CN111184773A

CN111184773A - Fructus Ligustri Lucidi extract and its application in preparation of 5 α -reductase inhibitor

Info

Publication number: CN111184773A
Application number: CN202010071785.7A
Authority: CN
Inventors: 沈志滨
Original assignee: Guangdong Pharmaceutical University
Current assignee: Guangdong Pharmaceutical University
Priority date: 2020-01-21
Filing date: 2020-01-21
Publication date: 2020-05-22
Anticipated expiration: 2040-01-21
Also published as: CN111184773B

Abstract

The invention discloses a glossy privet fruit extract and application thereof in preparation of a 5 α -reductase inhibitor, wherein the glossy privet fruit extract contains any one or more of salidroside, neoligustrazine, specnuezhenide, ligustrazine, oleuropein and ligustilide C, researches show that the glossy privet fruit extract and main components thereof have a remarkable inhibiting effect on 5 α -reductase and have good prevention and treatment effects on diseases related to 5 α -reductase dependent androgen action, and the glossy privet fruit extract is safe, non-irritant and free of side effects and can be used for development of related medicines.

Description

Fructus Ligustri Lucidi extract and its application in preparation of 5 α -reductase inhibitor

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a glossy privet fruit extract and application thereof in preparation of 5 α -reductase inhibitors and related disease prevention and treatment medicines.

Background

5 α -reductase is a key enzyme in androgen metabolism, and includes three isoenzymes of type I (5 α R1), type II (5 α R2) and type III (5 α R3), 5 α R1 is mainly distributed in androgen-independent organs such as liver and skin in human body, and the optimum pH value is 6-9, 5 α R2 is abundantly present in androgen-dependent organs such as hair follicle root sheath, prostate, epididymis, and the optimum pH value is 5.5, while the study on 5 α R3 is less at present, the enzyme-dependent reduced coenzyme II (NADPH) is a hydrogen donor which converts testosterone (T) irreversibly into more active Dihydrotestosterone (DHT), high levels of dihydrotestosterone cause androgen-dependent diseases such as acne, androgen-derived female alopecia, seborrheic dermatitis, hirsutism and benign prostatic hyperplasia and prostatic carcinoma, and the like, 5 α -reductase plays an important role in the above-mentioned diseases, wherein 5- α -5R 1 plays an important role in synthesizing androgen metabolism and causes benign prostatic hyperplasia and prostatic carcinoma, and the like, and the androgen metabolism causes the benign prostatic carcinoma.

For example, Benign Prostatic Hyperplasia (BPH) is a common disease of middle-aged and elderly men, and is clinically mainly manifested by symptoms of frequent micturition, urgent micturition, increased nocturia, interruption of urine and the like, and serious complications comprise repeated urinary tract infection, vesical calculus, renal failure and the like, and research has shown that II type 5 α -reductase converts T into DHT in prostatic tissue, the DHT accumulates in prostatic tissue and acts on stroma, and hyperplasia of glands is caused by indirect proliferation and differentiation of adjacent epithelial cells.

By way of further example, androgenic alopecia (AGA), the most common type of hair loss, is characterized by a reduced number of hair follicles and a gradual volume shrinkage until complete degeneration, hair follicles and sebaceous glands in human scalp, have been studied to show that 5 α -reductase activity and DHT content in the scalp of bald areas are higher than those in non-bald areas (e.g., occipital scalp) or in the same areas of non-bald areas, indicating that 5 α -reductase and dihydrotestosterone play a very important role in the process of inducing androgenic alopecia, thus inhibiting the activity of 5 α -reductase type ii has a great significance for preventing and treating androgenic alopecia, finasteride is a specific inhibitor of 5 α -reductase, is a modern drug commonly used for treating diseases related to 5 α -reductase, has a significant therapeutic effect, but also causes side effects such as erectile dysfunction, breast tenderness, testicular pain, ejaculatory abnormality, and thus, developing a novel, green, high-efficacy, reductase type ii 5 α -reductase inhibitor, which will have a promising clinical prospect.

Disclosure of Invention

The results of the invention show that the glossy privet fruit extract has stronger inhibition effect on 5 α -reductase and obvious prevention and treatment effect on the androgen-dependent 5 α -reductase, is safer and has no stimulation and side effect on human bodies, and can be used for preparing related medicaments.

The invention aims to provide a glossy privet fruit extract.

The invention also aims to provide the application of the glossy privet fruit extract in preparing the medicament for inhibiting the 5 α -reductase.

The invention also aims to provide the application of the glossy privet fruit extract in preparing medicines for treating diseases related to the androgen-dependent action of 5 α -reductase.

The above purpose of the invention is realized by the following technical scheme:

the research of the invention shows that the glossy privet fruit extract has a remarkable inhibition effect on 5 α -reductase, in particular to II type 5 α -reductase, so the invention provides the glossy privet fruit extract and the application thereof in inhibiting 5 α -reductase and further preventing and treating related diseases.

A fructus Ligustri Lucidi extract contains one or more of salidroside, neoligustrazine, specnuezhenide, ligustilide, oleuropein, and ligustilide C.

Preferably, the composition comprises salidroside, neoligustilide, specnuezhenide, ligustilide, oleuropein and ligustilide C, and the mass percentage is 10-14: 8-12: 18-22: 13-17: 15-19: 24-28;

more preferably, the mass percentage of salidroside, neoligustrin, specnuezhenide, ligustilide, oleuropein and ligustilide C is 12: 10: 20: 15: 17: 26.

the research of the invention finds that the glossy privet fruit extract has a remarkable inhibition effect on 5 α -reductase, and further the analysis on the components of the glossy privet fruit extract finds that salidroside, neoligustrazine, specnuezhenide, oleuropein and ligustrazine C in the main active components have inhibition effects of different degrees on 5 α -reductase.

The ethanol extract is 80% -99% ethanol extract, preferably the ethanol extract is 90% -95% ethanol extract

As an optional preferred scheme, the preparation method of the glossy privet fruit extract comprises the following steps: weighing glossy privet fruit powder, adding 80-99% ethanol for extraction, and concentrating to obtain the glossy privet fruit ethanol extract. More preferably, the extraction solvent is 90% to 95% ethanol, most preferably 95% ethanol.

More preferably, in the preparation process of the glossy privet fruit extract, ethanol is used for reflux extraction, and the material-liquid ratio is 1: 6-14, and the extraction time is 1-3 h.

Most preferably, the preparation method of the glossy privet fruit extract comprises the following steps: weighing dry powder of fructus Ligustri Lucidi, extracting with 95% ethanol for 2 times by reflux extraction at a ratio of 1:10 and 1:8 respectively; the extraction time is 2h and 1.5h respectively; mixing the two filtrates, and concentrating under reduced pressure to obtain 95% ethanol extract of fructus Ligustri Lucidi.

The research result shows that the glossy privet fruit extract has the same inhibition strength (81.49 +/-2.0%) to 5 α -reductase as finasteride (83.8 +/-2.02%) and has no toxic stimulation, and can be used for preparing 5 α -reductase inhibitors and medicines for preventing or treating related diseases.

Therefore, the application of the glossy privet fruit extract in preparing the medicament for inhibiting the 5 α -reductase, preferably the application in preparing the medicament for inhibiting the II type 5 α -reductase is all within the protection scope of the invention.

Meanwhile, the application of the glossy privet fruit extract in preparing the medicine for preventing and treating the 5 α -reductase-dependent androgen related diseases also belongs to the protection scope of the invention.

Moreover, a 5 α -reductase inhibitor or a medicament for preventing or treating 5 α -reductase-dependent androgen-related diseases, which comprises a prophylactically or therapeutically effective amount of the extract of fructus ligustri lucidi, shall also fall within the scope of the present invention.

Preferably, the 5 α -reductase-dependent androgen associated disease is prostatic hyperplasia, prostate cancer or androgenic alopecia.

More preferably, the 5 α -reductase-dependent androgen associated disorder is androgenic alopecia.

Wherein, preferably, the glossy privet fruit extract is any one of the glossy privet fruit extracts.

Preferably, the glossy privet fruit extract is a water extract or an ethanol extract of glossy privet fruit; preferably, the fructus ligustri lucidi extract is a dried fructus ligustri lucidi extract.

Preferably, the ethanol extract is 80% -99% ethanol extract, preferably the ethanol extract is 90% -95% ethanol extract, most preferably 95% ethanol extract.

The invention has the following beneficial effects:

the research of the invention shows that the glossy privet fruit extract and the main components thereof have obvious inhibition effect on 5 α -reductase, wherein the inhibition rate of the glossy privet fruit 95% ethanol extract on 5 α -reductase can reach 81.49 +/-2.0%, and the glossy privet fruit extract has stronger inhibition activity.

The invention not only provides a new application of the glossy privet fruit extract, but also provides a new safe and effective 5 α -reductase inhibitor, and simultaneously provides a new therapeutic medicine and a new approach for treating 5 α -reductase androgen-dependent diseases.

Drawings

FIG. 1 is a high performance liquid chromatogram of the chemical components of the glossy privet fruit extract of the present invention;

wherein, 1, salidroside; 2, new ligustilide; 3, specnuezhenide; 4, ligustilide; oleuropein 5; 6, ligustrum lucidum fruit glycoside C;

FIG. 2 is a total ion flow diagram of the fructus Ligustri Lucidi extract chemical components in negative ion mode;

FIG. 3 is a schematic diagram showing the hair growth scoring of the glossy privet fruit extract after acting on an androgenetic alopecia model C57BL/6 mouse;

FIG. 4 is a cross-sectional view of the skin of a mouse with the extract of Ligustrum lucidum of the present invention after acting on androgenetic alopecia model C57 BL/6;

wherein, (A) a normal group, (B) a model group, (C) minoxidil group, (D) a glossy privet fruit extract low-dose group, (E) a glossy privet fruit extract medium-dose group, (F) a glossy privet fruit extract high-dose group, (HE staining x 40);

FIG. 5 is a longitudinal section of the skin of a mouse with the extract of Ligustrum lucidum of the present invention acting on androgenetic alopecia model C57 BL/6;

FIG. 6 is a schematic representation of the vasoreactivity effect of the extract from Ligustrum lucidum of the present invention on chick embryo chorioallantoic membrane (CAM);

wherein (A) 0.9% NaCl solution (B) 1% Texpon ASV solution (C) fructus Ligustri Lucidi extract low dose group (D) fructus Ligustri Lucidi extract middle dose group (E) fructus Ligustri Lucidi extract high dose group; vascular response of each group of CAMs 5min after CAM exposure.

Detailed Description

The invention is further described with reference to the drawings and examples in the following description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

Example 1 preparation and composition analysis of Ligustrum lucidum extract

1. Preparation of glossy privet fruit extract

Weighing 20g of glossy privet fruit dry powder, and adding 95% ethanol for extraction for 2 times by adopting a reflux extraction 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, and concentrating under reduced pressure to obtain 95% ethanol extract of fructus Ligustri Lucidi.

2. Analysis of major ingredients of fructus Ligustri Lucidi extract

Chromatographic conditions are as follows: chromatographic column (HopuchromTM Cloud C18 column (4.6X 150mm, 5 μm), mobile phase of 0.1% formic acid in water (A), acetonitrile in water (B), gradient elution conditions of 0-10min, 10-20% B, 10-20min, 20-30% B, 20-30min, 30-35% B, 30-40min, 35-55% B, 40-45min, 55-65% B, 45-55min, 65-100% B, volume flow of 0.3 mL/min-1, sample amount of 5 μ l, column temperature of 25 ℃.

Mass spectrum conditions: electrospray ion source (ESI); detecting negative ions, wherein the scanning range m/z is 50-1500, the temperature of the drying gas is 300 ℃, the volume flow of the drying gas is 500L/hr, and the pressure of the atomizing gas is 0.24MPa (35.0 psi); the capillary voltage was 3 kV.

And (3) sample determination: dissolving 1.0g of fructus Ligustri Lucidi extract in 25.0mL of 80% methanol, and filtering with 0.22 μm filter membrane; UPLC-ESI-MS/MS analysis was performed.

The results of preliminarily determining 6 main components in the glossy privet fruit 95% ethanol extract are shown in table 1, wherein the high performance liquid chromatography of the glossy privet fruit extract is shown in figure 1, the total ion flow chromatography in the negative ion mode is shown in figure 2, and the results are shown in table 1.

TABLE 1 ethanol extract of Ligustrum lucidum

Example 2 preparation and composition analysis of Ligustrum lucidum extract

1. Preparation of glossy privet fruit extract

Weighing 20g of glossy privet fruit dry powder, and adding 95% ethanol for extraction for 2 times by adopting a reflux extraction method, wherein the material-liquid ratio is 1: 14. 1: 6; the extraction time is 3h and 1h respectively; mixing the two filtrates, and concentrating under reduced pressure to obtain 95% ethanol extract of fructus Ligustri Lucidi.

2. Analysis of major ingredients of fructus Ligustri Lucidi extract

The main components are salidroside, neoligustrazine, specnuezhenide, ligustilide, oleuropein and ligustilide C as in example 1, and the mass percentage is 10: 12: 22: 13: 15: 28.

example 3 preparation and composition analysis of Ligustrum lucidum extract

1. Preparation of glossy privet fruit extract

Weighing 20g of glossy privet fruit dry powder, and adding 95% ethanol for extraction for 2 times by adopting a reflux extraction method, wherein the material-liquid ratio is 1: 12. 1: 7; the extraction time is 2.5h and 1h respectively; (ii) a Mixing the two filtrates, and concentrating under reduced pressure to obtain 95% ethanol extract of fructus Ligustri Lucidi.

2. Analysis of major ingredients of fructus Ligustri Lucidi extract

The main components are salidroside, neoligustrazine, specnuezhenide, ligustilide, oleuropein and ligustilide C as in example 1, and the mass percentage is 14: 8: 18: 17: 19: 24.

EXAMPLE 4 assay of inhibitory Activity of Ligustrum lucidum extract on type II 5 α -reductase

1. Materials and instruments

Fructus Ligustri Lucidi (fructus Ligustri Lucidi) is purchased from Tongzhou Tongrentang pharmaceutical industry; coomassie brilliant blue G-250 (national pharmaceutical group chemical Co., Ltd.); bovine serum albumin (BSA, > 98%, Macklin); testosterone (T, > 98%, alatin corporation); finasteride (> 98%, Macklin Corp.); reducing coenzyme II (NADPH, > 98%, Macklin); dithiothreitol (DTT, > 98%, Macklin corporation); phenylmethylsulfonyl fluoride (PMSF, > 98%, Macklin Corp.); the methanol is chromatographically pure; the rest reagents are analytically pure, and the water is deionized water.

SD rats, 9 weeks old, 260-280g body weight, SPF rating, 6 males, provided by the Guangdong provincial animal center [ certification number: SCXK (yue) 2018-: SYXK (Yue) 2017-0125 ].

UV 2301-II UV spectrophotometer (Shanghai Tian scientific instruments, Inc.); LC-20A high performance liquid chromatograph (Shimadzu corporation, Japan); TGL-16 type high speed low temperature centrifuge (Hunan instrument laboratory Instrument development Co., Ltd.)

2. Preparation of extracts of Ligustrum lucidum of each experimental group

Preparing a glossy privet fruit aqueous extract: weighing 20g of glossy privet fruit dry powder, and adding deionized water for extraction for 2 times by adopting a 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 to obtain fructus Ligustri Lucidi water extract, and refrigerating at 4 deg.C;

preparing a glossy privet fruit 50% ethanol extract: weighing 20g of glossy privet fruit dry powder, and adding 50% ethanol for extraction for 2 times by adopting a reflux extraction method, wherein the material-liquid ratio is 1:10 and 1:8 respectively; the extraction time is 2h and 1.5h respectively; mixing the filtrates, concentrating under reduced pressure to obtain 50% ethanol extract of fructus Ligustri Lucidi, and refrigerating at 4 deg.C;

preparing a glossy privet fruit 75% ethanol extract: weighing 20g of glossy privet fruit dry powder, and adding 75% ethanol for extraction for 2 times by adopting a reflux extraction method, wherein the material-liquid ratio is 1:10 and 1:8 respectively; the extraction time is 2h and 1.5h respectively; mixing the filtrates, concentrating under reduced pressure to obtain 75% ethanol extract of fructus Ligustri Lucidi, and refrigerating at 4 deg.C;

preparing a glossy privet fruit 95% ethanol extract: the mixture was refrigerated at 4 ℃ for further use in the same manner as in example 1.

3. Preparation of type II 5 α -reductase

Taking 6 male SD rats, fasting, carrying out cervical dislocation after 12h without water prohibition, quickly taking out ventral prostate and epididymis, weighing, shearing tissues on an ice bench, preparing 1:5 homogenate (g/mL) in a glass homogenizer by using precooled homogenate (20mmol/L phosphate buffer (pH5.5), 1mmol/L DTT, 1mmol/L PMSF), lightly covering the homogenate on 0.34mol/L sucrose solution, centrifuging for 10min (4 ℃, 700 Xg), taking supernatant (1), adding 2mL of 0.25mol/L sucrose into a precipitate, mixing uniformly, centrifuging for 10min (4 ℃, 1000 Xg), taking supernatant (2), and combining the two supernatants to obtain supernatant (3); centrifuging the supernatant (3) for 10min (4 deg.C, 10000 Xg), collecting supernatant as crude enzyme extract, adding appropriate amount of glycerol, and storing in refrigerator at-80 deg.C for use.

4. Experimental procedure

A model for determining the in vitro activity of II type 5 α -reductase is established based on High Performance Liquid Chromatography (HPLC), testosterone (T) is used as a substrate, reduced coenzyme II (NADPH) is used as a hydrogen donor, the type II 5 α -reductase extracted from prostate and epididymis of male SD rats is used for catalyzing reaction, and the inhibition activity is characterized by measuring the change amount of the testosterone before and after the reaction system, wherein the inhibition activity is briefly described as follows, 50 mu L of glossy privet fruit ethanol extract test solution, 300 mu L of phosphate buffer solution (20mmol/L, pH5.5), 50 mu L of testosterone, 500 mu L of crude enzyme extract (PB buffer pH5.5 dilution) are sequentially added into a reaction tube, and finally 100 mu L of NADPH is added to start the reaction, the system is incubated at 37 ℃ for 60min, 2.5mL of dichloromethane is added to stop the reaction, the reaction is uniformly mixed, the mixture is centrifuged for 15min (5000r/min), an organic layer is decompressed, evaporated, methanol is added to dissolve to 1mL, and a filter membrane with 0.22 mu m is used for detecting the consumption of the testosterone.

The enzyme activity is expressed by T conversion rate, and the characterization method of the inhibition activity is shown as the formula (I):

in addition, the IC of the specific II type 5 α -reductase inhibitor finasteride is determined by using the system₅₀IC of finasteride₅₀The result is 242nmol/L, and the difference is not large compared with 237nmol/L measured by other high performance liquid chromatography systems, further illustrating the scientific effectiveness of the system, the measurement results of the inhibitory activity of finasteride on II type 5 α -reductase are shown in Table 2.

When the inhibitory activity of the glossy privet fruit extract on type II 5 α -reductase is measured, 1 mu mol/L finasteride is used as a positive control, the inhibitory rate of the glossy privet fruit ethanol extract and the finasteride is measured under the parallel operation condition, and the inhibitory rate range of the positive control group is 83.8 +/-2.02 percent, so that the measurement result is considered to be valid and credible.

The above procedure was repeated three times in parallel and the geometric mean of the inhibition rate was calculated. The results are shown in Table 3.

TABLE 2 in vitro inhibitory Activity of finasteride on type II 5 α -reductase

TABLE 3 in vitro inhibitory Activity of Ligustrum lucidum extract on type II 5 α -reductase

Note: p <0.05 compared to blank control group.

Experimental results show that when the concentration of crude drugs is 1g/L, the glossy privet fruit extract has a certain inhibition effect on II-type 5 α -reductase, wherein the inhibition rate of the glossy privet fruit 95% ethanol extract on II-type 5 α -reductase can reach 81.49 +/-2.0%, and the glossy privet fruit extract has obvious inhibition activity.

EXAMPLE 5 assay of inhibitory Activity of the major ingredient of Ligustrum lucidum extract on type II 5 α -reductase

The 6 main components measured in the glossy privet fruit extract prepared in example 1 are taken as research objects, the inhibition rate of II-type 5 α -reductase is measured, the preparation method and the inhibition activity measuring method of II-type 5 α -reductase are the same as those of example 4, the measurement is repeated three times in parallel, and the geometric mean of the inhibition rate is calculated, and the experimental results are shown in Table 4.

TABLE 4 in vitro inhibitory Activity of the major component of Ligustrum lucidum extract on type II 5 α -reductase

Note: p <0.05 compared to blank control group.

Experimental results show that the main components of the glossy privet fruit extract can obviously reduce the testosterone conversion rate and have better II-type 5 α -reductase inhibitory activity.

In the following, the mouse model of androgenetic alopecia is taken as an example to study the effect of glossy privet fruit extract in inhibiting 5 α -reductase and preventing and treating related diseases.

Example 6 Effect of Ligustrum lucidum extract on growth of mouse hair in androgen-induced alopecia model

1. Materials and instruments

Testosterone propionate (98%, Macklin corporation); oil for injection (Sigma company); 5% minoxidil tincture (xiamen mei merchant pharmaceuticals, ltd); 4% paraformaldehyde fixing solution (Biosharp); absolute ethanol (national pharmaceutical group chemical agents limited); xylene (national chemical group chemical Co., Ltd.); HE staining solution (servicebio corporation).

C57BL/6 mice, 6 weeks old, 18-25 g weight, SPF grade, 60 males, provided by the Guangdong provincial animal center [ certification No.: SCXK (yue) 2018-: SYXK (Yue) 2017-0125 ].

Model JJ-12J dehydrator (wuhan junjie electronics ltd); JB-P5 embedding machine (Wuhanjunjie electronics, Inc.); RM2016 type pathological microtome (shanghai come instrument limited); JB-L5 type jelly station (Wuhanjunjie electronics, Inc.); KD-P type tissue spreading machine (Kedi instruments, Inc., Jinhua, Zhejiang); GFL-230 type oven (Leiborui instruments, Inc., Tianjin); type BA310-T biomicroscopy (Miaodi practice group, Inc.).

2. Experimental procedure

In order to further study the effect of type II 5 α -reductase inhibitory activity of fructus Ligustri Lucidi extract on the growth of mouse hair in androgen-induced alopecia model, testosterone propionate was used to induce mouse androgen-induced alopecia model, and the growth status of hair in 35 days after administering fructus Ligustri Lucidi extract prepared in example 1 and the quality of hair and the number of hair follicles in the back of the mouse after 35 days were recorded and observed, as briefly described below, 60 male C57BL/6 mice were bred adaptively for 1 week, numbered according to body weight, randomly divided into 6 groups according to their body weight with Excel software, and then removed from their back by depilatory cream at about 3X 4cm²The hair follicles of the mice were confirmed to be in the resting stage, and the skin was pink and not damaged. Except for a blank control group, the skin of the hairless area on the back of each group of mice is injected with 0.02 ml/mouse/day of testosterone propionate suspension at multiple points under the skin. The test area on the back of each group of mice was smeared according to the corresponding method, once a day for 35 days continuously, and the grouping and smearing methods are shown in table 5. The skin and hair growth at the site of hair removal was observed daily for each group of mice and the time taken for the skin color to change from pink to black was recorded for each mouse hair removal areaAnd the time to start hair growth, and the effect of the drug on the hair growth cycle of mice was evaluated.

Each group was photographed at 0, 7, 14, 21, 28, 35 days of dosing and the hair growth scoring results were recorded, scoring criteria:

0 minute: non-growing, pink skin in the depilated area

1 minute: the depilated area skin appeared gray (less than 20% increase)

And 2, dividing: the skin in the depilated area is black (more than 20% and less than 40%)

And 3, dividing: the depilated area had black skin and little hair growth (greater than 40% and less than 60% growth)

And 4, dividing: the skin in the depilated area was black and had some hair growth (60-80% growth)

And 5, dividing: hair growth in the epilation zone was substantially complete (80% to 100% growth.

The mice are killed after neck removal, and the hair removal area of the back of each mouse is scraped by an electric hair scraping knife by 3 multiplied by 3cm²The area of the wool was weighed with an analytical balance and the average value was calculated. Scraping hair, drawing materials at a depilated part parallel to the spine, trimming skin into a strip shape with the width of 1cm, fixing in 4% paraformaldehyde for 24-36h, washing out paraformaldehyde, dehydrating with gradient alcohol, embedding paraffin, trimming, slicing, finally staining with hematoxylin-eosin, and observing the quantity and morphological difference of hair follicles under an optical microscope.

TABLE 5 grouping and methods of proofing groups

The hair growth and weight of the mice in each group are shown in Table 6, and the hair growth scores are shown in FIG. 3.

TABLE 6 influence of fructus Ligustri Lucidi extract on the growth of hair in androgen-induced alopecia model mouse

Note indicates significant differences compared to blank group (P < 0.05); # indicates significant difference compared to model group (P < 0.05).

Compared with the blank group, the skin discoloration time and the hair growing time of the model group mice are obviously prolonged (P <0.05), and the hair weight is obviously reduced (P <0.05) at the 35 th day. The results show that testosterone propionate subcutaneously injected has obvious inhibition effect on the hair growth of C57BL/6 mice.

Compared with the model group, the skin color changing time, the hair growing time and the hair quality of each group of the minoxidil group and the glossy privet fruit extract are remarkably different (P is less than 0.05), which indicates that the minoxidil and the glossy privet fruit extract can relieve the inhibition effect of testosterone propionate on the hair growth of mice to a certain extent.

The skin cross-section was used to determine the number of follicles, and the results are shown in FIGS. 4A-F, the number of follicles was observed under a 40-fold optical microscope in 6 different regions, the mean number of follicles in the selected region was 182. + -.5 in mice in the blank group, 74. + -.9 in the model group, the number of follicles in the model group was significantly reduced compared to the blank group (P <0.05), and the number of follicles in each of the minoxidil group and the glossy privet fruit extract group was 162. + -.22, 126. + -.18, 154. + -.21 and 145. + -.22, respectively, and significantly increased compared to the model group (P < 0.05).

The hair follicle morphology obtained from the longitudinal section of the skin is shown in FIGS. 5A-F, and the number of hair follicles in the mice in the blank group is large and the morphology is intact, at which time the hair follicles have entered the resting stage and melanin formation has ceased; the hair follicle shape of the model group is shorter and sparse, and the melanin is less formed; the minoxidil group and the glossy privet fruit extract have more hair follicles, the hair follicles are long and large, and melanin is obviously formed, so that the glossy privet fruit extract can improve the hair follicle miniaturization caused by testosterone propionate and maintain the normal shape of the hair follicles.

Example 7 chick embryo chorioallantoic Membrane (CAM) assay of Ligustrum lucidum extract

1. Lines and sources

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. HET-CAM assay

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.

The eye irritation of the glossy privet fruit extract is detected by adopting an end point evaluation method, the glossy privet fruit extract prepared in example 1 is used as a test group, physiological saline (0.9% NaCl, NS) is used as a negative control, and a 1% fatty alcohol ether sodium sulfate salt mixture (TexaponASV) solution is used as a positive control. 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 2.

TABLE 7 evaluation of end-points method eye irritation grading standards for test samples

No bleeding, angiolysis or clotting reactions were observed within 5 minutes after the 0.9% NaCl solution was contacted 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 a response score above 18 points, as shown in FIG. 6B. The glossy privet fruit extract dose groups did not show any vascular stimulation response after being contacted with CAM, as shown in figures 6C-E, the stimulation scores are all 0 points, and the eye irritation grading results are shown in Table 8. The glossy privet fruit extract has no toxic effect when acting on chick embryo chorioallantoic membrane, which indicates that the glossy privet fruit extract has no eye irritation and can be applied to medicaments as a functional component for preventing and treating alopecia.

TABLE 8 evaluation results of eye irritation in the HET-CAM test

The glossy privet fruit extract can obviously inhibit 5 α -reductase, has obvious prevention and treatment effects on androgen-dependent diseases of 5 α -reductase, is safe, has no stimulation and side effects, and can be applied to the development of 5 α -reductase inhibitors and medicines for preventing and treating androgen-related diseases.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. A fructus Ligustri Lucidi extract is characterized by comprising one or more of salidroside, neoligustrazine, specnuezhenide, ligustilide, oleuropein and ligustilide C.

2. The fructus ligustri lucidi extract as claimed in claim 1, comprising salidroside, neoligustrazine, specnuezhenide, nuezhenide, oleuropein, and nuezhenide C in a weight percentage of 10-14: 8-12: 18-22: 13-17: 15-19: 24-28;

preferably, the mass percentage of salidroside, neoligustilide, specnuezhenide, ligustilide, oleuropein and ligustilide C is 12: 10: 20: 15: 17: 26.

3. application of fructus Ligustri Lucidi extract in preparing medicine for inhibiting 5 α -reductase is provided.

4. Application of fructus Ligustri Lucidi extract in preparing medicine for preventing and treating 5 α -reductase androgen dependent diseases is provided.

5. The use of claim 4, wherein the 5 α -reductase-dependent androgen associated disorder is prostatic hyperplasia, prostate cancer or androgenic alopecia.

6. The use of any one of claims 3 to 5, wherein the extract of Ligustrum lucidum is the extract of Ligustrum lucidum as claimed in claim 1 or 2.

7. The use of any one of claims 3 to 5, wherein the extract of Ligustrum lucidum is an ethanol extract of Ligustrum lucidum.

8. Use according to claim 7, wherein the ethanol extract is 80-99% ethanol extract, preferably the ethanol extract is 90-95% ethanol extract.

9. A 5 α -reductase inhibitor, comprising a prophylactically or therapeutically effective amount of an extract of fructus ligustri lucidi, preferably the extract of fructus ligustri lucidi as defined in claim 1 or 2.

10. A medicament for preventing and treating 5 α -reductase androgen-dependent diseases, which is characterized by comprising a prophylactic or therapeutic effective amount of glossy privet fruit extract, preferably the glossy privet fruit extract is the glossy privet fruit extract in claim 1 or 2.