [(ABSsample-ABSblank)450nm-(ABSsample-ABSblank)650nm] /

[0039] [(ABScontrol-ABSblank)450nm-(ABScontrol-ABSblank)650nm], such as Figure 1 As shown, it was found that the proliferation rate increased with increasing dosage of gardenia extract. Furthermore, when using lower concentrations of gardenia extract and oxoglutaric acid preparation, the proliferation rate was not significantly different from the positive control group's 127.3%, indicating that gardenia extract and oxoglutaric acid preparation can effectively promote hair growth through synergistic effects. The variance of each of the five replicates was calculated, and the ratio of the mean variance to the mean was less than 5%.

[0040] 2. RT-PCR quantitative detection of mRNA of VEGF, TGF-β, and Wnt / β-catenin factors in human dermal papilla cells

[0041] Human dermal papilla cells were divided into 10 5 Cells were seeded at a density of 10 cells / well in 6-well tissue culture plates and cultured overnight. Cells were treated with the formulations and minoxidil solution provided in different examples, with a blank control included. cDNA was synthesized from 40 nanograms of total RNA using a two-step RT-PCR kit. The density of RT-PCR products was measured using a gel recording and system analysis machine to obtain data on the mRNA of VEGF, TGF-β, and Wnt / β-catenin factors. The results are shown in the figures below. Figure 2 , 3 4. It was found that as the concentration of gardenia extract increased, it could promote the expression of Wnt / β-catenin and VEGF, while inhibiting the expression of TGF-β. Overall, it was beneficial to the activity of dermal papilla cells and promoted hair growth.

[0042] Example 5 provides a composition and formulation, and measures the changes in mouse hair follicle tissue through mouse experiments.

[0043] Considering that the effect of the drug on cells is better than that of actual application to humans or animals, Example 5 provides a higher dosage composition and formulation to observe the performance of the present invention and minoxidil in actual application. The composition includes gardenia extract and oxoglutaric acid in a weight ratio of 100:1. The formulation, by weight percentage, includes 1% gardenia extract, 0.01% oxoglutaric acid, and the balance being water.

[0044] like Figure 5 As shown, the formulation provided in Example 5 ( Figure 5 (Right), 2.0% minoxidil aqueous solution ( Figure 5 85% alcohol (medium) Figure 5 (Left) These were used as the experimental group, positive control group, and negative control group, respectively. The solution was sprayed onto the treatment site of the mice at a rate of 200 μL per day for 35 days. Changes in mouse hair follicle tissue were measured and compared using HE staining.

[0045] (1) Sample collection: Cut off the back skin of the mouse administration site, rinse it multiple times in PBS, trim it into a rectangular shape, and quickly fix it in 4% paraformaldehyde for 18 hours;

[0046] (2) Gradient dehydration: 50% ethanol for 2h → 75% ethanol for 2h → 85% ethanol for 2h → 95% ethanol for 2h → 100% ethanol for 1h → 100% ethanol for 1h → xylene for 15min → xylene for 15min.

[0047] (3) Embedding: The tissue was immersed in paraffin overnight and then embedded the next day.

[0048] (4) Sectioning: 5μm sections were baked at 60℃ for 3.5h;

[0049] (5) Gradient dehydration: First, xylene is applied for 15 min → xylene is replaced and applied for 15 min → 100% ethanol is applied for 3 min → 95% ethanol is applied for 5 min → 85% ethanol is applied for 3 min → 75% ethanol is applied for 5 min → 50% ethanol is applied for dehydration for 4 min → then ddH2O is used for washing for 3 min.

[0050] (6) Staining: Stain with hematoxylin for 1-2 minutes, then rinse with water. The water flow should be small and not too strong until the slice turns blue, then rinse the other side.

[0051] (7) Color separation: Use hydrochloric acid ethanol solution for 3-5 seconds to separate the color (multiple lifting and inserting) until the slice turns red and the red is relatively light. The purpose of this step is to remove the staining of the cytoplasm and deepen the staining of the cell nucleus.

[0052] (8) Blueing: Soak in water for 15 minutes;

[0053] (9) Counterstain: Shake off excess water, then apply eosin solution for 2 minutes;

[0054] (10) Dehydration: Dehydrate with ethanol of different purities. The steps are: 50% ethanol for 3 min, 75% ethanol for 3 min, 85% ethanol for 4 min, 95% ethanol for 3 min, 100% ethanol for 5 min, 100% ethanol for 5 min, remove excess ethanol with filter paper, and then dehydrate with xylene for 4 min. Repeat once.

[0055] (11) Mounting: Take the slide directly from the xylene, wipe the xylene around the tissue clean, quickly drop a drop of neutral resin onto the tissue, clean the coverslip, heat it in alcohol for 3-5 seconds, and gently cover the other side onto the resin, ensuring that there are no air bubbles during this process.

[0056] (12) Observe the slides under a microscope and take photos for recording, such as Figure 6 As shown, a blank control group can be found ( Figure 6 Left) Hair follicles are small, while the positive control group ( Figure 6 (in Chinese) and the composition provided by the present invention (in Chinese) Figure 6 (Right) The hair follicles are thick and robust, which can promote hair growth similar to the positive control group.

Claims

1. A hair loss prevention and hair growth preparation, characterized in that, Including gardenia extract and oxoglutaric acid; The gardenia extract is a gardenia fruit extract; The gardenia fruit extract accounts for 0.5-2.1 wt% of the preparation; The oxoglutaric acid constitutes 0.005-0.01 wt% of the formulation; The weight ratio of the gardenia fruit extract to oxoglutaric acid is (50-210):

1.

2. The hair loss prevention and hair growth preparation according to claim 1, characterized in that, The preparation is at least one of powder, tablet, granule, liquid preparation, and capsule.

Images