TRADITIONAL CHINESE MEDICINE COMPOUND COMPOSITION WITH EFFECT OF IMPROVING MALE SEXUAL FUNCTION AND APPLICATION THEREOF

Abstract

A traditional Chinese medicine compound composition with effect of improving male sexual function and its application are disclosed. The disclosure prepares the traditional Chinese medicine compound composition by compounding Semen Allii Tuberosi with Radix Morindae Officinalis, Epimedii Folium, Cistanche deserticola Ma and Semen Cuscutae. The traditional Chinese medicine compound composition has a good effect on improving the sexual function of kidney Yang deficiency model mice. While improving sexual function, it can improve the abundance of spermatogenic cells in testicular seminiferous tubules, ensure that spermatogenic cells can continuously produce new sperm, and improve the number and morphology of sperm in the lumen. Moreover, the disclosed traditional Chinese medicine compound composition can be used as a raw material of dietary supplement or health food for the male population with low sexual function and easy fatigue. Therefore, the disclosed traditional Chinese medicine compound composition is suitable for market promotion and application.

Claims

1. A traditional Chinese medicine compound composition with effect of improving male sexual function, wherein the traditional Chinese medicine compound composition is prepared by mixing following raw materials after separate extraction, and the raw materials comprises: 1˜10 parts by weight of Semen Allii Tuberosi; 1˜10 parts by weight of Radix Morindae Officinalis; 1˜10 parts by weight of Epimedii Folium; or 1˜10 parts by weight of Semen Allii Tube rosi; 1˜10 parts by weight of Radix Morindae Officinalis; 1˜10 parts by weight of Cistanche deserticola Ma; or 1˜10 parts by weight of Semen Allii Tuberosi; 1˜10 parts by weight of Lycium barbarum; 1˜10 parts by weight of Semen Cuscutae; or 1˜10 parts by weight of Semen Allii Tuberosi; 1˜10 parts by weight of Lycium barbarum; 1˜10 parts by weight of Semen Cuscutae; 1˜10 parts by weight of Radix Morindae Officinalis; 1˜10 parts by weight of Cistanche deserticola Ma; 1˜10 parts by weight of Epimedii Folium.

2. An application of the traditional Chinese medicine compound composition with effect of improving male sexual function of claim 1 in a preparation of health food.

3. The application of claim 2, wherein the traditional Chinese medicine compound composition is prepared by mixing Semen Allii Tuberosi, Radix Morindae Officinalis, Epimedii Folium, Cistanche deserticola Ma, Lycium barbarum and Semen Cuscutae after separate extraction.

4. The application of claim 2, wherein the traditional Chinese medicine compound composition can be made into capsules, tablets or granules for improving male sexual function.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0053] In order to explain the embodiments of the present disclosure or the technical solutions in the prior art more clearly, the following drawings that need to be used in the description of the embodiments or the prior art will be briefly introduced. Obviously, the drawings in the following description are only embodiments of the present disclosure. For those of ordinary skill in the art, other drawings can be obtained based on the drawings disclosed without creative work.

[0054] FIG. 1 shows the histomorphological observation of mouse testes (HE staining, ×100, ×400).

[0055] FIG. 2 is the histomorphological observation of mouse epididymides (HE staining, ×100, ×400).

[0056] FIG. 3 is the histomorphological observation of mouse kidneys (HE staining, ×100, ×400).

[0057] FIG. 4 is the histomorphological observation of mouse adrenal glands (HE staining, ×100, ×400).

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0058] Technical solutions of the present disclosure will be clearly and completely described below with reference to the embodiments. Obviously, described below are merely some embodiments of the disclosure, which are not intended to limit the disclosure. Other embodiments made by those skilled in the art without sparing any creative effort should fall within the scope of the disclosure.

[0059] The embodiments of disclosure provide a traditional Chinese medicine composition, which has the effect of improving male sexual function and can be used as a dietary supplement or health food raw material.

[0060] In order to better understand the present disclosure, the present disclosure will be further specifically described by the following embodiments, but it should not be construed as a limitation of the present disclosure. Some non-essential improvements and adjustments made by those skilled in the art according to the above-mentioned contents of the disclosure are also considered to fall within the protection scope of the present disclosure.

[0061] The disclosed technical solutions will be further described below with reference to specific embodiments.

Embodiment 1

[0062] The raw materials of a traditional Chinese medicine compound composition for improving male sexual dysfunction and improving fatigue included 1 part of Semen Allii Tuberosi, 1 part of Lycium barbarum and 1 part of Semen Cuscutae.

[0063] The preparation method of the traditional Chinese medicine compound composition included the following steps.

[0064] (1) Semen Allii Tuberosi was subjected to extraction with water in a volume ratio of 1:8 under heating and refluxing for 2 h and filtration to obtain a residue. The residue was subjected to extraction with deionized water in a volume ratio of 1:6 under heating and refluxing twice, for 1 h each time. Then the extracting solutions were combined, purified, concentrated, sprayed dry at 200° C. and sieved with a sieve of 80 mesh to obtain the Semen Allii Tuberosi extract.

[0065] (2) Lycium barbarum was subjected to extraction with water in a volume ratio of 1:8 under heating and refluxing for 2 h and filtration to obtain a residue. The residue was subjected to extraction with deionized water in a volume ratio of 1:6 under heating and refluxing twice, for 1 h each time. Then the extracting solutions were combined, purified, concentrated, sprayed dry at 200° C. and sieved with a sieve of 80 mesh to obtain the Lycium barbarum extract.

[0066] (3) Semen Cuscutae was subjected to soak with 80% ethanol solution in a volume ratio of 1:8 for 0.5 h, extraction under heating and refluxing for 2 h and filtration to obtain a residue. The residue was subjected to extraction with 80% ethanol solution in a volume ratio of 1:6 under heating and refluxing twice, for 1 h each time. Then the extracting solutions were combined, purified, concentrated, sprayed dry at 200° C. and sieved with a sieve of 80 mesh to obtain the Semen Cuscutae extract.

[0067] The extracts obtained by the above preparation were uniformly mixed to obtain the traditional Chinese compound composition.

Embodiment 2

[0068] The raw materials of a traditional Chinese medicine compound composition for improving male sexual dysfunction and improving fatigue included 2 parts of Semen Allii Tuberosi, 1 part of Epimedii Folium and 2 parts of Radix Morindae Officinalis.

[0069] The preparation method of the traditional Chinese medicine compound composition included the following steps.

[0070] (1) Semen Allii Tuberosi was subjected to extraction with water in a volume ratio of 1:8 under heating and refluxing for 2 h and filtration to obtain a residue. The residue was subjected to extraction with deionized water in a volume ratio of 1:6 under heating and refluxing twice, for 1 h each time. Then the extracting solutions were combined, purified, concentrated, sprayed dry at 200° C. and sieved with a sieve of 80 mesh to obtain the Semen Allii Tuberosi extract.

[0071] (2) Epimedii Folium was subjected to extraction with 80% ethanol solution in a volume ratio of 1:8 under heating and refluxing for 2 h and filtration to obtain a residue. The residue was subjected to extraction with 80% ethanol solution in a volume ratio of 1:6 under heating and refluxing for 2 times, 2 h each time. Then the extracting solutions were combined, passed through the column and concentrated to obtain eluents. The eluents were collected, concentrated, sprayed dry at 200° C. and sieved with a sieve of 80 mesh to obtain the Epimedii Folium extract.

[0072] (3) Radix Morindae Officinalis was subjected to extraction with 80% ethanol solution in a volume ratio of 1:8 under heating and refluxing for 2 h and filtration to obtain a residue. The residue was subjected to extraction with 80% ethanol solution in a volume ratio of 1:6 under heating and refluxing twice, for 2 h each time. Then the extracting solutions were combined, purified, concentrated, sprayed dry at 200° C. and sieved with a sieve of 80 mesh to obtain the Morindae Officinalis extract.

[0073] The extracts obtained by the above preparation were uniformly mixed to obtain the traditional Chinese compound composition.

Embodiment 3

[0074] The raw materials of a traditional Chinese medicine compound composition for improving male sexual dysfunction and improving fatigue included 2 parts of Semen Allii Tuberosi, 1 part of Epimedii Folium and 1 part of Radix Morindae Officinalis.

[0075] The preparation method of the traditional Chinese medicine compound composition included the following steps.

[0076] (1) Semen Allii Tuberosi was subjected to extraction with water in a volume ratio of 1:8 under heating and refluxing for 2 h and filtration to obtain a residue. The residue was subjected to extraction with deionized water in a volume ratio of 1:6 under heating and refluxing twice, for 1 h each time. Then the extracting solutions were combined, purified, concentrated, sprayed dry at 200° C. and sieved with a sieve of 80 mesh to obtain the Semen Allii Tuberosi extract.

[0077] (2) Cistanche deserticola Ma was subjected to extraction with 50% ethanol solution in a volume ratio of 1:8 under heating and refluxing for 2 h and filtration to obtain a residue. The residue was subjected to extraction with 50% ethanol solution in a volume ratio of 1:6 under heating and refluxing twice, for 2 h each time. Then the extracting solutions were combined, concentrated, sprayed dry at 200° C. and sieved with a sieve of 80 mesh to obtain the Cistanche deserticola Ma extract.

[0078] (3) Radix Morindae Officinalis was subjected to extraction with water in a volume ratio of 1:8 under heating and refluxing for 2 h and filtration to obtain a residue. The residue was subjected to extraction with deionized water in a volume ratio of 1:6 under heating and refluxing twice, for 2 h each time. Then the extracting solutions were combined, purified, concentrated, sprayed dry at 200° C. and sieved with a sieve of 80 mesh to obtain the Morindae Officinalis extract.

[0079] The extracts obtained by the above preparation were uniformly mixed to obtain the traditional Chinese compound composition.

Embodiment 4

[0080] Preparation of Capsules:

[0081] The raw materials of a traditional Chinese medicine compound composition for improving male sexual dysfunction and improving fatigue included 2 parts of Semen Allii Tuberosi, 1 part of Epimedii Folium, 1 part of Radix Morindae Officinalis, 2 parts of Lycium barbarum, 2 parts of Semen Cuscutae, and 1 part of Cistanche deserticola Ma.

[0082] The preparation method of the traditional Chinese medicine compound composition included the following steps.

[0083] (1) Semen Allii Tuberosi was subjected to extraction with water in a volume ratio of 1:8 under heating and refluxing for 2 h and filtration to obtain a residue. The residue was subjected to extraction with deionized water in a volume ratio of 1:6 under heating and refluxing twice, for 1 h each time. Then the extracting solutions were combined, purified, concentrated, sprayed dry at 200° C. and sieved with a sieve of 80 mesh to obtain the Semen Allii Tuberosi extract.

[0084] (2) Epimedii Folium was subjected to extraction with 80% ethanol solution in a volume ratio of 1:8 under heating and refluxing for 2 h and filtration to obtain a residue. The residue was subjected to extraction with 80% ethanol solution in a volume ratio of 1:6 under heating and refluxing twice, for 2 h each time. Then the extracting solutions were combined, passed through the column and concentrated to obtain eluents. The eluents were collected, concentrated, sprayed dry at 200° C. and sieved with a sieve of 80 mesh to obtain the Epimedii Folium extract.

[0085] (3) Cistanche deserticola Ma was subjected to extraction with 50% ethanol solution in a volume ratio of 1:8 under heating and refluxing for 2 h and filtration to obtain a residue. The residue was subjected to extraction with 50% ethanol solution in a volume ratio of 1:6 under heating and refluxing twice, for 2 h each time. Then the extracting solutions were combined, concentrated, sprayed dry at 200° C. and sieved with a sieve of 80 mesh to obtain the Cistanche deserticola Ma extract.

[0086] (4) Lycium barbarum was subjected to extraction with water in a volume ratio of 1:8 under heating and refluxing for 2 h and filtration to obtain a residue. The residue was subjected to extraction with deionized water in a volume ratio of 1:6 under heating and refluxing twice, for 1 h each time. Then the filtrates were combined, purified, concentrated, sprayed dry at 200° C. and sieved with a sieve of 80 mesh to obtain the Lycium barbarum extract.

[0087] (5) Semen Cuscutae was subjected to soak with 80% ethanol solution in a volume ratio of 1:8 for 0.5 h, extraction under heating and refluxing for 2 h and filtration to obtain a residue. The residue was subjected to extraction with 80% ethanol solution in a volume ratio of 1:6 under heating and refluxing twice, for 1 h each time. Then the filtrates were combined, purified, concentrated, sprayed dry at 200° C. and sieved with a sieve of 80 mesh to obtain the Semen Cuscutae extract.

[0088] (6) Radix Morindae Officinalis was subjected to extraction with 80% ethanol solution in a volume ratio of 1:8 under heating and refluxing for 2 h and filtration to obtain a residue. The residue was subjected to extraction with 80% ethanol solution in a volume ratio of 1:6 under heating and refluxing twice, for 2 h each time. Then the extracting solutions were combined, passed through the column and concentrated to obtain eluents. The eluents were collected, concentrated, sprayed dry at 200° C. and sieved with a sieve of 80 mesh to obtain the Radix Morindae Officinalis extract.

[0089] The extracts prepared above were mixed according to the ratio of Semen Allii Tuberosi: Lycium barbarum: Semen Cuscutae (18:10:72) to obtain mixture A, Semen Allii Tuberosi: Epimedii Folium: Radix Morindae Officinalis (18:10:72) to obtain mixture B, and Semen Allii Tuberosi: Cistanche deserticola Ma: Radix Morindae Officinalis (20:45:35) to obtain mixture C.

[0090] Pregelatinized starch, talc powder and magnesium stearate were added into the prepared traditional Chinese medicine compounds, then mixed evenly, and put into capsules to prepare capsules.

Embodiment 5

[0091] Preparation of Tablets:

[0092] The raw materials of a traditional Chinese medicine compound composition for improving male sexual dysfunction and improving fatigue included 2 parts of Semen Allii Tuberosi, 1 part of Epimedii Folium, 1 part of Radix Morindae Officinalis, 2 parts of Lycium barbarum, 2 parts of Semen Cuscutae, and 1 part of Cistanche deserticola Ma.

[0093] The preparation method of the traditional Chinese medicine compound composition included the following steps.

[0094] (1) Semen Allii Tuberosi was subjected to extraction with water in a volume ratio of 1:8 under heating and refluxing for 2 h and filtration to obtain a residue. The residue was subjected to extraction with deionized water in a volume ratio of 1:6 under heating and refluxing twice, for 1 h each time. Then the extracting solutions were combined, purified, concentrated, sprayed dry at 200° C. and sieved with a sieve of 80 mesh to obtain the Semen Allii Tuberosi extract.

[0095] (2) Epimedii Folium was subjected to extraction with 80% ethanol solution in a volume ratio of 1:8 under heating and refluxing for 2 h and filtration to obtain a residue. The residue was subjected to extraction with 80% ethanol solution in a volume ratio of 1:6 under heating and refluxing twice, for 2 h each time. Then the extracting solutions were combined, passed through the column and concentrated to obtain eluents. The eluents were collected, concentrated, sprayed dry at 200° C. and sieved with a sieve of 80 mesh to obtain the Epimedii Folium extract.

[0096] (3) Cistanche deserticola Ma was subjected to extraction with 50% ethanol solution in a volume ratio of 1:8 under heating and refluxing for 2 h and filtration to obtain a residue. The residue was subjected to extraction with 50% ethanol solution in a volume ratio of 1:6 under heating and refluxing twice, for 2 h each time. Then the extracting solutions were combined, concentrated, sprayed dry at 200° C. and sieved with a sieve of 80 mesh to obtain the Cistanche deserticola Ma extract.

[0097] (4) Lycium barbarum was subjected to extraction with water in a volume ratio of 1:8 under heating and refluxing for 2 h and filtration to obtain a residue. The residue was subjected to extraction with deionized water in a volume ratio of 1:6 under heating and refluxing twice, for 1 h each time. Then the extracting solutions were combined, purified, concentrated, sprayed dry at 200° C. and sieved with a sieve of 80 mesh to obtain the Lycium barbarum extract.

[0098] (5) Semen Cuscutae was subjected to soak with 80% ethanol solution in a volume ratio of 1:8 for 0.5 h, extraction under heating and refluxing for 2 h and filtration to obtain a residue. The residue was subjected to extraction with 80% ethanol solution in a volume ratio of 1:6 under heating and refluxing twice, for 1 h each time. Then the extracting solutions were combined, purified, concentrated, sprayed dry at 200° C. and sieved with a sieve of 80 mesh to obtain the Semen Cuscutae extract.

[0099] (6) Radix Morindae Officinalis was subjected to extraction with 80% ethanol solution in a volume ratio of 1:8 under heating and refluxing for 2 h and filtration to obtain a residue. The residue was subjected to extraction with 80% ethanol solution in a volume ratio of 1:6 under heating and refluxing twice, for 2 h each time. Then the extracting solutions were combined, passed through the column and concentrated to obtain eluents. The eluents were collected, concentrated, sprayed dry at 200° C. and sieved with a sieve of 80 mesh to obtain the Radix Morindae Officinalis extract.

[0100] The extracts prepared above were mixed according to the ratio of Semen Allii Tuberosi: Lycium barbarum: Semen Cuscutae (18:10:72) to obtain mixture A, Semen Allii Tuberosi: Epimedii Folium: Radix Morindae Officinalis (18:10:72) to obtain mixture B, and Semen Allii Tuberosi: Cistanche deserticola Ma: Radix Morindae Officinalis (20:45:35) to obtain mixture C.

[0101] Pregelatinized starch, talc powder and magnesium stearate were added to the prepared traditional Chinese medicine compounds, then mixed evenly, pressed into tablets, coated with film, and made into tablets.

Embodiment 6

[0102] Preparation of Granules:

[0103] The raw materials of a traditional Chinese medicine compound composition for improving male sexual dysfunction and improving fatigue included 2 parts of Semen Allii Tuberosi, 1 part of Epimedii Folium, 1 part of Radix Morindae Officinalis, 2 parts of Lycium barbarum, 2 parts of Semen Cuscutae, and 1 part of Cistanche deserticola Ma.

[0104] The preparation method of the traditional Chinese medicine compound composition included the following steps.

[0105] (1) Semen Allii Tuberosi was subjected to extraction with water in a volume ratio of 1:8 under heating and refluxing for 2 h and filtration to obtain a residue. The residue was subjected to extraction with deionized water in a volume ratio of 1:6 under heating and refluxing twice, for 1 h each time. Then the extracting solutions were combined, purified, concentrated, sprayed dry at 200° C. and sieved with a sieve of 80 mesh to obtain the Semen Allii Tuberosi extract.

[0106] (2) Epimedii Folium was subjected to extraction with 80% ethanol solution in a volume ratio of 1:8 under heating and refluxing for 2 h and filtration to obtain a residue. The residue was subjected to extraction with 80% ethanol solution in a volume ratio of 1:6 under heating and refluxing twice, for 2 h each time. Then the extracting solutions were combined, passed through the column and concentrated to obtain eluents. The eluents were collected, concentrated, sprayed dry at 200° C. and sieved with a sieve of 80 mesh to obtain the Epimedii Folium extract.

[0107] (3) Cistanche deserticola Ma was subjected to extraction with 50% ethanol solution in a volume ratio of 1:8 under heating and refluxing for 2 h and filtration to obtain a residue. The residue was subjected to extraction with 50% ethanol solution in a volume ratio of 1:6 under heating and refluxing twice, for 2 h each time. Then the extracting solutions were combined, concentrated, sprayed dry at 200° C. and sieved with a sieve of 80 mesh to obtain the Cistanche deserticola Ma extract.

[0108] (4) Lycium barbarum was subjected to extraction with water in a volume ratio of 1:8 under heating and refluxing for 2 h and filtration to obtain a residue. The residue was subjected to extraction with deionized water in a volume ratio of 1:6 under heating and refluxing twice, for 1 h each time. Then the extracting solutions were combined, purified, concentrated, sprayed dry at 200° C. and sieved with a sieve of 80 mesh to obtain the Lycium barbarum extract.

[0109] (5) Semen Cuscutae was subjected to soak with 80% ethanol solution in a volume ratio of 1:8 for 0.5 h, extraction under heating and refluxing for 2 h and filtration to obtain a residue. The residue was subjected to extraction with 80% ethanol solution in a volume ratio of 1:6 under heating and refluxing twice, for 1 h each time. Then the filtrates were combined, purified, concentrated, sprayed dry at 200° C. and sieved with a sieve of 80 mesh to obtain the Semen Cuscutae extract.

[0110] (6) Radix Morindae Officinalis was subjected to extraction with 80% ethanol solution in a volume ratio of 1:8 under heating and refluxing for 2 h and filtration to obtain a residue. The residue was subjected to extraction with 80% ethanol solution in a volume ratio of 1:6 under heating and refluxing twice, for 2 h each time. Then the extracting solutions were combined, passed through the column and concentrated to obtain eluents. The eluents were collected, concentrated, sprayed dry at 200° C. and sieved with a sieve of 80 mesh to obtain the Radix Morindae Officinalis extract.

[0111] The extracts prepared above were mixed according to the ratio of Semen Allii Tuberosi: Lycium barbarum: Semen Cuscutae (18:10:72) to obtain mixture A, Semen Allii Tuberosi: Epimedii Folium: Radix Morindae Officinalis (18:10:72) to obtain mixture B, and Semen Allii Tuberosi: Cistanche deserticola Ma: Radix Morindae Officinalis (20:45:35) to obtain mixture C.

[0112] (7) The above mixture A, B or C was mixed with a certain proportion of lactose monohydrate evenly, and 95% ethanol was added to produce a mixture. The mixture was stirred until it was kneaded into a dough and dispersed when touched, and then granulation was started. Granules that can not pass through the 20-mesh sieve and 80-mesh sieve but can pass the 10-mesh sieve were collected and dried in an oven in time for use.

[0113] (8) The prepared granules were put into a vacuum dryer for drying so that the moisture content of the granules was controlled at 4%. The compound granules with the effect of improving male sexual dysfunction and fatigue were prepared.

[0114] The content of the disclosure is not limited to the above embodiments, and one or the combination of several embodiments can also achieve the purpose of the disclosure.

[0115] Further, the following test was performed to evaluate the efficacy of the traditional Chinese medicine compound composition prepared herein.

Efficacy Test

I. Test Purpose

[0116] The Efficacy test was carried out to explore the effects of several traditional Chinese medicines to be studied on the sexual function of kidney Yang deficiency model mice induced by hydrocortisone, to observe the effects of the above traditional Chinese medicines on the general external performance, sexual behavior (capture, mount, ejaculation latency and times), kidney, adrenal gland, testis, epididymis and seminal vesicle organ index, tissue morphology, and various sex hormone levels in serum of the kidney Yang deficiency mice, to explore the mechanism of the above-mentioned traditional Chinese medicines to improve the sexual function of kidney Yang deficiency model mice, so as to provide data support for the prevention and treatment of sexual dysfunction of kidney Yang deficiency mice by relevant traditional Chinese medicines.

II. Test materials and methods

1. Animals, Reagents and Instruments

1.1 Animals

[0117] 100 male and 100 female clean ICR mice, 4-6 weeks old, 18-22 g, were purchased from SiPeiFu (Beijing) Biotechnology Co., Ltd. with certificate No. SCXK (Beijing) 2019-0010. All tests of the disclosure were carried out in accordance with the ethical code of experimental animals.

1.2 Drugs and Reagents

[0118]

TABLE-US-00001 TABLE 1 Test drugs and reagents Reagent name Manufactor Article Number Hydrocortisone injection Changzhou SiYao License No.: Pharmaceutical GUOYAOZHUNZI Co., Ltd H20093293 Estradiol valerate tablets Bayer Healthcare License No.: Company Limited GUOYAOZHUNZI Guangzhou Branch Testosterone (T) ELISA Kit Beijing GongGeBoYe DG30457M-96T, Biotechnology Co., Ltd Folliclestimulating Beijing GongGeBoYe DG30497M-96T, Hormone Biotechnology DG30497M-48T (FSH) ELISA Kit Co., Ltd Luteinizing Hormone (LH) Beijing GongGeBoYe DG30426M-96T, ELISA Kit Biotechnology DG30426M-48T Co., Ltd Superoxidedismutase Nanjing Jiancheng A001-3-1, (SOD) Bioengineering A001-3-2 Biochemical Kit Institute Mmalondialdehyde (MDA) Nanjing Jiancheng A003-1-1, Biochemical Kit Bioengineering A003-1-2 Institute Glutathion peroxidase Nanjing Jiancheng A005-1-2 (GSH-PX) Bioengineering Assay Kit Institute Nitricoxidesynthase Nanjing Jiancheng A014-2-1, (NOS) Bioengineering A014-2-2 Biochemical Kit Institute indicates data missing or illegible when filed

1.3 Test Instrument

[0119]

TABLE-US-00002 TABLE 2 Test instrument Name Manufactor Model Centrifuge Beckman Coulter AllgraX-15R Microplate Reader BioTek Biotek Epoch2 Sartorius Electronic Sartorius BP211D Analytical Balance Dehydrator Wuhan Junjie Electronics Co., Ltd JJ-12J Embedding machine Wuhan Junjie Electronics Co., Ltd JB-P5 Pathological microtome Shanghai Leica Instrument Co., Ltd RM2016 Freezing table Wuhan Junjie Electronics Co., Ltd JB-L5 Tissue spreader Zhejiang Jinhua Kedi Instrument KD-P Equipment Co., Ltd Oven Tianjin Labotery Instrument GFL-230 Equipment Co., Ltd Glass slide Servicebio — Orthostatic optical Nikon Nikon Eclipse E100 microscope Imaging system Nikon NIKON DS-U3

2. Model Establishment and Group Administration

[0120] Male mice were randomly divided into normal group, model group, sample 1 group, sample 2 group, sample 3 group and sample 4 group. Each administration group was given the corresponding dose by gavage. The normal group and the model group were given the same volume of normal saline (1 mL/10 g) by gavage once a day for 4 weeks. The model was established on the 7th day of the second week after gavage. Except the normal group, the kidney Yang deficiency model was established by injecting hydrocortisone (37.5 mg/kg/d) intramuscularly for 14 days. At the same time, the model was continuously administered. On the 7th day of the 4th week, the general external performance and behavioral performance of the animals were observed and recorded one hour after administration, and then the mice were anesthetized and the test materials were taken.

TABLE-US-00003 TABLE 3 Test grouping and dosage Group Tab Dosage Normal group Normal / Model group Model / Compound of Semen Allii Tuberosi, Sample 1 200 mg/kg Lycium barbarum and Semen Cuscutae Compound of Semen Allii Tuberosi, Sample 2 200 mg/kg Radix Morindae Officinalis and Epimedii Folium Compound of Semen Allii Tuberosi, Sample 3 200 mg/kg Radix Morindae Officinalis and Cistanche deserticola Ma Positive control - Aphrodyne Tablets Sample 4  83 mg/kg

3. Index Detection

(1) General External Performance

[0121] The weight, body temperature, hair color and shape, eating state and other main performance of male mice before and after treatment were observed and recorded.

(2) Sexual Behavior of Male Mice Observed by Mating Test

[0122] On the 7th day of the 4th week of administration of male mice, all animals were subjected to mating experiments according to groups. Female mice were given estradiol valerate (1 mg/kg) by gavage once a day on the 24th to 27th days to synchronize the female mice with estrus. The male mice caged with their corresponding female mice 1 h after the last administration. The specific operations were as follows. The male mice were put in each group separately into the cage. The surrounding environment was kept dark and quiet. The red dim light was used to make them adapt to the environment for 5mins, and then the corresponding female mice were put into the cage for cage combining test. The following indexed were observed within 30 mins of cage combining.

[0123] a. Capture latency (CL): the time from the female mouse being put into the observation box to the male mouse capturing the female mouse for the first time (unit: s). Capture behavior: the behavior of catching and chasing the female mouse after the male mouse sniffed the base of the female mouse's tail.

[0124] b. Capture times (CT): the number of times that male mouse captured female mouse within 30 min.

[0125] c. Mount latency (ML): the time interval from when the female mouse was put into the observation box to the first mount behavior (unit: s). Mount behavior: the sexual behavior of the male mouse's waist doing back and forth motions, while the hind limbs repeatedly do flexion and extension movements, with or without insertion counted.

[0126] d. Mount times: the number of mount times before male mouse ejaculated.

[0127] e. Ejaculation latency: time interval from the time the female mouse was put into the observation box to the first ejaculation of the male mouse (unit: s).

[0128] f. Ejaculation times (ET).

(3) Organ Index Determination

[0129] 24 h after the last administration, the male mice were weighed and killed. The kidneys, adrenal glands, testes, epididymides and seminal vesicles of the male mice were quickly taken out, weighed accurately, and the organ index was calculated, where organ index=organ weight (mg)/body mass of the mouse (g).

(4) HE Staining for Observing Histomorphology of Kidneys, Adrenal Glands, Testes and Epididymides

[0130] The kidneys, adrenal glands, testes and epididymides of male mice were fixed with 10% formalin. The tissues were dehydrated, embedded in wax and sectioned. Five of them were taken from each group for HE staining.

[0131] 1) Dewaxing paraffin sections to water: the sections were successively placed in xylene I for 20 min—xylene II for 20 min—anhydrous ethanol I for 5 min—anhydrous ethanol II for 5 min—75% ethanol solution for 5 min, and washed with tap water.

[0132] 2) Hematoxylin staining: the sections were stained with hematoxylin staining solution for 3-5 min, washed with tap water, differentiated with differentiation solution, washed with tap water, promoted to blue with blue promoting solution, and rinsed with running water.

[0133] 3) Eosin staining: the sections were dehydrated with 85% and 95% gradient ethanol solution for 5 min respectively, and stained with eosin staining solution for 5 min.

[0134] 4) Dehydration sealing: the sections were successively placed in anhydrous ethanol I for 5 min—anhydrous ethanol II for 5 min—anhydrous ethanol III for 5 min—xylene I for 5 min—xylene II for 5 min in order to be transparent, and sealed by neutral gum.

[0135] 5) Microscopic examination, image acquisition and analysis, pathological observation of tissue morphology and magnification of corresponding times (100ט400×) for taking photos were performed successively.

(5) ELISA to Detect the Levels of Various Sex Hormones in Serum

[0136] Orbital blood was collected from male mice, left standing for 1 h, centrifuged at 3000 r/min for 10 min to separate serum, and the levels of serum T, E2, FSH, LH and GnRH were determined by Elisa method according to the kit instructions.

(6) Detection of Peroxide Level in Testes by Biochemical Method

[0137] The testis tissues of male mice were frozen and stored in −80° C. refrigerator. The tissues were ground when used, and the expressions of testis SOD, MDA, GSH-PX and penile NOS were detected according to the instructions of the biochemical kit.

4. Statistical Analysis

[0138] SPSS 19.0 software was used for statistics, measurement data were expressed as mean±standard deviation (mean±SD), and one-way-ANOVA was used to compare more than two groups. P<0.05 means the difference was statistically significant.

III. Test Results

1. General State

[0139] Compared with the normal mice, the mice in the model group had kidney Yang deficiency symptoms, such as aversion to cold, decreased autonomous activity, dull fur, and some of the mice were depressed in mental state and slow in activity. The general state of mice in each sample group was improved to some extent.

2. Sexual Behavior

[0140] Compared with the normal group, the capture times, mount times and ejaculation times of the model group were significantly reduced (P<0.05). Compared with the model group, the capture latency of sample 1 group was shorter, and the capture times, mount times and ejaculation times were significantly increased (P<0.05), the capture times of sample 2 group and sample 4 group were significantly increased (P<0.05), the capture times, mount times and ejaculation times of sample 4 group were significantly increased (P<0.05), and the capture latency of sample 4 group was shorter, and the capture times of sample 7 group were significantly increased (P<0.05), which were shown in Table 4 and FIG. 1. The results suggest that sample 2 and sample 4 can improve the sexual behavior of kidney Yang deficiency model mice.

TABLE-US-00004 TABLE 4 Comparison of sexual behavior of mice in each group after administration ({grave over ( )}x ± s) Group Normal Model Sample 1 Sample 2 Sample 3 Sample 4 CL(s) 7.00 ± 4.69 8.42 ± 7.81 6.70 ± 2.45 8.00 ± 2.00 6.50 ± 3.21 5.40 ± 2.22 CT (times) 39.60 ± 21.46 16.58 ± 6.52* 24.70 ± 10.06 27.60 ± 9.67# 24.30 ± 9.23   36.80 ± 14.99# ML(s) 205.00 ± 124.72 254.73 ± 321.17 158.70 ± 148.63 171.70 ± 106.92 185.22 ± 100.39 145.70 ± 131.50 MT (times) 18.60 ± 12.30  8.36 ± 6.79* 13.00 ± 5.87  13.80 ± 4.69  14.44 ± 5.48  18.40 ± 9.03# EL(s) 328.43 ± 125.57 364.11 ± 101.73 268.50 ± 90.41  278.33 ± 82.99  250.00 ± 101.09 256.25 ± 124.14 ET (times) 8.57 ± 5.91  3.67 ± 2.83* 4.25 ± 2.92 5.00 ± 2.87 5.33 ± 3.44  8.63 ± 3.54# Note: *compared with the normal group, P < 0.05. #Compared with the model group, P < 0.05.

3. Tissue Morphology

[0141] The tissue structure of each specimen of normal mice was normal. There were abundant layers of spermatogenic cells in the seminiferous tubule of testes, sperm were found in the lumen, and the epididymal tubes were closely arranged with abundant sperm in the tubule. Although all layers of spermatogenic cells in the seminiferous tubules of the testes of the model group were visible, the local layers were reduced, no intact sperm was found in the lumen, denatured sperm were visible, the arrangement of epididymal tubules was loose, and the number of sperms in the tubules was reduced. After administration, the layers of testicular seminiferous epithelium of mice in each group increased in varying degrees, the number of sperms cells and sperm increased, the arrangement of epididymal tubes improved compared with the model group, and the number of sperms in the lumen increased. In the sample 2 group and sample 4 group, the epithelium of testicular seminiferous tubules was significantly improved, spermatogenic cells in all layers were visible, the epididymides were closely arranged, and the number of sperms in the lumen was increased. No obvious pathological changes were found in adrenal histomorphology of mice in each group, as shown in FIG. 2 to FIG. 4.

4. Sex Hormones

[0142] Compared with the normal group, the levels of T, E2, FSH, LH and GnRH in the model group decreased (P<0.05). Compared with the model group, the levels of serum T, E2, FSH, LH and GnRH of mice in sample 4 group increased significantly (P<0.05), the levels of T, E2, FSH and GnRH in sample 2 group increased significantly (P<0.05), the levels of LH, T and E2 in sample 1 group increased (P<0.05), and the levels of FSH, T and E2 in sample 3 group increased (P<0.05), as shown in Table 5. The results showed that each sample had different regulating effects on the level of sex hormones, and sample 2 and sample 4 had the most obvious effect on the improvement of sex hormones in kidney Yang deficiency model mice.

TABLE-US-00005 TABLE 5 Levels of sex hormones in serum of mice in each group after administration ({grave over ( )} x ± s) T E2 FSH LH GnRH Group (ng/mL) ( (mIU/mL) (mU/mL) (mIU/mL) Normal 3.42 ± 0.42  6.97 ± 1.49  9.81 ± 1.84 4.65 ± 0.79  13.46 ± 2.38  Model 2.81 ± 0.61* 5.26 ± 0.96*  7.93 ± 1.95* 3.69 ± 0.66* 11.32 ± 1.95* Sample 1 3.28 ± 0.46# 6.66 ± 0.62# 9.35 ± 1.69 4.60 ± 0.52# 13.04 ± 1.80  Sample 2 3.45 ± 0.59# 7.18 ± 0.86#  9.71 ± 1.50# 4.32 ± 0.71  14.10 ± 0.37# Sample 3 3.39 ± 0.49# 7.06 ± 1.20#  9.81 ± 1.49# 4.25 ± 1.03  13.05 ± 1.77  Sample 4 3.46 ± 0.59# 7.25 ± 0.86# 10.33 ± 1.18# 4.60 ± 0.52# 14.28 ± 0.43# Note: *Compared with the normal group, P<0.05. #Compared with the model group, P < 0.05. indicates data missing or illegible when filed

5. Levels of Oxidative Stress Related Factors

[0143] Compared with the normal group, the testicular SOD decreased, MDA increased, and the penile NOS activity decreased in the model group (P<0.05). Compared with the model group, the testicular SOD and GSH-Px activities in sample 2 group and sample 4 group increased significantly (P<0.05), and penile NOS activities in sample 1 group and sample 4 group increased significantly (P<0.05), as shown in Table 6. The results showed that sample 2 and 4 could improve the oxidative damage of testes, and sample 1 and 4 could also improve the erectile function of penises.

TABLE-US-00006 TABLE 6 Levels of oxidative stress related factors in testes and penises of mice in each group after administration ({grave over ( )} x±s) Testicular SOD Testicular MDA Testicular activity content GSH-Px Penile NOS Group (U/mgprot) (nmol/mgprot) activity (U/mgprot) Normal 131.78 ± 35.76  5.57 ± 1.52 136.87 ± 38.75 2.20 ± 0.88 Model 82.47 ± 7.23*  7.03 ± 1.23*  94.97 ± 23.64*  0.80 ± 0.40* Sample 1 81.83 ± 9.09  6.13 ± 0.96 128.11 ± 51.79  2.1 ± 1.27# Sample 2 104.98 ± 17.47# 6.08 ± 1.05  137.47 ± 59.37# 1.48 ± 0.50 Sample 3 85.85 ± 4.67  6.72 ± 1.17 130.96 ± 72.67 1.00 ± 0.77 Sample 4 102.27 ± 11.04# 6.17 ± 1.41  146.40 ± 42.26#  2.47 ± 1.57# Note: *compared with the normal group, P < 0.05. #compared with the model group, P < 0.05.

IV. Conclusion

[0144] The test results of the disclosure show that, compared with the normal group, the capture times, mount times and ejaculation times of the model group mice were significantly reduced, the testicular index was significantly reduced, the levels of serum GnRH, T, E2, FSH and LH were reduced, the testicular SOD and GSH-Px were reduced, MDA was increased, and the penis NOS activity was reduced. Compared with the model group, the levels of LH, T and E2 in sample 1 group were increased, and the penile NOS activity was increased. The capture times of sample 2 group were significantly increased, the levels of serum GnRH, FSH, T and E2 were significantly increased, and the testicular SOD activity and GSH-Px activity were significantly increased. The levels of serum FSH, T and E2 in sample 3 group were increased. The capture times, mount times and ejaculation times, the testicular index, the levels of serum GnRH, T, E2, FSH and LH, the testicular SOD activity, GSH-Px activity and penile NOS activity were significantly increased in the sample 4 group.

[0145] Mouse mating test is a common method to evaluate sexual function. The latency and frequency of sexual behaviors such as capture, mount, and ejaculation can reflect the strength of sexual function. When one of the results is positive, it indicates that the drug has a certain promoting effect on sexual function. The test of the disclosure finds that sample 2 and sample 4 can increase the capture times of kidney Yang deficiency model mice. It can be seen that sample 2 and sample 4 can improve the mating ability and sexual function of mice. They are good sexual function enhancers and can improve the sexual desire and sexual activity ability of mice to a certain extent.

[0146] Atrophy and pathological changes of reproductive organs can lead to sexual dysfunction to some extent. The results of HE staining showed that the epithelium of seminiferous tubules in the testes of the sample 2 group was significantly improved, spermatogenic cells in all layers were visible, the epididymides were closely arranged, and the number of sperms in the lumen was increased. Therefore, sample 2 can improve the pathological changes of testes.

[0147] Sexual behavior is regulated by sex hormones, and the hypothalamus-pituitary-gonad axis (HPG axis) is the main regulatory pathway in the body. GnRH is synthesized in hypothalamus, which promotes pituitary gonadotropin cells to secrete LH and FSH, and then regulates the synthesis and secretion of sex hormones T and E2, while sex hormones reversely regulate pituitary and hypothalamus to form a feedback pathway to maintain endocrine homeostasis. The test of the disclosure shows that sample 2 can increase the levels of GnRH, FSH, T and E2, sample 1 can increase the levels of LH, T and E2, and sample 3 can increase the levels of FSH, T and E2, suggesting that each sample has the effect of regulating the serum sex hormones of kidney Yang deficiency model mice to varying degrees. Sample 2 may improve sexual function by regulating hypothalamic-pituitary-gonadal axis function, mainly acting on FSH. Sample 1 mainly acts on LH. Sample 3 mainly acts on FSH. LH and FSH mainly coregulate a series of processes related to male growth, development, puberty sexual maturity and reproduction. Among them, LH mainly stimulates interstitial cells to synthesize and secrete T, providing a hormone environment for spermatogenesis. FSH mainly promotes spermatogenesis and germ cell differentiation. Therefore, sample 1 focuses on stimulating the synthesis and secretion of T by interstitial cells, and samples 2 and 3 focus on promoting spermatogenesis and germ cell differentiation.

[0148] NOS is the rate limiting enzyme for the synthesis of nitric oxide (NO), and its activity changes have a great impact on the functional state of the neuroendothelial dependent NO erectile system. If the activity of NOS in penile cavernosum tissue decreases, it will inevitably affect the normal play of erectile function. It is found in the test of the disclosure that sample 1 can increase the penile NOS activity, suggesting that these samples can improve the erectile function of the penis.

[0149] In the current research on male infertility, the view that oxidative stress is the cause of infertility is recognized by most scholars. When the strong oxidants produced in the body are too much and exceed the normal scavenging capacity of the antioxidant enzymes in the body under the original physiological state, the physiological function of the body's normal antioxidation will be abnormal, resulting in tissue damage. SOD and GSH-Px are important antioxidant enzymes to eliminate oxidants in the body. They are also important substances to enhance immunity and prevent diseases in the human body. They can remove free radicals and protect cells from oxidative damage. MDA is the oxidation end product of the oxidation reaction of lipids by free radicals in living organisms, and its content can reflect the degree of oxidative stress damage. The test of the disclosure finds that sample 2 can increase the testicular SOD activity and GSH-Px activity, reduce oxidative stress damage by enhancing the ability to scavenge oxidative free radicals in mice, reduce MDA level, and has better effect in improving oxidative stress damage.

[0150] In conclusion, different samples can improve male sexual function to varying degrees. Sample 1, Sample 2 and Sample 3 can improve the sexual behavior, serum sex hormones and testicular antioxidant capacity of kidney Yang deficiency model mice in varying degrees, and can improve the level of sex hormones by inhibiting oxidative stress, so as to improve male sexual function.

[0151] The above description of the disclosed embodiments enables the skilled in the art to achieve or use the disclosure. Multiple modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be achieved in other embodiments without departing from the spirit or scope of the disclosure. The present disclosure will therefore not be restricted to these embodiments shown herein, but rather to comply with the broadest scope consistent with the principles and novel features disclosed herein.