CN111700899A

CN111700899A - Application of safflower yellow in preparation of II-type 5 alpha-reductase inhibitor

Info

Publication number: CN111700899A
Application number: CN202010389591.1A
Authority: CN
Inventors: 沈志滨; 胡锡昌; 张衍湖; 邓榕榕
Original assignee: Guangdong Huaxia Youmei Biotechnology Co ltd
Current assignee: Guangdong Huaxia Youmei Biotechnology Co ltd
Priority date: 2020-05-08
Filing date: 2020-05-08
Publication date: 2020-09-25

Abstract

The invention discloses application of safflower yellow in preparing a II-type 5 alpha-reductase inhibitor, and meanwhile, the safflower yellow can also be applied to preparing a medicament for treating androgen-dependent diseases, wherein the androgen-dependent diseases comprise prostatic hyperplasia, hirsutism, androgen-induced alopecia and acne. The safflower yellow has stronger II-type 5 alpha-reductase inhibition activity, and the minimum effective inhibition concentration in vitro can reach 69 mu M; among a plurality of traditional Chinese medicine active ingredients, the safflower yellow has better docking effect with II type 5 alpha-reductase protein, and the effect is higher than that of the currently common II type 5 alpha-reductase inhibitor, namely finasteride, which shows that the safflower yellow has strong binding capacity with II type 5 alpha-reductase and better II type 5 alpha-reductase inhibition activity.

Description

Application of safflower yellow in preparation of II-type 5 alpha-reductase inhibitor

Technical Field

The invention relates to the technical field of medicines, in particular to application of safflower yellow in preparing a II-type 5 alpha-reductase inhibitor.

Background

Safflower (Carthamus tinctorius L.), alias: carthamus tinctorius flower and Carthamus spinosus flower belonging to Compositae and Carthamus. Carthami flos has effects of promoting blood circulation, dredging channels, removing blood stasis, and relieving pain, and can be used for treating amenorrhea, dysmenorrhea, lochiorrhea, thoracic obstruction, cardialgia, blood stasis, abdominal pain, pricking pain in chest and hypochondrium, traumatic injury, pyocutaneous disease, and swelling and pain.

Safflower is a Chinese herbal medicine with high safety and multiple active potentials. Safflower contains many active chemical components including flavonoids, lignans, alkaloids, linoleic acids, etc. The safflower yellow is a flavonoid compound in traditional Chinese medicine safflower, and has the effects of anticoagulation, antithrombotic, antioxidation, immunoregulation, tumor resistance and the like (Liu Shi Jun, Tang Shi Shu, Cuichui, and the like, research on chemical components of traditional Chinese medicine safflower is advanced [ J ]. Henan traditional Chinese medicine, 2017 (1)). The chemical structural formula of the carthamus tinctorius yellow colour is as follows:

5 alpha-reductase (5 alpha-reductase, 5 alpha R) is a microsomal, membrane protease that catalyzes the reduction of steroid hormones by means of a reducing coenzyme II, and includes both type I (5 alpha R1) and type II (5 alpha R2) isozymes. Among them, 5 α R1 is mainly distributed in androgen-independent organs in the human body, such as liver and brain; 5 α R2 is found in large amounts in androgen-dependent organs such as the hair follicle root sheath, prostate and epididymis (Inui S, Itami S. android actions on the human hairpin: perspectives [ J ]. Experimental Dermatology,2013,22(3): 168-. In human body, 5 alpha-reductase is a key enzyme for catalyzing testosterone (T) to be more active Dihydrotestosterone (DHT), and high-level DHT is easy to cause androgen-dependent diseases (Pansheng, Wangyongcheng, Chuilimin. the role of 5 alpha-reductase in related diseases [ J ] medical review, 2011,17(17): 2571-. The reduction of androgen levels by inhibition of 5 α -reductase has become an important approach to the treatment of androgen-dependent diseases such as benign prostatic hyperplasia, androgenetic alopecia and the like (Liuben, Mirabilis, Chenguli. research on 5 α -reductase as a target for natural drug screening [ J ] drug biology, 2006(06): 468-.

At present, the 5 alpha-reductase inhibitors commonly used in clinic are mainly steroid drugs such as finasteride and dutasteride, but all of them have strong adverse reactions such as erectile dysfunction, sexual dysfunction, decreased sexual libido, feminization of male breasts, etc. (Banday AH, shamemem S a, Jeelani S. step pyrazolines and pyrazoles potential5 alpha-reduction enzyme inhibitors: Synthesis and biological evaluation [ J ]. steps, 2014,92:13-19), so that it is especially necessary to search for novel inhibitors with high activity and less side effects from Chinese herbal medicines. The inhibitory effect of safflower yellow on type II 5 alpha-reductase has not been reported at present.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art. Therefore, the research shows that the safflower yellow has good inhibiting effect on the II type 5 alpha-reductase.

In a first aspect of the invention, there is provided the use of safflower yellow for the preparation of a type II 5 α -reductase inhibitor.

According to some embodiments of the invention, safflor yellow is capable of inhibiting the biological activity of type II 5 α -reductase in vitro.

In a second aspect of the invention, there is provided the use of safflower yellow in the manufacture of a product for the treatment of androgen dependent diseases.

According to some embodiments of the invention, the androgen-dependent disease comprises at least one of prostatic hyperplasia, hirsutism, androgenic alopecia and acne.

According to some embodiments of the invention, the product is in the form of a capsule, tablet, oral preparation, microcapsule, injection, suppository, spray or ointment.

In a third aspect of the present invention, there is provided the use of safflower yellow for the manufacture of a hair loss preventing and hair setting product.

Type II 5 alpha-reductase inhibitors are commonly used in the treatment of androgen-dependent diseases such as prostatic hyperplasia, hirsutism, androgenic alopecia and acne. Among them, androgenetic alopecia (also called seborrheic alopecia) is alopecia caused by androgen secretion, because a large amount of 5 alpha-reductase in hair follicles can convert male hormones to generate a large amount of metabolite dihydrotestosterone, and the hair follicles begin to atrophy and degenerate to begin to fall. At present, the treatment mode and the treatment effect of androgenetic alopecia are limited, and the requirements of people on alopecia treatment cannot be met; the invention discovers that the safflower yellow has better II-type 5 alpha-reductase inhibition, so the anti-hair loss and hair-fixing product containing the safflower yellow is prepared by taking the safflower yellow as an active ingredient for treating the androgenetic alopecia, can be widely applied to the prevention and treatment of the androgenetic alopecia and has better anti-hair loss and hair-fixing effects.

In a fourth aspect of the present invention, there is provided a type II 5 α -reductase inhibitor, the active ingredient of which comprises safflor yellow.

In a fifth aspect of the present invention, there is provided a medicament for treating androgen-dependent diseases, the active ingredient of which comprises safflower yellow.

According to some embodiments of the invention, the safflower yellow is proved to have stronger II-type 5 alpha-reductase inhibitory activity and application prospect in the treatment of related androgen-dependent diseases; according to the safety data in the embodiment of the invention, the safflor yellow is proved to have high safety and low toxicity, and meets the requirements of preparing related medicines.

In a sixth aspect of the present invention, there is provided a pharmaceutical composition for treating androgen-dependent diseases, the active ingredient of which comprises safflor yellow.

In a seventh aspect of the present invention, there is provided an anti-hair loss and hair-fixing product, wherein the active ingredient comprises safflor yellow.

According to some embodiments of the invention, the safflower yellow is present in an amount of 0.01 to 0.5% by weight.

In the present invention, the product includes a drug or a cosmetic.

In the present invention, the term "treating" includes alleviating, inhibiting or ameliorating the symptoms or conditions of a disease; inhibiting the generation of complications: ameliorating or preventing underlying metabolic syndrome; inhibiting the development of a disease or condition, such as controlling the development of a disease or condition; alleviating the disease or symptoms; regression of the disease or symptoms; alleviating a complication caused by the disease or symptom, or preventing or treating a symptom caused by the disease or symptom. As used herein, administration can result in an improvement in a disease, symptom, or condition, particularly an improvement in severity, delay in onset, slow progression, or decrease in duration of a condition. Whether fixed or temporary, sustained or intermittent, can be attributed to conditions associated with administration.

The invention has the beneficial effects that:

1. the safflower yellow has stronger II-type 5 alpha-reductase inhibition activity, and the minimum effective inhibition concentration in vitro can reach 69 mu M; among a plurality of traditional Chinese medicine active ingredients, the safflower yellow has better docking effect with II type 5 alpha-reductase protein, and the effect is higher than that of the currently common II type 5 alpha-reductase inhibitor, namely finasteride, which shows that the safflower yellow has strong binding capacity with II type 5 alpha-reductase and better II type 5 alpha-reductase inhibition activity;

2. the safflower yellow is an active ingredient derived from natural plant safflower, and has high safety, low toxicity and little side effect;

3. the prepared product containing the safflower yellow has good effects of preventing hair loss and fixing hair on the treatment of the alopecia caused by androgen.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

FIG. 1 is a pull chart of an optimum model in example 1 of the present invention;

FIG. 2 is a 3D model structure diagram in embodiment 1 of the present invention;

FIG. 3 is a diagram showing a binding pattern of safflower yellow to type II 5. alpha. -reductase in example 1 of the present invention;

FIG. 4 shows RMSD values of amino acid backbone atoms of type II 5. alpha. -reductase in example 2 of the present invention as a function of time;

FIG. 5 is a diagram showing RMSF value fluctuation of amino acid skeleton atoms of a ligand-protein complex in example 2 of the present invention;

FIG. 6 is a graph showing the results of a chicken chorioallantoic membrane (CAM) vasoreactivity assay with safflor yellow and related products of example 5 in accordance with the present invention;

FIG. 7 is a graph showing the patch test results of the safflor yellow pigment in example 5 of the present invention.

Detailed Description

In order to explain technical contents, achieved objects, and effects of the present invention in detail, the following description is made with reference to the accompanying drawings in combination with the embodiments.

Materials and instruments: LC-20A high performance liquid chromatograph (Shimadzu corporation, Japan); chromatography column (COSMOSIL 5C18-MS-II column (250mm x 2.6, 5 μm)); TGL-16 type high speed low temperature centrifuge (Hunan instruments laboratory development Co., Ltd., Hunan); model SB-1200 constant temperature water bath (shanghai alexander instruments ltd).

Safflower yellow (CAS: 36383-96-2, 98%, Chengdu Egya method Biotech Co., Ltd., lot number AF 8030707); reducing coenzyme II (NADPH, > 98%, Shanghai Michelin Biotechnology Ltd., batch No. C10526802); dithiothreitol (DTT, > 98%, Shanghai Michelin Biochemical technology Ltd., lot No. C10182317); phenylmethylsulfonyl fluoride (PMSF, > 98%, Shanghai Michelin Biochemical technology Co., Ltd., batch No. C10090911); the methanol is chromatographically pure; the rest reagents are analytically pure, and the water is deionized water.

SD rats, 9 weeks old, weight 260-: SCXK (yue) 2018-: SYXK (Yue) 2017-0125 ].

And (3) statistical data analysis: the data obtained in this example were analyzed using the software SPASS 23.0. Each group of data is expressed as 'x +/-s', single-factor analysis of variance is adopted for comparison among groups, if the homogeneity of variance is detected by an LSD method, the Dunnett T3 method is adopted for detection when the variance is not uniform, and P <0.05 indicates that the difference has statistical significance.

Example 1: II type 5 alpha-reductase inhibitor for screening traditional Chinese medicine sources based on molecular docking technology

The molecular docking technique (molecular docking method) is a virtual high-throughput screening means, mainly studies the interaction between ligand and receptor molecules, and predicts the binding mode, binding strength and binding effect of ligand receptors. The research is that the technology is adopted to screen the activity of Chinese medicine chemical components for inhibiting II type 5 alpha-reductase, then Molecular dynamics Simulation (MD) technology is carried out on compounds with better activity for activity evaluation, and finally an in vitro enzyme activity measuring system is adopted for experimental verification.

1. Establishment of small molecule database

The method comprises the following steps of carrying out II-type 5 alpha-reductase inhibitory activity screening research on 20 traditional Chinese medicines in earlier work in a laboratory, and displaying that cortex dictamni, radix aucklandiae, radix ophiopogonis, safflower, radix sophorae flavescentis, garden balsam stem and ginseng have certain inhibitory activity.

The active ingredients (monomer compounds) in the selected traditional Chinese medicine comprise: carthamin yellow, hydroxycarthamin yellow, myricetin, ginsenoside, luteolin, diosmetin, matrine, costunolide, kaempferol, quercetin, methyl radix Ophiopogonis flavanone, Han baicalein, Fraxinellone, ferulic acid, radix Ophiopogonis homoisoflavonoid, pregnenolone, betulin alcohol and linoleic acid.

Homologous modeling of type II 5 alpha-reductase structures

1) The homologous modeling method comprises the following steps: since the crystal structure of the type II 5 alpha-reductase protein is unknown, the 3D crystal structure of the receptor protein is obtained by a homologous modeling method according to a reference (Wangyi, Luxuan, Caohong, and the like. Urtica compound anti-benign prostatic hyperplasia active receptor molecular docking and pharmacokinetic property research thereof [ J ]. Chinese journal of clinical pharmacology, 2019 (18)). Sequence similarity search is carried out in a Protein Data Bank (PDB) database by using a BLAST tool, according to the amino acid sequence of II type 5 alpha-reductase, Protein with highest sequence homology is selected as a template to carry out homology modeling by using MODELLER 9.23 software, and the optimization of the Protein is completed by a Protein preparation Wizard program in Schrodinger software under an OPLS-2005 force field.

2) And (3) homologous modeling results: five homology models (GA341 score: 0.122794; GA341 score: 0.257079; GA341 score: 0.113738; GA341 score: 0.125828; GA341 score: 0.110722) were generated in MODELLER using 3CMF protein as a template, and the model with the highest GA341score was selected for optimization, Ramachandranplot is shown in FIG. 1, 96.7% of residues in the model are within a reasonable range, which indicates that the constructed protein structure is reliable and reasonable, and the optimized model is shown in FIG. 2 and used for molecular docking.

3. Molecular docking

1) The experimental method comprises the following steps: the docking procedure was performed according to Maestro 10.2 in Schrodinger Glide 2015-2. Determining a docking area by using a Receptor Grid Generation program, setting Grid according to the size of a default frame, carrying out 1.0 scaling on the Van der Waals radius of protein atoms with partial atomic charges less than or equal to 0.25, adopting a Glide XP (high-precision mode Glide algorithm) docking scheme after the Grid file is set, sequentially docking ligands (monomeric compounds in a traditional Chinese medicine) into an active pocket of a Receptor protein, and fixing the conformation of the II type 5 alpha-reductase protein in the docking process, wherein the conformation of the ligands is flexible and variable.

Scoring function G-score 0.065 × vdW +0.130 × Coul + Lipo + Hbond + Metal + BuryP + RotB + Site

Wherein G-score: glide score (composite); vdW: intermolecular interaction energy scores; coul: coulomb effect energy scoring; lipo: a lipophilicity score; hbond: hydrogen bond scoring; metal: a metal coordination bond score; BuryP: hidden polar group penalties; RotB: rotational bond energy penalties; site: protein active site polar interaction scores.

2) Molecular docking scoring results:

the monomeric compounds and finasteride (positive control) in the selected traditional Chinese medicine are butted with II type 5 alpha-reductase protein, and the scoring results are shown in table 1. In Glide docking, the score G-score is negative, the absolute value of the score G-score indicates the acting force of the ligand and the receptor, and the larger the absolute value is, the stronger the binding capacity is, and the better the docking effect is. The scoring result of finasteride is-6.112 kcal/mol, and in the selected traditional Chinese medicine monomer compounds, the safflower yellow G-score is higher than finasteride, which shows that the safflower yellow has strong binding capacity with II-type 5 alpha-reductase and has potential II-type 5 alpha-reductase inhibition activity.

Table 1 results of docking scoring of Compounds with type II 5 alpha-reductase protein

3) Molecular docking pattern analysis: according to the docking scoring results, a binding pattern diagram of the safflor yellow and the protein is made, and the binding pattern of the active compound and the target and the interaction with the surrounding amino acid residues can be visually observed, and the result is shown in FIG. 3.

Carthamin yellow occupies an active pocket formed by residues Arg233, Ala117, Leu226, Ile112, Thr120, Asn122, Pro108, Glu57, Trp201, Phe61, Agr227, Pro106, Asn160, Glu197, Asn193, Phe194, Asn102, Arg105, Thr95, Phe92, etc., when interfacing with the receptor protein, and the compounds are analyzed for interfacing with the receptor protein as shown in Table 2.

TABLE 2 analysis of the docking of Compounds with type II 5 alpha-reductase protein

The binding mode of the safflower yellow and the protein is that the safflower yellow is combined with 5 amino acids by hydrogen bond interaction, and the-OH in the molecule forms the hydrogen bond interaction with the O in the amino acids Glu197 and Asn122, besides, the safflower yellow can form the hydrogen bond interaction with Glu57, Tyr91 and Arg227 by H in the molecule or O and O or H in the amino acids; in the hydrophobic interaction, safflower yellow is bound to Phe92, Phe61, Ile112, Leu226, Phe219 and Phe118 by hydrophobic interaction, respectively. In the binding process with the protein, safflower yellow is bound to the protein mainly by forming a large number of hydrogen bonds and hydrophobic interactions, and therefore, the binding to the protein is strong and its possible 5. alpha. -reductase inhibitory activity is strong.

Example 2: molecular dynamics research on II-type 5 alpha-reductase inhibition of safflower yellow

1. Molecular dynamics simulation

1) The experimental method comprises the following steps: molecular Dynamics simulations were performed using GROMACS 5.1.1 (Davood A, HarcheganiAB, Khamesipor A, et al. molecular Dynamics Simulation and Docking students of Selenocyanite Derivatives as Anti-Leishmanial Agents [ J ]. combinatorial chemistry & High Throughput Screening,2016,19 (10)). The compound topology file was generated from Acpype (http:// bio2byte. be/Acpype /). The topology of the type II 5 α -reductase protein was obtained by the PDB2GMX tool, the water model was the SPC/E solvent model, and sodium ions neutralized the system charge, making the system electrically neutral. The method comprises the steps of firstly performing 1000-step energy minimization on a protein and small molecular ligand complex at the temperature of 300K, then respectively performing 100ps NVT ensemble balance and 100ps NPT ensemble balance on an optimized system, limiting the position of the system in the process, finally keeping the temperature of the whole system at 300K, performing 50ns dynamic simulation, and storing a coordinate trajectory file every 10ps, wherein the time interval is 2 fs. 150 frames of kinetic trajectory files are extracted from the stable kinetic trajectories of 30ns-50ns, and the MM/PBSA method is used for calculating the binding free energy of the system after the system is stabilized.

2) Molecular dynamics simulation results: in the research, safflower yellow is selected to carry out molecular dynamics simulation of 50ns, and the stability of the system is tested by calculating the Root Mean Square Deviation (RMSD) value of a simulation track within 50 ns. As a result, as shown in FIG. 4, after 20ns of the safflower yellow system, the RMSD values were almost unchanged and almost reached the equilibrium state; the RMSD fluctuation value of all the systems is stabilized below 0.2nm, and the simulated analysis dynamics simulation system is balanced.

The study analyzed the volatility of the II-type 5 alpha-reductase and the skeleton amino acids in the three compounds in the simulation process by calculating the root mean square fluctuation value (RMSF) of the skeleton atoms of the safflower yellow and receptor protein complex amino acids in the kinetic simulation process. The results are shown in FIG. 5: the safflower yellow-II type 5 alpha-reductase system has low fluctuation (RMSF <0.2nm) in amino acids 40-57,80-95,100-125, 140-195, and the amino acids with small RMSF fluctuation in the system are matched with key amino acids, which is probably related to the generation of stable hydrogen bond, hydrophobic interaction or pi-pi stacking interaction between the compound and the II type 5 alpha-reductase, and the compound and the amino acids around the binding pocket are mutually connected to form a stable compound. After the safflower yellow is combined with the II type 5 alpha-reductase, the key amino acid is more stable, which is beneficial to the interaction of the ligand and the receptor.

2. Binding affinity assay

The difference in binding affinity between safflor yellow and the type II 5 alpha-reductase receptor was further investigated, the binding free energy of the complex of safflor yellow and the type II 5 alpha-reductase receptor was calculated using the MM/PBSA method, and the effect of binding of safflor yellow to the type II 5 alpha-reductase receptor was analyzed. The results are shown in Table 3:

TABLE 3 binding free energy of safflor yellow to type II 5 alpha-reductase protein

From the results of Table 4, it is found that Van der Waals force and electrostatic potential energy are most favorable for the binding of safflower yellow to the type II 5. alpha. -reductase receptor, and that surface solvation is favorable for the binding, but weak in comparison with the values of Van der Waals force and electrostatic potential energy, and polar solvation is unfavorable for the system binding. During the process of combining the safflower yellow with the protein, the polar interaction in the solvent largely offsets the electrostatic potential energy, so the non-polar interaction of the system is more favorable for the combination of the ligand and the receptor, wherein Van der Waals force plays a leading role and influences the free energy of the compound combined with the II type 5 alpha-reductase protein. The smaller the value of the binding free energy, the lower the energy required for binding, and the more favorable the binding of the ligand to the protein.

Example 3: verification of 5 alpha-reductase inhibitory activity of safflower yellow

1. Type II 5 alpha-reductase inhibitory Activity test of safflower yellow

1) The experimental steps are as follows: safflor yellow was selected for bioactivity validation by reference to the reference method (Bei Zhang, Rong-weng Zhang, Xi-quan, equivalent. inhibition activities of biological Chinese herbaceous rostasterone 5. alpha. -products and effects of cancer platypladi on hair-growth testasterone-treated microorganism [ J ]. Journal of ethipharacology, 2016,177). The experimental steps are as follows: taking 10 male SD rats, fasting, keeping the water for 12h, removing the neck, killing, taking out the ventral prostate and epididymis, weighing, shearing on an ice bench, preparing 1:5 homogenate (g/mL) in a glass homogenizer by using precooled homogenate (20mmol/L phosphate buffer solution (pH5.5), 1mmol/L DTT and 1mmol/L PMSF), centrifuging, and combining supernate to obtain the II type 5 alpha-reductase crude enzyme. The enzyme reaction system is formed by adding type II 5 alpha-reductase crude enzyme and reduced coenzyme II (NADPH) by taking testosterone as a substrate, and the composition of the system is shown in a table 4:

TABLE 4 inhibitory Activity of type II 5 alpha-reductase study reaction System

In the table, "-" indicates that the item is not added.

Incubating at 37 ℃ for 1h, adding 2.5mL of dichloromethane, terminating the reaction, mixing, centrifuging for 15min (5000r/min), taking the organic layer, spin-drying, adding methanol to dissolve to a constant volume of 1mL, filtering with a 0.22-micron filter membrane, and detecting the consumption of testosterone by high performance liquid chromatography. The type II 5. alpha. -reductase activity is expressed as the amount of testosterone converted [ mu. mol/L (mg protein. multidot.60 min) ] per mg of enzyme solution in 60 min; formula for the inhibition of type II 5 α -reductase: inhibition rate ═ change in testosterone concentration in placebo-change in testosterone concentration in test drug group/change in testosterone concentration in placebo × 100%. The experiment was repeated three times and the relationship between the logarithm of the concentration and the inhibition ratio was analyzed linearly using the software SPASS 23.0.

Chromatographic conditions are as follows: chromatography column (COSMOSIL 5C18-MS-II column (4.6X 250mm, 5 μm)); the column temperature is 30 ℃; the mobile phase is methanol-water (70/30, v/v); the flow rate is 1 mL/min; the sample injection amount is 20 mu L; the detector is an ultraviolet detector and has a detection wavelength of 242 nm.

2) The experimental results are as follows:

the results of the bioactivity verification of the safflower yellow are shown in the table 5:

TABLE 5 inhibition of type II 5 α -reductase by safflor yellow (II)

n＝3)

P <0.01 compared to blank group; p < 0.001.

The inhibiting rate of safflower yellow on the activity of II type 5 α -reductase is improved along with the increase of the concentration, which shows that the inhibiting activity of safflower yellow on the enzyme presents obvious dose-effect relationship in a certain concentration range₅₀The value shows that the inhibition activity is consistent with the conclusion obtained by molecular docking and molecular dynamics simulation, which indicates that the monomer component with potential inhibition on the activity of II type 5 α -reductase can be rapidly screened out by molecular docking and molecular dynamics simulation, and the inhibition activity is shown to a certain degree in a biological activity verification experiment.

The safflower yellow mainly forms hydrogen bond and hydrophobic effect with amino acid in II type 5 alpha-reductase through-OH and-O-in molecule, and generates stronger combination effect; the molecular dynamics simulation result shows that the safflower yellow and the II-type 5 alpha-reductase can be stably combined, and the combination strength of the system is consistent with the molecular docking result; in vitro experiments, safflower yellow showed strong inhibitory activity against type II 5 α -reductase.

2. Control experiment of safflower extract type II 5 alpha-reductase Activity

1) The experimental steps are as follows:

preparation of safflower extract: weighing 20g of traditional Chinese medicine dry powder, placing in a round-bottom flask, adding deionized water for extraction for 2 times by adopting a water decoction method, wherein the material-liquid ratio is 1:10 and 1:8 respectively; the extraction time is 2h and 1.5h respectively; mixing the two filtrates, concentrating, lyophilizing to obtain lyophilized powder, and refrigerating at 4 deg.C.

Selecting 1mg/mL of the safflower extract as a sample concentration to be determined according to a pre-experimental result, and carrying out II type 5 alpha-reductase inhibitory activity test, wherein the test method is the same as the above, so as to obtain the inhibitory activity result of the safflower extract on II type 5 alpha-reductase, and the inhibitory activity result is shown in the following table 6:

TABLE 6 inhibitory Effect of safflower extract on type II 5 α -reductase: (

n＝3)

Note: p <0.05, P <0.01 compared to the blank control group.

From the control results of the above safflower extract, it was found that safflower yellow IC₅₀69.06 +/-6.35 mu mol/L, the safflower extract has about 65% inhibition rate under the concentration of 1mg/mL, therefore, the inhibition efficiency of II type 5 α -reductase using the safflower yellow is far higher than that of the safflower extract, and the safflower yellow is proved to be an important active ingredient which plays a role in inhibiting II type 5 α -reductase in the safflower.

Example 4: application of safflower yellow in preparation of related products

An anti-hair loss and hair-fixing product containing safflower yellow comprises the following components in percentage by weight as shown in the following table 7:

TABLE 7 formula of hair loss preventing and hair strengthening product containing safflor yellow

The hair care essential oil product for preventing hair loss and fixing hair is prepared according to the formula, the formula is only an accurate formula used in experiments, and the mass ratio of the safflower yellow serving as a main hair loss preventing and fixing active ingredient in the cosmetic can be 0.01-0.5%, so that the hair care essential oil product has a good effect; other components in the formula are commonly added components and auxiliary materials in the cosmetic components, and the auxiliary materials with similar functions can be used for conventional combination or replacement.

Human use experiments: the anti-hair loss and hair-fixing product prepared by the method is distributed to 20 volunteers for product use effect experiments, frequency analysis results of the product use effects are shown in table 8, and the effects of anti-hair loss, oil control and the like of the product are analyzed:

TABLE 8 frequency analysis results of the product use effects

According to the results in the table, the alopecia improving effect of the product prepared in the embodiment is as high as 85%, the product can take effect within 15 days of most of use, and the product does not generate allergy or anaphylactic reaction in the use process; therefore, the product has better anti-hair loss and hair-fixing effects and higher safety.

Example 5: safety test of safflower yellow and related products

Test drugs: safflower yellow (CAS: 36383-96-2, 98%, Chengdei method Biotech Co., Ltd.), the hair loss preventing and hair fixing product prepared in example 4; in the following experiment, safflower yellow was prepared as a 0.4mg/ml solution.

1. Chick embryo chorioallantoic membrane test

The chorioallantoic membrane (CAM) test is one of the widely used models in the alternative to the eye irritation test. The chorioallantoic membrane of chick embryo is an extracorporeal circulation system of chick embryo, has complete tissue rich in blood vessels, and can evaluate the possible irritation of chemical substances by observing the change of blood vessels (hyperemia, hemorrhage and blood coagulation) after the CAM is exposed to the chemical substances and detecting the damage of the chemical substances to the CAM by utilizing the characteristic that the CAM is similar to the conjunctiva structure.

1) Strain and source:

the fertilized chick embryo of White Lei Hangzhou chicken (White egg) should be SPF chick embryo, the supplier should have qualification of SPF chick (egg) fixed-point production enterprise for veterinary drug production and inspection, which is approved by agricultural departments, and the chick embryo quality should meet the requirements of national standard.

The chick embryo should be fresh, clean and intact, and the weight of the chick embryo is 50 g-60 g. When the eggs are hatched to 9 days old, egg examination is carried out, unfertilized, inactive or defective chick embryos are discarded, and seriously deformed, broken or thin-shelled chick embryos cannot be used.

Hatching conditions: the room temperature is 20-25 ℃, and the relative humidity is 45-70%. The incubation temperature is 37.5 +/-0.5 ℃, the relative humidity is 55-70 percent, and the turntable frequency is 3-6 times/h. The chick embryos of 9d age do not need to rotate when hatching.

2) Chick embryo allantoic membrane (HET-CAM) test procedure:

performing egg-lighting inspection on 9 d-old chick embryos, and marking the positions of air chambers on the surfaces of the eggshells; peeling off the eggshell part by using a dental serrated curved forceps knife, completely exposing a white egg membrane, dripping 1-2 mL of 0.9% NaCl solution by using a suction pipe to moisten the egg membrane, carefully removing the inner membrane by using the curved forceps, exposing a chorioallantoic membrane (CAM), and avoiding the vascular membrane from being damaged, wherein the eggshell part is not used for experiments if the eggshell part is damaged.

According to the commercial inspection standard of SNT 2329-. Taking 0.3mL of a test sample to directly act on the CAM, ensuring that at least 50% of the CAM surface is covered by the test sample, slightly washing the CAM with physiological saline after acting for 5min, observing the bleeding, angiolysis and coagulation reaction of the CAM after 30s, and respectively recording the bleeding, the angiolysis and the coagulation reaction according to light, medium and heavy degree for 1 minute, 2 minutes and 3 minutes, wherein the highest integral of each chick embryo is 9 minutes. Each test sample was tested with 6 chick embryos, and the total of the stimulus response scores (ES) of the 6 chick embryos was calculated, and the eye irritation of the test sample was evaluated in a graded manner according to the grading standards shown in table 9.

TABLE 9 evaluation of end-points method eye irritation grading Standard for test samples

3) The experimental results are as follows:

the results of the experiment are shown in table 10 below:

TABLE 10 evaluation results of eye irritation in the HET-CAM test

The experimental results show that: no bleeding, angiolysis or clotting reactions were seen in the 0.9% NaCl solution within 5 minutes after contact with the CAM and blood flow was clearly visible as shown in FIG. 6A. Bleeding and vessel fusion occurred after 1% Texpon ASV solution contacted the CAM, with response scores above 18 points, as shown in fig. 6B. After the safflower yellow group and the anti-hair loss and hair-fixing product group are contacted with the CAM, no vascular stimulation reaction occurs, as shown in figures 6C and 6D, the stimulation scores are 0 score, and the results of the stimulation grading are shown in Table 9. The safflower yellow group and the anti-hair loss and hair-fixing product group have no toxic effect when acting on chick chorioallantoic membranes, which indicates that the safflower yellow group and the anti-hair loss and hair-fixing product group have no irritation, and can be applied to medicines or cosmetics as functional external components for preventing and treating alopecia.

2. Patch test

The sensitization of the yellow-red pigment is only tested in the experiment, and the experimental concentration is 0.4 mg/ml.

Patch Test is a simple and reliable method for determining cosmetic allergens. The patch test is to artificially prepare a certain concentration of a suspected allergen, place the allergen in a special chamber, apply the allergen to a covered part of a human body (usually on the back and the flexed side of the forearm), and determine whether a test object is an allergen (i.e., a sensitizer) according to the presence or absence of a positive reaction after a certain period of time.

1) Test sites: normal skin on both sides of the upper back spine.

2) Removing the protective paper of the spot tester, and sequentially placing the prepared allergens in an aluminum spot tester. The sequence of spot test material is from top to bottom, from left to right and marked. The spot reagent dosage, 25. mu.l of the ointment preparation, was directly put into the spot tester, and 25. mu.l of the liquid preparation was dropped on the filter paper sheet put into the spot tester. The test article should be added with the spot-test article to avoid sticking to the edge of the spot-test apparatus.

3) The spot tester adhesive tape with the spot test substance is firmly and flatly attached from bottom to top and lightly pressed by a palm so as to exhaust air.

4) Patch test time: for 48 hours.

5) Observation time: 48 hours after application, the patch tester is removed first, and in order to avoid possible reactions caused by the patch tester pressing the skin, the results should be observed at least 30 minutes after the patch tester is removed.

FIG. 7 is a photograph showing the test result of the patch test, wherein the left side shows the beginning of the test and the right side shows the end of the test; the results showed that safflower yellow showed no irritation or characteristic manifestation of allergy, and it was judged that the sample was safe and non-irritant.

The test results of this example prove that the yellow-red pigment and the hair loss preventing and fixing product prepared in example 4 have the advantages of low irritation, low anaphylactic reaction and the like, and therefore, both have higher safety.

In summary, the safflower yellow has potential activity of inhibiting II type 5 alpha-reductase, can be used for preparing II type 5 alpha-reductase inhibitors, preparing medicines or cosmetics for inhibiting II type 5 alpha-reductase, is used for treating and preventing androgen-dependent diseases, has good effect, high safety and small side effect, and particularly has good effect on treating alopecia caused by androgen.

The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent changes made by using the contents of the present specification and the drawings, or applied directly or indirectly to the related technical fields, are included in the scope of the present invention.

Claims

1. Use of safflower yellow in preparing type II 5 alpha-reductase inhibitor is provided.

2. Use of safflower yellow in the preparation of a product for the treatment of androgen dependent diseases.

3. The use of claim 2, wherein the androgen-dependent disease comprises at least one of prostatic hyperplasia, hirsutism, androgenic alopecia, and acne.

4. Use according to claim 2 or 3, wherein the product is in the form of capsules, tablets, oral preparations, microcapsules, injections, suppositories, sprays or ointments.

5. Application of safflower yellow in preparing hair loss preventing and hair strengthening product is provided.

6. A type II 5 alpha-reductase inhibitor characterized in that the active ingredient thereof comprises safflor yellow.

7. A medicine for treating androgen-dependent diseases is characterized in that the active component of the medicine contains safflower yellow.

8. A pharmaceutical composition for treating androgen-dependent diseases, characterized in that its active ingredient comprises safflower yellow.

9. An anti-hair loss and hair-fixing product is characterized in that the active component of the product contains safflower yellow.

10. The product according to claim 9, wherein the safflower yellow is contained in an amount of 0.01 to 0.5% by weight.