Radix Aucklandiae extract, preparation method thereof and application of the extract in preventing hair loss

Abstract

The present application provides a costus root extract, a preparation method thereof and an application thereof in preventing hair loss. The costus root extract contains costunolide and dehydrocostus lactone, and is obtained by supercritical fluid extraction with costus as raw material. The costus root extract can inhibit the opening of TRPV3 channel, reduce Ca 2+ inflow, promote hair growth and ORS cell proliferation, and achieve the effect of preventing hair loss.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic application development, specifically relating to a root extract of Costus Root, its preparation method, and its application in preventing hair loss. Background Technology

[0002] With increasing social pressure, a faster pace of life, environmental degradation, and unhealthy dietary habits, the prevalence of unhealthy and sub-health conditions is rising, leading to a surge in hair loss sufferers. Patent CN 111329910 A, titled "Anti-Hair Loss and Hair Strengthening Care Essence and Its Production Process," describes a care essence with anti-hair loss and hair strengthening effects, containing ingredients such as costus root, safflower, purslane, dictamnus root bark, ursolic acid, oleanolic acid, and seaweed extract. This patented product primarily stimulates vellus hair growth in mice by inhibiting 5α-reductase activity. Patent CN 102727855B, titled "A Traditional Chinese Medicine Composition for Treating Hair Loss and Gray Hair," describes a traditional Chinese medicine composition consisting of kudzu root, scutellaria root, honeysuckle, burnet root, and costus root, which is effective in treating hair loss and gray hair. Currently, patents related to the treatment of hair loss using plant or traditional Chinese medicine compositions generally lack the mechanisms and targets of hair loss, making it impossible to provide scientific data to support their claimed hair loss effects. Furthermore, there are few commercially available products that can ultimately alleviate or treat hair loss problems.

[0003] Therefore, there is an urgent need in this field to develop a product that has good therapeutic effects and fundamentally solves the problem of hair loss from the perspective of its mechanism. Summary of the Invention

[0004] The present invention aims to provide an extract for preventing hair loss, specifically relating to a root extract of Costus root, its preparation method, and its application in preventing hair loss.

[0005] In a first aspect of the invention, a use is provided for a costus root extract for preparing a product that inhibits the opening of TRPV3 channels, wherein the costus root extract contains costunolide and dehydrocostunolide, and the costus root extract is obtained by supercritical fluid extraction using costus root as raw material.

[0006] In another preferred embodiment, the content of costus lactone in the costus root extract is 10%-20%, more preferably 11%-18%, based on the total mass of the costus root extract.

[0007] In another preferred embodiment, the content of dehydrocostane lactone in the costus root extract is 10%-20%, more preferably 11%-18%, based on the total mass of the costus root extract.

[0008] In another preferred embodiment, the costus root extract has the effect of inhibiting the opening of TRPV3 channels.

[0009] In another preferred embodiment, the TRPV3 channel is a 2-APB activated TRPV channel.

[0010] In another preferred embodiment, the inhibition rate of TRPV3 channels in the costus root extract is 1%-30%.

[0011] In another preferred embodiment, the root extract of Saussurea costus has an inhibition rate of 4.5%-30% on TRPV3 channels, more preferably 20%-28%, and even more preferably 23.11±3.66%.

[0012] In another preferred embodiment, the costus root extract has the effect of promoting hair growth and ORS cell proliferation.

[0013] In a second aspect of the present invention, a method for preparing the root extract of *Saussurea costus* as described in the first aspect is provided, the method comprising the steps of:

[0014] (S1) provides costus root raw materials; and

[0015] (S2) The raw material of Saussurea costus is subjected to supercritical extraction to obtain Saussurea costus root extract.

[0016] In another preferred embodiment, the method further includes (S3) diluting the Saussurea costus root extract.

[0017] In another preferred embodiment, the raw material of *Saussurea costus* is *Saussurea costus* root.

[0018] In another preferred embodiment, the costus root raw material is either pulverized or uncrushed.

[0019] In another preferred embodiment, the extraction temperature of the supercritical extraction in step (S2) is 40-60°C, more preferably 45-55°C.

[0020] In another preferred embodiment, the supercritical CO2 flow rate in step (S2) is 50-70 kg / h.

[0021] In another preferred embodiment, the supercritical extraction time in step (S2) is 2-3 hours.

[0022] In another preferred embodiment, the extraction pressure of the supercritical extraction in step (S2) is 10-30 MPa, more preferably 12-25 MPa, and even more preferably 13-20 MPa.

[0023] In another preferred embodiment, the separation pressure of the supercritical extraction in step (S2) is 7-8 MPa.

[0024] In another preferred embodiment, the separation pressure of the supercritical extraction in step (S2) is 5.5 to 6 MPa.

[0025] In another preferred embodiment, the separation temperature of the supercritical extraction described in step (S2) is 40-50°C.

[0026] In another preferred embodiment, the step (S3) is further included: diluting the costus root extract to 0.0001 wt / v%-0.005 wt / v, more preferably 0.0002 wt / v%-0.004 wt / v, and even more preferably 0.0005 wt / v%-0.002 wt / v.

[0027] In another preferred embodiment, the dilution described in step (S3) is to dilute the Saussurea costus root extract to a concentration of 0.005 wt / v%.

[0028] In another preferred embodiment, the dilution in step (S3) is to dilute the Saussurea costus root extract to a concentration of 0.0001 wt / v%.

[0029] In another preferred embodiment, the product may also include: lotion, serum, essence, shampoo, conditioner, etc.

[0030] In another preferred embodiment, the product has the function of inhibiting the opening of the TRPV3 channel.

[0031] In another preferred embodiment, the concentration of the costus root extract in the product is 0.0001 wt / v%.

[0032] 0.005wt / v%.

[0033] In another preferred embodiment, the TRPV3 channel is a 2-APB activated channel.

[0034] In another preferred embodiment, the inhibition rate of TRPV3 channels in the costus root extract is 1%-30%.

[0035] In another preferred embodiment, the product has the effect of promoting hair growth and ORS cell proliferation.

[0036] In another preferred embodiment, the product is a daily chemical product.

[0037] In another preferred embodiment, the daily chemical product also contains additional ingredients that can be used in daily chemical products.

[0038] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0039] Figure 1 The diagram shows the relationship between the TRPV3 pathway and ORS cells.

[0040] Figure 2 The high-performance liquid chromatogram of the root extract of Aucklandia lappa is shown. The target components of the root extract were detected using costenolide and dehydrocostenolide as standards. Figure 2 A is the chromatogram of the standard. Figure 2 B is the chromatogram of the root extract of Costus root (E1). Figure 2 C is the chromatogram of the root extract of Saussurea costus (E2).

[0041] Figure 3 The study demonstrated the inhibitory effect of the root extract of Costus root on TRPV3, among which... Figure 3 A shows the inhibition rate of different concentrations of Costus root extract on TRPV3 channels. Figure 3 B shows the inhibition rate of 0.0001% concentration of Saussurea costus root extract and forsythoside B on TRPV3 channels. Detailed Implementation

[0042] Through extensive and in-depth research and numerous screenings, the inventors of this invention have unexpectedly developed, for the first time, a root extract of *Saussurea costus* that inhibits TRPV3. This *Saussurea costus* root extract contains costunolide and dehydrocostunolide, and is obtained by supercritical fluid extraction of *Saussurea costus* raw materials. Experiments show that this *Saussurea costus* root extract can promote hair growth and ORS cell proliferation by inhibiting TRPV3 channel activation, thus achieving an anti-hair loss effect. This invention was completed based on this.

[0043] Terminology Explanation

[0044] In this invention, the "Saussurea costus root extract" and "Saussurea costus root extract paste" can be used interchangeably.

[0045] Transient receptor potential channel (TRP)

[0046] Transient receptor potential channels (TRPs) are non-selective cation channels. The TRP family consists of six homologous but functionally different TRPV channels (TRPV1-TRPV6), which are divided into temperature-sensitive TRPV1-4 and calcium-sensitive TRPV5 and TRPV6. Among them, TRPV3 is mainly distributed in keratinocytes of the skin and can respond to non-noxious thermal stimuli (≥33℃), exhibiting unique sensitization properties.

[0047] TRPV3 is expressed in many tissues and organs, such as the brain, tongue, cornea, and cardiomyocytes. TRPV3 is highly expressed in perifollicular cells; activation of TRPV3 leads to excessive calcium increase within ORS cells, which in turn activates an unknown calcium... 2+ Dependent kinases lead to the death of ORS cells. Since ORS cells are essential for maintaining the integrity of hair follicle structure and regulating hair growth, their death inhibits hair axis growth, reduces the number and size of hair follicles, and delays the hair growth cycle, thereby inhibiting hair growth or causing hair loss.

[0048] Studies have shown that carvacrol and 2-APB can act as activators of TRPV3 channels, with 2-APB being the most widely used TRPV3 activator. TRPV3 activation induces ORS cell death in a dose-dependent manner, primarily by inhibiting hair growth through suppression of hair axis elongation and reduction of hair follicles. Forsythoside B, as a TRPV3 inhibitor, can inhibit the current induced by carvacrol-activated TRPV3 channels in ORS cells. The specific inhibition of TRPV3 by natural forsythoside B reverses the carvacrol-induced inhibition of hair growth and ORS cell apoptosis.

[0049] like Figure 1 This diagram illustrates the relationship between TRPV3 and hair growth. 2-APB is a TRPV3 channel activator, while forsythoside B is a TRPV3 channel inhibitor. Activation of the TRPV3 channel leads to ORS cell death and hair growth inhibition. In the diagram, ARD (Ankyrin Repeat Domain) represents the ankyrin repeat domain, which is the binding site for calcium ions and calmodulin.

[0050] TRPV3 channels in hair follicles play an important regulatory role in hair growth, and inhibition of TRPV3 channels may be a promising treatment strategy for preventing or treating hair follicle-related alopecia or skin diseases.

[0051] Yunmuxiang

[0052] Yunnan costus root (Saussurea costus (Falc.) Lipech.) is the dried root of a perennial herbaceous plant in the genus Saussurea. Its main production area in China is northwestern Yunnan, with some cultivation in Sichuan, Hubei, and Guizhou. The Yunnan-grown roots are considered the best quality and are also one of Yunnan's authentic medicinal herbs, hence the name "Yunnan costus root." Yunnan costus root is primarily composed of sesquiterpenes and possesses various biological activities, including relieving smooth muscle spasms, lowering blood pressure, promoting qi circulation and relieving pain, and strengthening the spleen and aiding digestion. Currently, it is commonly used in clinical medicine, as a fragrance ingredient, and in cosmetics.

[0053] Costus root extract

[0054] The costus root extract of this invention uses costus root as raw material and employs supercritical fluid extraction to extract costunolide and dehydrocostunolide to obtain a costus root extract paste. The prepared costus root extract paste was used to test its efficacy in inhibiting TRPV3.

[0055] The terms "Saussurea costus root extract" and "Saussurea costus root extract paste" used in this invention are interchangeable.

[0056] Preparation method of Saussurea costus root extract

[0057] The preparation method of the root extract of Costus root of the present invention includes the following steps:

[0058] (S1) Provides raw materials for costus root;

[0059] (S2) The raw material of Saussurea costus is subjected to supercritical extraction to obtain Saussurea costus root extract.

[0060] In this invention, the method further includes (S3) diluting the extract of Costus root.

[0061] Preferably, the raw material of *Saussurea costus* in this invention is *Saussurea costus* root.

[0062] In this invention, the raw material of *Saussurea costus* is either pulverized or uncrushed.

[0063] Preferably, the dilution in step (S3) is to dilute the Saussurea costus root extract to a concentration of 0.005 wt / v%.

[0064] Preferably, the dilution in step (S3) is to dilute the Saussurea costus root extract to a concentration of 0.0001 wt / v%.

[0065] In this invention, the root extract of Costus root has the effect of inhibiting the opening of TRPV3 channels.

[0066] Preferably, the TRPV3 channel is a 2-APB activated TRPV channel.

[0067] Preferably, the inhibition rate of TRPV3 channels in the Costus root extract is 1%-30%.

[0068] Uses of Costus Root Extract

[0069] The root extract of Costus root of the present invention can reduce calcium by inhibiting the opening of TRPV3 channels. 2+ It promotes hair growth and the proliferation of outer root sheath (ORS) cells in the hair follicle, thus achieving the effect of preventing hair loss.

[0070] The root extract of *Saussurea costus* of the present invention can be used to prepare products containing *Saussurea costus* root extract.

[0071] In this invention, the products also include: lotions, serums, essences, shampoos, conditioners, etc.

[0072] In another preferred embodiment, the concentration of the costus root extract in the product is 0.0001 wt / v% to 0.005 wt / v.

[0073] In this invention, the product has the function of inhibiting the opening of the TRPV3 channel.

[0074] Preferably, the TRPV3 channel is a 2-APB activated channel.

[0075] Preferably, the inhibition rate of TRPV3 channels in the Costus root extract is 1%-30%.

[0076] In this invention, the product promotes hair growth and ORS cell proliferation.

[0077] In this invention, the product is a daily chemical product.

[0078] In this invention, the daily chemical product also contains additional ingredients that can be used in daily chemical products.

[0079] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0080] (1) The content of costus lactone in the root extract of the present invention is 10%-20% and the content of dehydrocostus lactone is 10%-20%, based on the total mass of the root extract of the present invention.

[0081] (2) The root extract of the present invention can promote hair growth and ORS cell proliferation by inhibiting the opening of TRPV3 channel, thereby achieving the effect of preventing hair loss.

[0082] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.

[0083] Example 1: Preparation and content determination of Costus root extract

[0084] In this experiment, costus lactone and dehydrocostus lactone were extracted from the root of *Saussurea costus* to obtain *Saussurea costus* root extract. The extraction process is as follows:

[0085] 1.1 Supercritical fluid extraction of costus lactone and dehydrocostus lactone from costus root

[0086] According to the conditions in Table 1, the supercritical CO2 flow rate was 50-70 kg / h. Supercritical extraction was performed on costane lactone and dehydrocostane lactone in Aquilaria sinensis. Different extraction pressures were used for extraction. The Aquilaria sinensis root extract obtained at an extraction pressure of 15 MPa was designated as E1, and the Aquilaria sinensis root extract obtained at an extraction pressure of 25 MPa was designated as E2.

[0087] Table 1 Supercritical Extraction Conditions

[0088]

[0089] 1.2 Determination of the content of costus lactone and dehydrocostus lactone

[0090] Using octadecylsilane-bonded silica gel as the filler and methanol-water (65:35) as the mobile phase, the detection wavelength was 225 nm. The theoretical plate number, calculated based on the costone lactone peak, should be no less than 3000.

[0091] Preparation of standard solutions of costunolide and dehydrocostunolide: Take appropriate amounts of costunolide and dehydrocostunolide reference standards, accurately weigh them, and add methanol to prepare a mixed solution containing 0.05-0.5 mg of each per 1 mL.

[0092] Sample solution preparation: Take an appropriate amount of the root extract of Saussurea costus, accurately weigh it, dilute the extract with methanol, shake well, filter, and collect the filtrate to obtain the sample solution.

[0093] Table 2. Results of determination of content of costus lactone and dehydrocostus lactone

[0094] sample Costus lactone Dehydroauric acid lactone Extraction of the extract at 15 MPa (E1) 14.28％ 14.77％ Extraction of the extract at 25 MPa (E2) 11.43％ 11.52％

[0095] As shown in Table 2, in the *Saussurea costus* root extract obtained using supercritical fluid extraction, the average content of costane lactones in the extract obtained at 15 MPa extraction pressure was 14.28% (calculated as the mass percentage of the target component in the total mass of the extract), the average content of dehydrocostane lactones was 14.77%, and the average content of total lactones was 29.05%; the average content of costane lactones in the extract obtained at 25 MPa extraction pressure was 11.43%, the average content of dehydrocostane lactones was 11.52%, and the average content of total lactones was 22.95%. Therefore, the *Saussurea costus* extract obtained at 15 MPa extraction pressure (E1) was selected for subsequent anti-hair loss efficacy testing. The liquid chromatography chromatograms of the standard, *Saussurea costus* root extract (E1), and *Saussurea costus* root extract (E2) are shown in [Table 2]. Figure 2 .

[0096] Example 2: Test on the anti-hair loss efficacy of Costus root extract

[0097] In this experiment, the root extract E1 of *Saussurea costus* prepared in Example 1 was used. E1 was diluted for anti-hair loss efficacy testing. The diluted concentrations were 0.005 wt / v% and 0.0001 wt / v, respectively. The sample diluent used was a 0.1-0.5% DMSO aqueous solution. The testing procedure is as follows:

[0098] 2.1 Inhibition experiment of Costus Root Extract on TRPV3

[0099] 1) Cell Culture: HEK-293 cells were cultured in DMED medium containing 10% fetal bovine serum. When the cell density was ≥70%, the medium was discarded, 5 mL of PBS was added to wash the cells, and then 4 mL of DMEM basal medium was added again. The cells were resuspended by pipetting and transferred to 2 mL centrifuge tubes. The cells were centrifuged at 1000 rpm for 4 minutes, and the supernatant was discarded. 1 mL of complete medium was added again, and the cells were mixed by pipetting. The cells were then diluted appropriately and counted. Based on the count results, the cells were seeded into 6-well plates, and the cell density per well was adjusted to 5 × 10⁶ cells / well. 5 Cells / mL, returned to the incubator for incubation (37℃, 5% CO2).

[0100] 2) After 24 hours of cell culture, transfect cells using Lipofectamine 3000 transfection reagent at a ratio of 1 μg plasmid to 2 μL. The total amount of plasmid used per well is 3 μg. Take two sterile centrifuge tubes and add 100 μL of Opti-MEM to each. Add 6 μL of Lipofectamine 3000 to one tube and mix well; add 3 μg of plasmid to the other tube, mix well, then add 6 μL of P3000 and mix well. Then add the diluted plasmid DNA to the diluted Lipofectamine 3000 and incubate at room temperature for 10-15 min. Drop the DNA-liposome complex into the cells, gently shake to mix, and incubate in an incubator. Change the medium after 4-6 hours and return the cells to the incubator (37℃, 5% CO2).

[0101] 3) After 24 hours, digest the cells and seed them into 24-well plates pre-positioned with coverslips, 8 × 10⁸ cells per well. 3 Each cell was placed back into the incubator for incubation (37°C, 5% CO2).

[0102] 4) Patch-clamp assay: After whole-cell sealing, the cell membrane voltage is clamped at -80mV. The membrane potential is first recorded at 0mV, then the voltage is commanded to depolarize from -100mV to 100mV within 100ms using a ramp function, and finally returned to 0mV. Data is collected every 5s to observe the inhibitory effect of the drug on this current peak. Experimental data are acquired by an EPC 10 amplifier (HEKA) and stored in PatchMaster (HEKA) software. A capillary glass tube is drawn into a recording electrode using a microelectrode drawing device. The electrode, filled with intracellular fluid, is placed into a microelectrode holder. Under an inverted microscope, the microelectrode manipulator is manipulated to immerse the electrode in the extracellular fluid, and the electrode resistance (Rpip) is recorded. The electrode is brought into contact with the cell surface, and negative pressure is applied to aspirate and form a high-resistance seal (GΩ). Fast capacitance compensation is then performed, and negative pressure is continued to rupture the cell membrane, forming a whole-cell recording mode. Slow capacitance compensation was then performed, and experimental parameters such as membrane capacitance (Cm) and series resistance (Rs) were recorded. No leakage compensation was applied. Once the TRPV3 current recorded in whole cells stabilized, drug administration began. Each drug concentration was administered for 5 minutes (or until the current stabilized) before moving to the next concentration. Multiple concentrations were measured for each test compound, and the experimental groups are shown in Table 3. A coverslip containing cells was placed in a recording bath under an inverted microscope. Blank control solution and working solution of the test compound were perfused sequentially from low to high concentration through the recording bath using gravity perfusion, acting on the cells. A peristaltic pump was used for fluid exchange during recording. The current detected in the solution without the compound served as the control group for each cell. Each concentration was measured twice independently using at least two cells. All electrophysiological experiments were performed at room temperature.

[0103] 5) Inhibition rate calculation: The peak current intensity I (nA) of each sample is detected, and the inhibition rate is calculated according to the following formula. The magnitude of the inhibition rate indicates the inhibition effect of the sample on the 2-APB activated TRPV3 channel.

[0104]

[0105] Table 3 Experimental Groups

[0106]

[0107] 2.2 Inhibitory effect of Costus root extract on TRPV3

[0108] Table 4. Inhibitory effect of Costus root extract on TRPV3

[0109]

[0110] As shown in Table 4 and Figure 3 As shown, forsythoside B was used as a positive control to detect the inhibition rate of Saussurea costus root extract on 2-APB-activated TRPV3 channels. Figure 3 As shown in Figure A, the root extract of *Saussurea costus* exhibits inhibitory activity against TRPV3. At a concentration of 0.005%, the inhibition rate against 2-APB-activated TRPV3 channels was 23.11 ± 3.66%, and at a concentration of 0.0001%, the inhibition rate was 6.56 ± 1.62%. Figure 3 As shown in Figure B, at a concentration of 0.0001%, the inhibition rate of the root extract of Aucklandia lappa on the 2-APB-activated TRPV3 channel was more than twice that of forsythoside B. This suggests that the root extract of Aucklandia lappa has a significant inhibitory effect on the 2-APB-activated TRPV3 channel, thus achieving the effect of preventing hair loss.

[0111] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A use of a root extract of Aucklandia lappa, characterized in that, The product is used to prepare a product that inhibits the opening of TRPV3 channels. The root extract of Costus root contains costunolide and dehydrocostunolide. The root extract of Costus root is obtained by supercritical fluid extraction using Costus root as raw material. The product is used to prevent hair loss. The costus root extract mentioned above is prepared by the following method: (S1) provides costus root raw materials; and (S2) The raw material of Costus Root is subjected to supercritical extraction to obtain Costus Root Extract; In the supercritical extraction described in step (S2), the flow rate of supercritical CO2 is 50-70 kg / h; The extraction pressure of the supercritical extraction described in step (S2) is 10-30 MPa.

2. The use as described in claim 1, characterized in that, The content of costus lactone in the root extract of *Saussurea costus* is 10%-20%, based on the total mass of the root extract.

3. The use as described in claim 1, characterized in that, The content of dehydrocostane lactone in the root extract of *Saussurea costus* is 10%-20%, based on the total mass of the root extract.

4. The use as described in claim 1, characterized in that, The TRPV3 channel is a 2-APB activated TRPV channel.

5. The use as described in claim 1, characterized in that, The content of dehydrocostane lactone in the root extract of *Saussurea costus* is 11%-18%.

6. The use as described in claim 1, characterized in that, The supercritical extraction temperature described in (S2) is 40-60℃.

7. The use as described in claim 1, characterized in that, In the supercritical extraction described in step (S2), the flow rate of supercritical CO2 is 50-70 kg / h.

8. The use as described in claim 1, characterized in that, The extraction time for supercritical extraction described in step (S2) is 2-3 hours.

9. The use as described in claim 1, characterized in that, The extraction pressure of the supercritical extraction described in step (S2) is 10-30 MPa.

10. The use as described in claim 1, characterized in that, The method further includes step (S3): diluting the costus root extract to 0.0001 wt / v% - 0.005 wt / v%.

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