Preparation for improving male sexual function and preparation method and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a preparation for improving male sexual function and a preparation method and application thereof. The preparation is prepared from the following raw materials: a lactobacillus extracellular vesicle solution and a procyanidine microemulsion, the volume ratio of the lactobacillus extracellular vesicle solution to the procyanidine microemulsion is 1: (3-5); the lactobacillus extracellular vesicle solution is derived from fermentation liquor of lactobacillus; the lactobacillus comprises any one of lactobacillus reuteri, lactobacillus jensenii, lactobacillus acidophilus, lactobacillus delbrueckii and lactobacillus gasseri; the procyanidine micro-emulsion is a micro-emulsion prepared by taking procyanidine as a main raw material. The preparation provided by the invention has a better improvement effect on glycolipid metabolism, vascular endothelial function and erectile function of diabetic erectile dysfunction rats, and provides a new direction for treating and / or improving diabetic erectile dysfunction diseases.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a preparation for improving male sexual function and a preparation method and application thereof. Background Art

[0002] Penile erection requires the coordination of complete molecular and neural, endocrine, and vascular system pathways, and is also affected by psychological factors, systemic diseases, nutrition, and drugs. If any of these processes is interrupted, erectile dysfunction (ED) may occur. ED is a common type of male sexual dysfunction, which means that the penis cannot maintain a full erection and it is difficult to complete satisfactory sexual intercourse. ED not only causes great psychological damage to patients, but also seriously reduces the quality of life of patients.

[0003] Diabetes is a common chronic metabolic disorder characterized by elevated blood sugar levels, which can affect one or more organs throughout the body, leading to acute and chronic complications. ED is a common complication of diabetes. Continuous high blood sugar levels in diabetic patients can cause persistent inflammation of the corpus cavernosum, leading to damage to the vascular endothelial cells of the corpus cavernosum. Continuous endothelial damage can further cause fibrosis, which in turn affects the normal structure and function of the corpus cavernosum. As diabetes becomes younger, diabetic erectile dysfunction (DMED) is clinically manifested by the inability to complete normal sexual life due to the hardness or hardness duration of the corpus cavernosum during erection being lower than normal.

[0004] As the number of patients with diabetes gradually increases, the number of patients with DMED will inevitably increase as well.

[0005] In the prior art, the first choice of intervention for DMED is oral or injection treatment in addition to lifestyle and psychological intervention. Phosphodiesterase 5 (PDE5) inhibitors are commonly used drugs for first-line oral treatment. PDE5 inhibitors include sildenafil, tadalafil and vardenafil, all of which inhibit high concentrations of PDE5 in the corpus cavernosum, thereby preventing the cleavage of 3'5'-cGMP in the corpus cavernosum and promoting penile erection. However, some studies have shown that the efficacy of PDE5 inhibitors in male DMED patients is lower than that in non-diabetic patients, because the effect of PDE5 inhibitors depends on the formation of endogenous NO. Severe endothelial dysfunction or nerve damage can lead to insufficient endogenous bioavailable NO. PDE5 inhibitors may not be able to increase cGMP levels above the necessary threshold, thereby affecting the efficacy. In addition, PDE5 inhibitors currently have many adverse reactions, such as headache, myalgia, back pain, indigestion, blurred vision, facial flushing, nasal congestion and dizziness. In addition, intracavitary injection of papaverine, phentolamine and alprostadil, and transurethral administration of alprostadil as second-line or combined administration methods also have obvious side effects: the former requires a complicated injection procedure and may cause adverse reactions such as penile pain, prolonged erection, abnormal penile erection and fibrosis, while the latter may also cause urethral discomfort.

[0006] In summary, the current drug treatments for DMED have poor effects and significant side effects. Therefore, seeking a drug for DMED that reduces side effects and enhances therapeutic effects is an urgent problem to be solved. Summary of the invention

[0007] In order to solve the problem that the drugs used for DMED in the prior art have poor therapeutic effects and large side effects, the present invention provides a preparation for improving male sexual function and its preparation method and application. To achieve the above purpose, the present invention adopts the following technical solution.

[0008] The invention provides a preparation for improving male sexual function, which is prepared from the following raw materials: lactobacillus extracellular vesicle solution and proanthocyanidin microemulsion.

[0009] The volume ratio of the lactobacillus extracellular vesicle solution to the proanthocyanidin microemulsion is 1:3-5.

[0010] The lactobacillus extracellular vesicle solution is derived from the fermentation broth of lactobacillus; the lactobacillus includes any one of Lactobacillus reuteri, Lactobacillus jensenii, Lactobacillus acidophilus, Lactobacillus delbrueckii and Lactobacillus gasseri. Studies have found that these strains have better effects in regulating the gut-brain axis.

[0011] The proanthocyanidin microemulsion is a uniformly mixed emulsion containing proanthocyanidin, which is composed of proanthocyanidin, water phase, oil phase, surfactant, co-surfactant and auxiliary stabilizer.

[0012] A preparation for improving male sexual function provided by the present invention is a preparation prepared by using lactobacillus extracellular vesicle solution and proanthocyanidin microemulsion as raw materials, and through the synergistic effect of lactobacillus and proanthocyanidin, an antioxidant effect is synergistically exerted, an anti-inflammatory effect is enhanced, and the production of certain neurotransmitters and hormones in the intestine is affected, thereby affecting the hypothalamus-pituitary-gonad axis (HPGA) through the gut-brain axis, and then indirectly regulating the secretion of male reproductive hormones such as testosterone. The preparation provided by the present invention has clear ingredients, no side effects, and improves male erectile dysfunction from multiple angles, including restoring normal sugar and lipid metabolism levels and hormone levels, thereby improving inflammation, restoring the structure and function of the cavernous vascular endothelium, and finally achieving erectile dysfunction improvement and treatment, with good therapeutic effect.

[0013] Preferably, the Lactobacillus extracellular vesicle solution is obtained by dispersing Lactobacillus extracellular vesicles in physiological saline.

[0014] The preparation method of the lactobacillus extracellular vesicles comprises the following steps:

[0015] The fermented liquid of the lactobacillus is centrifuged at low temperature and low speed at 1°C to 9°C and 2500rpm to 5500rpm, and the lower sediment is collected to obtain a bacterial sediment; after the bacterial sediment is resuspended, it is placed at 36°C to 38°C for redissolution after being frozen at -80°C for 1h to 2h; after repeating 3 to 5 times, the first centrifugation is performed to collect the first supernatant; after filtering the first supernatant, the filtrate is collected, and then the second ultrafiltration centrifugation is performed to collect the second supernatant to obtain the lactobacillus extracellular vesicle. This preparation method does not require complex and precise equipment, nor does it require cumbersome operating procedures, and can easily achieve high-yield recovery of extracellular vesicles, while ensuring the purity. Increase the overall recovery of extracellular vesicles.

[0016] Preferably, the conditions for low-temperature and low-speed centrifugation are 2°C to 8°C, 3000 rpm to 5000 rpm, and centrifugation for 10 min to 15 min.

[0017] The conditions of the first centrifugation are 4500rpm-5500rpm for 20min-30min; the method of the second ultrafiltration centrifugation is: the filtrate is placed in a 100KD ultrafiltration tube, at 2℃-8℃, at 2500rpm-3500rpm for 10min-20min.

[0018] Preferably, the proanthocyanidin microemulsion is made of the following ingredients by weight: 2% to 10% proanthocyanidin, 40% to 90% aqueous phase, 5% to 40% oil phase, 10% to 40% surfactant, 2% to 10% co-surfactant and 1% to 4% auxiliary stabilizer; the sum of the mass percentages of the above ingredients is 100%.

[0019] Preferably, the proanthocyanidin is an oligomeric proanthocyanidin with a degree of polymerization between 2 and 5. The oligomeric proanthocyanidin has a lower degree of polymerization, has more active phenolic hydroxyl groups exposed on the molecular surface in the chemical structure, has more active binding sites, and thus has a stronger free radical scavenging ability, and can better play an antioxidant role. In addition, the oligomeric proanthocyanidin generally has a smaller molecular weight, can better penetrate cells, and has a higher bioavailability.

[0020] Preferably, the aqueous phase is water or physiological saline.

[0021] The oil phase is a mixture of any one of isopropyl myristate, isopropyl palmitate, isopropyl octanoate, trioctanoin, soybean oil, peanut oil and olive oil and any one of nitrooleic acid and nitrolinoleic acid.

[0022] Preferably, the surfactant is made of any two materials selected from polyoxyethylene 40 castor oil, polyoxyethylene 35 castor oil, Tween 80, Tween 20 and Span 80; the mass ratio of any two materials is 0.15-5:1.

[0023] The co-surfactant is any one of ethanol, propanol, 1,2-propylene glycol and glycerol.

[0024] The mass ratio of the surfactant to the co-surfactant is 1 to 4:1.

[0025] The mass ratio of the total mass of the surfactant and the co-surfactant to the mass of the oil phase is 0.4-5:1.

[0026] Preferably, the auxiliary stabilizers are PEG-100 and vitamin E.

[0027] The mass ratio of the vitamin E to the proanthocyanidins is 1:5-10.

[0028] The PEG-100 accounts for 0.5% to 5% of the total mass of the proanthocyanidin microemulsion.

[0029] The present invention also provides a method for preparing the preparation for improving male sexual function, comprising the following steps:

[0030] At a rotation speed of 200 rpm to 500 rpm, the lactobacillus extracellular vesicle solution is added to the proanthocyanidin microemulsion at a rate of 0.5 mL / min to 2 mL / min to obtain the preparation for improving male sexual function. The preparation can enhance the stability of the effective ingredients on the one hand, and on the other hand, the microemulsion is used as a carrier system to achieve a sustained release effect, further improving the absorption and bioavailability of the effective ingredients.

[0031] For ease of understanding, the advantages of the preparation provided by the present invention are described below with nitrooleic acid as an example. Due to its fat solubility, nitrooleic acid alone needs to interact with substances such as bile acid to form mixed micelles before it can be effectively absorbed by the gastrointestinal tract. And in the water environment of the gastrointestinal tract, agglomeration may occur, further affecting its absorption efficiency. Furthermore, nitrooleic acid may be directly partially degraded by enzymes in the gastrointestinal tract, resulting in loss. In addition, under conditions such as light, oxygen or high temperature, nitrooleic acid is prone to oxidation reactions, resulting in changes in its chemical structure, thereby losing activity. And as a microemulsion component, the emulsification properties of the microemulsion can make nitrooleic acid better dispersed in the aqueous phase environment of the gastrointestinal tract to form tiny oil droplets. These oil droplets have a smaller particle size and a larger specific surface area, which is conducive to the contact and absorption of nitrooleic acid with the gastrointestinal mucosa. The surfactant and cosurfactant in the microemulsion can reduce the oil-water interfacial tension and help nitrooleic acid penetrate the barrier of the gastrointestinal mucosa. In the microemulsion system, the stability of nitrooleic acid is enhanced. The oil phase components in the microemulsion can provide a relatively stable environment for nitrooleic acid, reducing its contact with external factors (such as oxygen and light). At the same time, the antioxidant components in the microemulsion (such as vitamin E, etc.) can prevent the oxidation of nitrooleic acid and protect the integrity of its chemical structure. The interfacial film in the microemulsion (formed by surfactants and cosurfactants) can serve as a physical barrier to prevent harmful substances such as oxygen from contacting nitrooleic acid. Microemulsion can also achieve the slow release of nitrooleic acid and prolong its action time.

[0032] The present invention also provides an application of a preparation for improving male sexual function in preparing a product for improving male sexual function. The product is an oral capsule or liquid health product prepared by using the preparation for improving male sexual function as a raw material and supplemented with pharmaceutically acceptable excipients.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] The present invention provides a preparation for improving male sexual function. The preparation of the present invention has clear ingredients and no side effects, and improves male erectile dysfunction from multiple angles, including restoring normal glucose and lipid metabolism levels and hormone levels, thereby improving inflammation, restoring the structure and function of the cavernous vascular endothelium, and ultimately achieving improvement and treatment of erectile dysfunction, with good therapeutic effects. Specifically embodied in:

[0035] (1) Proanthocyanidin microemulsion is rich in nitrooleic acid or nitrolinoleic acid, which, as electrophilic small molecule nitroene compounds, directly targets the cysteine ​​proteome. These cysteine ​​residues mainly play a role in regulating pro-inflammatory and adaptive gene expression responses, immune cell activation and the production of oxidative inflammatory mediators in protein regulation, and ultimately show anti-fibrosis and inflammatory effects through multiple signaling effects. Lactobacillus itself can regulate the balance of intestinal flora, and intestinal flora is closely related to the body's immune response. By regulating the intestinal microecology, the Lactobacillus extracellular vesicle solution can affect the body's immune status to a certain extent, inhibit the growth of harmful bacteria, and reduce the triggering factors of inflammatory response. The former acts directly, and the latter acts indirectly, cooperating with each other to enhance the anti-inflammatory effect of the preparation.

[0036] (2) Nitrooleic acid or nitrolinoleic acid has antioxidant, hypoglycemic, anti-apoptotic and lipid metabolism regulating effects. Vitamin E and proanthocyanidins in proanthocyanidin microemulsion can synergistically exert antioxidant effects, protect vascular endothelial cells, and improve blood circulation, which is very important for maintaining male erectile function. The regulation of intestinal flora and immunity by Lactobacillus extracellular vesicle solution may also indirectly affect the blood circulation and function of the reproductive system through the neuro-endocrine-immune network. It works together through multiple mechanisms such as anti-inflammatory and improved blood circulation in improving male sexual function, reflecting a synergistic effect.

[0037] (3) Vitamin E can directly promote androgen secretion, and Lactobacillus extracellular vesicles can affect the production of certain neurotransmitters and hormones in the intestine, such as serotonin (5-hydroxytryptamine), thereby affecting the hypothalamus-pituitary-gonadal axis through the gut-brain axis, and then indirectly regulating the secretion of male reproductive hormones such as testosterone. In addition, the improvement of intestinal flora balance can also reduce the entry of harmful substances such as endotoxins into the blood circulation, avoid these substances from interfering with the endocrine system, and thus maintain a normal hormone secretion environment.

[0038] In addition, the preparations for improving male sexual function are all made of food-grade raw materials, are safe and effective, easy to use, and have no obvious toxic side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a particle size distribution diagram of the extracellular vesicles of Lactobacillus in the present invention.

[0040] Figure 2 It is a transmission electron micrograph of the extracellular vesicles of Lactobacillus in the present invention.

[0041] Figure 3 The graphs of particle size and potential changes of the proanthocyanidin microemulsion stored at different temperatures for 3 months in the present invention are shown; wherein, Figure 3 A in the figure is the particle size change diagram; Figure 3 B in the figure is the potential change diagram.

[0042] Figure 4 This is a graph showing changes in blood sugar levels in the present invention.

[0043] Figure 5 The sugar metabolism in the present invention: serum insulin level and liver glycogen level; wherein, Figure 5 A in it is the serum insulin level; Figure 5 B in the figure represents the liver glycogen level; where * indicates p < 0.05, and ** indicates p < 0.01.

[0044] Figure 6 The blood lipid level in the present invention is: total cholesterol (TC); triglyceride (TG); high-density lipoprotein cholesterol (HDL-C); low-density lipoprotein cholesterol (LDL-C); wherein, Figure 6 A in is TC; Figure 6 B in it is TG; Figure 6 The C in it is HDL-C; Figure 6 D in the figure represents LDL-C; * represents p<0.05, ** represents p<0.01, and *** represents p<0.001.

[0045] Figure 7 For the erectile function evaluation of the present invention; wherein, Figure 7 A in it is the maximum intracavernous pressure (Max ICP); Figure 7 B in is mean arterial pressure (MAP); Figure 7 C in the figure is the Max ICP / MAP ratio; * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001.

[0046] Figure 8 The endothelial function in the present invention is affected by: NO expression level; ET-1 expression level; LOX-1 expression level; wherein, Figure 8 A in the figure represents the expression level of nitric oxide (NO); Figure 8 B in the figure is the expression level of endothelin 1 (ET-1); Figure 8 C in the figure represents the expression level of lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1); among them, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0047] Fig. 9 The expression level of sex hormones in the present invention: serum testosterone (Testosterone, T) expression level; follicle stimulating hormone (FSH) expression level; luteinizing hormone (LH) expression level; wherein, Fig. 9 A in is the expression level of T; Fig. 9 B in the figure is the expression level of FSH; Fig. 9C in the figure represents the expression level of LH; among them, * represents p<0.05, ** represents p<0.01, and *** represents p<0.001. DETAILED DESCRIPTION

[0048] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0049] Example 1: Preparation of a preparation for improving male sexual function

[0050] 1. Preparation of Lactobacillus extracellular vesicles

[0051] 1. Bacterial recovery and culture

[0052] The Lactobacillus reuteri glycerol storage tube stored at -80°C was placed in a 37°C water bath and gently shaken to thaw, and then 1 mL of bacterial solution was aspirated with a pipette under sterile conditions, inoculated into a test tube containing 10 mL of MRS medium, and activated and cultured in an anaerobic incubator at 37°C for 24 hours to obtain a seed fermentation liquid. Subsequently, the Lactobacillus reuteri seed fermentation liquid was inoculated into 100 mL of MRS medium at a volume ratio of 1%, and was allowed to stand in an anaerobic incubator at 37°C for 24 hours for expansion culture to obtain a Lactobacillus reuteri fermentation liquid.

[0053] Among them, Lactobacillus reuteri was purchased from China Industrial Microbiological Culture Collection Center.

[0054] The full English name of MRS medium is Man-Rogosa-Sharpe medium, which comes from Guangdong Huankai Microbiological Technology Co., Ltd.; phosphate buffer.

[0055] 2. Vesicle Collection

[0056] After the cultivation is completed, the obtained Lactobacillus reuteri fermentation liquid is collected and centrifuged at low temperature (4°C) and low speed (5000rpm) for 10 minutes to make the bacteria precipitate at the bottom of the centrifuge tube, discard the supernatant, and collect the lower bacterial precipitate. Then wash the bacterial precipitate with sterile saline, repeat the centrifugation step to remove the impurities in the culture medium, and obtain the bacterial precipitate. Then add sterile saline to the bacterial precipitate again, mix it evenly, place it in a -80°C refrigerator and freeze it for 2 hours, and then quickly transfer it to a 37°C water bath to thaw. Repeat the freezing and thawing 3 times. Then transfer the thawed liquid to a centrifuge tube, centrifuge it at 5000rpm for 10 minutes, discard the precipitate, and collect the supernatant. The supernatant was filtered with a 0.22 μm filter membrane, and the filtrate was transferred to a 100 KD ultrafiltration tube and centrifuged at 3000 rpm for 15 min at 4°C. This process was repeated until the concentrated volume of the liquid was 1 / 4 of the original solution. An equal volume of physiological saline was added again and centrifuged under the same conditions to obtain a concentrated solution, which was the Lactobacillus extracellular vesicle solution. This was frozen and stored at -80°C for subsequent use.

[0057] 2. Characterization of Lactobacillus extracellular vesicles

[0058] 1. Particle size determination

[0059] The Lactobacillus extracellular vesicle solution was diluted 100 times with physiological saline, and then 500 μL was loaded into the NS300 nanoparticle tracking analyzer for detection. Five different fields of view were detected for each sample, and the acquired data were analyzed using NTA 3.2 software.

[0060] Among them, NS300 nanoparticle tracking analyzer: Malvern, Worchestershire, UK.

[0061] The instrument detection parameters were set as follows: Camera Level was 16, Time of Video Records was 60 s, Detect Threshold was 7, and other parameters were set to default.

[0062] Test results are shown in Figure 1 ..

[0063] Depend on Figure 1 It can be seen that the particle size of Lactobacillus extracellular vesicles is between 75nm and 225nm, and the vesicles with a particle size of 105nm are the most numerous.

[0064] 2. Transmission electron microscopy observation

[0065] According to the particle size determination results, the concentration of Lactobacillus extracellular vesicles was adjusted to about 1×10 10particles / mL, use a pipette to draw 10μL of sample suspension and carefully drop it onto a 200-mesh copper grid with a support membrane; let it stand at room temperature for 3 minutes, carefully absorb the excess liquid with filter paper, and when it is almost completely dry, use a pipette to draw a drop of 2% (w / v) sodium phosphotungstate dye solution, dye for 2 minutes, and then use filter paper to absorb the excess dye solution, let it stand at room temperature, and observe it on a microscope after it is dry.

[0066] The prepared vesicles were observed and photographed using a JEM1011 transmission electron microscope. The acceleration voltage of the transmission electron microscope was set to 100 kV, and other parameters were set to default.

[0067] Wherein, sodium phosphotungstate dye solution: pH = 7.0, purchased from Solebol, product number G1870.

[0068] JEM1011 transmission electron microscope: JEOL, Tokyo, Japan.

[0069] Transmission electron microscopy results Figure 2 shown.

[0070] The transmission electron microscopy results show that the size of the separated vesicles ranges from 50nm to 150nm, which is consistent with the size measurement results. By observing the structure of these vesicles, it was found that most of them are elliptical and contain double-layer membrane bubble structures (indicated by the arrows).

[0071] 3. Protein concentration determination

[0072] The protein content of the sample (Lactobacillus extracellular vesicle solution) was determined using a BCA protein quantification kit. A standard curve for protein quantification was first established according to the method provided in the kit instructions, and then the protein content of the sample was calculated based on the OD value of the sample.

[0073] The experimental results show that 1×10 10 The protein content of particles / mL was 24.4 μg / mL.

[0074] Among them, BCA protein quantification kit: Solebao, catalog number: PC0020.

[0075] 3. Preparation of proanthocyanidin microemulsion

[0076] Weigh 0.3g proanthocyanidin, 1.45g polyoxyethylene 35 castor oil, 0.48g Span-80, 0.64g glycerol, 0.85g isopropyl octanoate, 0.15g nitrooleic acid, 0.03g vitamin E, 0.1g PEG-100, and 6.0g distilled water. Stir polyoxyethylene 35 castor oil, Span-80, PEG-100, isopropyl octanoate, nitrooleic acid, and vitamin E at 500rpm / min to mix them evenly, then take another beaker to mix glycerol and distilled water evenly at 60℃, then slowly add the liquid in the previous beaker to the above beaker under the stirring state of 1000rpm / min until a uniform liquid is formed, finally cool to room temperature and then add proanthocyanidin, stir well, and obtain a dark red, clear and transparent proanthocyanidin microemulsion, which is sealed and stored away from light.

[0077] Among them, the proanthocyanidins are oligomeric proanthocyanidins purchased from Guilin Rhine Biotechnology Co., Ltd., and the effective content of proanthocyanidins is 95%.

[0078] 4. Structural Characterization of Proanthocyanidin Microemulsion

[0079] 1. Appearance observation

[0080] The appearance of the proanthocyanidin microemulsion was observed to be a clear dark red transparent liquid, and there was no stratification, precipitation or flocculation after being left for 1 hour.

[0081] 2. Particle size and distribution determination

[0082] The particle size distribution and potential of the microemulsion were regularly tested using a nanoparticle size analyzer to ensure that the particle size was stable and had no significant changes during storage, thereby ensuring the stability and physical properties of the microemulsion.

[0083] Results Figure 3 .

[0084] 3. Stability inspection

[0085] The prepared microemulsion was stored at different temperatures: 4°C, 25°C, and 40°C for 3 months, and its appearance changes were observed. The particle size and potential changes were regularly tested to evaluate the stability of the microemulsion.

[0086] Results Figure 3 .

[0087] Regular observation of the microemulsion showed no stratification or flocculation. Figure 3 From the particle size and potential change diagram, it can be seen that the particle size and potential do not change much when stored at different temperatures (such as 4°C, 25°C, and 45°C) for 3 months. Combined with the appearance observation of the emulsion, they are all clear without stratification and precipitation, which proves that the emulsion has good stability.

[0088] 5. Preparation of preparations for improving male sexual function

[0089] Under stirring at 300 rpm / min, 20 volumes of Lactobacillus extracellular vesicle solution were added to 80 volumes of proanthocyanidin microemulsion at a drop rate of 1 mL / min and stirred evenly to obtain a preparation for improving male sexual function.

[0090] Example 2: Evaluation of the efficacy of a preparation for improving male sexual function

[0091] 1. Methods

[0092] 1. Establishment of diabetic erectile dysfunction model (DMED model)

[0093] Six-week-old male Sprague-Dawley rats with an average body weight of 200 ± 20 g were purchased from the Experimental Animal Center of Xi'an Jiaotong University.

[0094] Ten rats were randomly selected as the control group (CON, injected with the same dose of citric acid buffer), and the remaining rats were intraperitoneally injected with streptozotocin to establish the diabetic model.

[0095] Among them, streptozotocin: STZ, purchased from Solebao, product number: S8050.

[0096] STZ preparation method: 10 mg / mL, dissolved in citric acid buffer, prepared and used immediately, stored in an ice bath away from light, used immediately after filtering with a 0.22 μm filter membrane, and injection completed within 30 minutes, STZ injection dose is 65 mg / kg, injection method is intraperitoneal injection, injection times are single injections. Citric acid buffer was purchased from Solebao, item number: C1013.

[0097] Blood glucose was tested by sampling from the tip of the tail 72 hours after injection, and the diabetes model was considered to be successfully established if the blood glucose value was >16.7mmol / L as measured by a blood glucose meter.

[0098] DMED model establishment: 2 weeks after the diabetic model was established, apomorphine (APO) solution was injected into the loose skin of the rat neck at a dose of 80 μg / kg. The rats were observed for erectile reactions (full glans penis, and the end of the penis was exposed) within 30 minutes. Those without erection were considered to have successfully established the DMED model.

[0099] Among them, vitamin C was purchased from Solebao, item number: A8100. Physiological saline (0.9%, sterile) was purchased from Sichuan Kelun Pharmaceutical Co., Ltd.

[0100] Apomorphine (APO): purchased from Sigma, USA, product number: PHR2621. Apomorphine (APO) solution consists of 5 mg APO, 250 mg vitamin C and 500 mL normal saline.

[0101] 2. Preparation processing and grouping

[0102] The rats with successful DMED model were randomly divided into model group (MOD), positive control group (TAD, tadalafil, 0.52 mg / kg / d, gavage), and experimental group (EXP, preparation, 0.80 mg / kg / d)

[0103] 3. Erectile function evaluation

[0104] After 4 weeks of drug administration, the maximum intracavernous pressure (Max ICP), mean arterial pressure (MAP) and Max ICP / MAP ratio of rats in each group were measured to evaluate erectile function. The specific method is as follows: Before measurement, the rats were anesthetized, the right common carotid artery was carefully separated, and a PE-50 tube containing 250IU / mL heparin saline was inserted, and a pressure sensor was attached to the tail end to facilitate the measurement of MAP. The rat's abdominal perineum was cut inward to expose the corpus cavernosum, a 25-gauge needle was connected to the PE-50 tube, and the corpus cavernosum was inserted to measure ICP, and Max ICP was calculated. Both were connected to the Powerlab work platform (5V voltage, 15HZ frequency, 1.2ms wave width, 1 minute duration, 3 minutes interval electrical stimulation) to observe ICP / MAP and calculate Max ICP / MAP.

[0105] Among them, it contains 250IU / mL heparin saline: Beijing Coolaber Technology Co, Ltd, Beijing, China.

[0106] Powerlab workbench: ADInstruments, Sydney, Australia.

[0107] 4. Blood glucose measurement

[0108] Blood glucose was tested by sampling blood from the tip of the tail and measuring blood glucose level using a blood glucose meter.

[0109] 5. Serological indicators

[0110] The levels of serum insulin, liver glycogen, TG, TC, HDL-C and LDL-C of rats in each group were measured to evaluate the effects on glucose and lipid metabolism.

[0111] The levels of T, FSH, LH, NO, ET-1 and LOX-1 in rats of each group were measured to evaluate the effects on sex hormone levels and vascular endothelial function.

[0112] The method of collecting samples from each group of rats was as follows: 2% wt sodium pentobarbital was intraperitoneally injected to anesthetize the rats (0.25 mL / 100 g body weight). After the rats were anesthetized, they were quickly cut open from the abdomen upwards to expose the entire abdominal cavity and fixed with hemostatic forceps. The abdominal aorta was separated with forceps, and blood was drawn from the abdominal aorta with a 20 mL syringe and injected into a disposable centrifuge tube. The tube was left to stand at room temperature for 2 hours, centrifuged (3000 r / min) for 15 minutes, and the supernatant was separated and stored at -80°C for testing. Serum insulin, glycogen, TG, TC, HDL, and LDL levels were detected using detection kits (insulin, glycogen detection kit, TG detection kit, TC detection kit, HDL detection kit, and LDL detection kit, respectively), and the detection methods were carried out according to the instructions. Among them, insulin, glycogen detection kit, TG detection kit, TC detection kit, HDL detection kit, and LDL detection kit were all purchased from Nanjing Jianjian Bioengineering Research Institute, and the article numbers are H203-1-1, A043-1-1, A110-1-1, A111-1-1, A112-1-1, and A113-1-1, respectively.

[0113] At the same time, ELISA was used to quantitatively measure the expression levels of sex hormones such as FSH, LH, T, and vascular endothelial function indicators such as NO, ET-1, and LOX-1. The operation methods were strictly followed in accordance with the kit.

[0114] The purchase information of FSH, LH, T, NO, ET-1 and LOX-1 assay kits is as follows:

[0115] Rat FSH ELISA Kit, catalog number: E-EL-R0391.

[0116] Rat LH ELISA Kit, catalog number: E-EL-R0026.

[0117] QuicKey Pro-Rat T ELISA Kit, Cat. No.: E-OSEL-R0003.

[0118] NO colorimetric test kit (nitrate reductase method), item number: E-BC-K135-M.

[0119] Rat ET-1 ELISA kit, catalog number: E-EL-R1458. All of the above were purchased from Elabscience.

[0120] Rat lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1) ELISA kit, Wuhan Aidikang Biotechnology Co., Ltd., catalog number: AD31676.

[0121] 2. Experimental results:

[0122] 1. Blood glucose measurement

[0123] The results are as follows Figure 4 shown.

[0124] From the blood sugar level change graph, it can be seen that the blood sugar level of rats in the DMED group was significantly increased compared with the control group. Compared with the DMED group, the treatment group was able to reduce the blood sugar level, while the positive control group did not show any obvious hypoglycemic effect.

[0125] The above results show that the preparation has a significant hypoglycemic effect.

[0126] 2. Effects on glucose and lipid metabolism

[0127] Insufficient serum insulin secretion is an important pathogenic factor in the development of diabetes. The liver is the center of glucose and lipid metabolism. Glycogen is the main source of blood sugar. Reduced glycogen synthesis and increased decomposition will lead to increased blood sugar. The balance between glycogen synthesis and catabolism is the key determinant for maintaining stable blood sugar levels. By testing serum insulin levels and glycogen levels, it can be seen that the serum insulin and glycogen levels of rats in the model group were lower than those in the control group ( Figure 5 A and Figure 5 B). Compared with the model group, the serum insulin and liver glycogen levels of rats treated with the preparation were significantly increased ( Figure 5 A and Figure 5 B).

[0128] 3. Blood lipid levels

[0129] The results are as follows Figure 6 As shown. TC is the sum of cholesterol contained in various lipoproteins in the blood and is one of the important indicators of blood lipids. Increased TC means increased blood lipid levels. TG is a lipid formed by the combination of glycerol and fatty acids in the blood. Increased TG levels are also a common manifestation of dyslipidemia. LDL is called "bad cholesterol". It transports cholesterol from the liver to peripheral tissues. When LDL levels increase, it is easy to deposit on the blood vessel walls, increasing the risk of dyslipidemia and cardiovascular disease. On the contrary, HDL is called "good cholesterol". It can transport cholesterol from peripheral tissues to the liver for metabolism. Increased HDL levels help reduce the risk of dyslipidemia and cardiovascular disease. Lipid metabolism and sugar metabolism influence and restrict each other. Compared with the control group, the TC, TG, and LDL levels of the model group rats increased, and the HDL level decreased. After treatment, the preparation group reduced the TC, TG and LDL levels and increased the HDL level. TAD had no effect on the blood lipids of DMED rats. It can be seen that the preparation has a good regulatory effect on the blood glucose level, serum insulin, liver glycogen and blood lipids of DMED rats. This suggests that it can significantly improve the glucose and lipid metabolism of DMED rats.

[0130] 4. Erectile function

[0131] The ICP / MAP ratio is a reliable indicator for evaluating erectile function. Our results showed that the ICP and ICP / MAP ratio of rats in the DMED group were significantly lower than those in the control group ( Figure 7 ). Compared with untreated rats, ICP and ICP / MAP ratio were significantly improved in both formulation-treated and positive drug-treated rats ( Figure 7 ).

[0132] 5. Endothelial function

[0133] Penile erection is a neuroendocrine-regulated vasopressor response characterized by changes in relaxation, which can be regulated by endothelial function. NO, ET-1, and LOX1 are important biomarkers of endothelial function. NO is the most important molecule involved in penile erection. After the body is physically or centrally stimulated, NO is released from the endothelial cells of the cavernous nerves, activating soluble guanylate cyclase (sGC), which increases the level of cyclic guanosine monophosphate (cGMP), leading to smooth muscle relaxation and arteriolar dilation, thereby achieving penile erection. LOX-1 is mainly expressed on endothelial cells and is involved in the recognition and internalization of oxidized low-density lipoprotein (ox-LDL). The main function of LOX-1 is to mediate the uptake of ox-LDL by endothelial cells, leading to damage to endothelial cell function. The higher the expression level of LOX-1, the more serious the damage to endothelial cell function. Overexpression of LOX-1 further reduces NO synthesis and increases ET-1 synthesis. Excessive ET-1 can promote fibrosis of the corpus cavernosum, reduce the elasticity and compliance of the corpus cavernosum, affect its normal relaxation and contraction function, and impair erectile function. The results of enzyme-linked immunosorbent assay (ELISA) showed that the serum NO level of rats in the model group was significantly reduced ( Figure 8 A) ET-1 and LOX-1 levels were significantly increased ( Figure 8 B and Figure 8 C). Compared with the model group, the NO level increased significantly after treatment with the preparation, while the ET-1 and LOX-1 levels decreased, especially the ET-1 level was closer to the normal control group.

[0134] 6. Sex hormone levels

[0135] Endothelial function in male rats is regulated by sex hormones. T can promote the expression and activity of endothelial nitric oxide synthase (eNOS), increase NO levels, and reduce ET-1 levels. It is a key hormone for maintaining male erection. LH and FSH can both indirectly affect testosterone secretion. Fig. 9 As shown, the T level of rats in the model group was significantly lower than that in the control group. Compared with the model group, the T level in the preparation treatment group was significantly increased. However, the analysis showed that there was no statistically significant difference in LH and FSH levels between the two groups.

[0136] From the above experimental results, it can be seen that the present invention provides a preparation for improving male sexual function. The preparation of the present invention has clear ingredients, no side effects, and improves male erectile dysfunction from multiple angles, including restoring normal glucose and lipid metabolism levels and hormone levels, thereby improving inflammation, restoring the structure and function of the cavernous vascular endothelium, and ultimately achieving improvement and treatment of erectile dysfunction with good therapeutic effect.

[0137] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0138] Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they understand the basic inventive concepts, and all such changes and modifications fall within the scope of the present invention.

Claims

1. A preparation for improving male sexual function, characterized in that Made from the following raw materials: Lactobacillus extracellular vesicle solution and proanthocyanidin microemulsion; The volume ratio of the lactobacillus extracellular vesicle solution to the proanthocyanidin microemulsion is 1:3-5; The lactobacillus extracellular vesicle solution is derived from the fermentation broth of lactobacillus; the lactobacillus includes any one of Lactobacillus reuteri, Lactobacillus jensenii, Lactobacillus acidophilus, Lactobacillus delbrueckii and Lactobacillus gasseri; The proanthocyanidin microemulsion is a uniformly mixed emulsion containing proanthocyanidin, which is composed of proanthocyanidin, water phase, oil phase, surfactant, co-surfactant and auxiliary stabilizer.

2. A preparation for improving male sexual function according to claim 1, characterized in that: The lactobacillus extracellular vesicle solution is obtained by dispersing lactobacillus extracellular vesicles in physiological saline; The preparation method of the lactobacillus extracellular vesicles comprises the following steps: The fermentation liquid of the lactobacillus is centrifuged at low temperature and low speed under the conditions of 1°C to 9°C and 2500rpm to 5500rpm, and the lower layer precipitate is collected to obtain a bacterial precipitate; after the bacterial precipitate is resuspended, it is frozen at -80°C for 1h to 2h and then placed at 36°C to 38°C for redissolution; after repeating 3 to 5 times, the first centrifugation is performed to collect the first supernatant; after filtering the first supernatant, the filtrate is collected, and then a second ultrafiltration centrifugation is performed to collect the second supernatant to obtain the lactobacillus extracellular vesicles.

3. A preparation for improving male sexual function according to claim 2, characterized in that: The conditions for low-temperature low-speed centrifugation are 2°C to 8°C, 3000rpm to 5000rpm, and centrifugation for 10min to 15min; The conditions of the first centrifugation are 4500rpm-5500rpm for 20min-30min; the method of the second ultrafiltration centrifugation is: the filtrate is placed in a 100KD ultrafiltration tube, at 2℃-8℃, at 2500rpm-3500rpm for 10min-20min.

4. A preparation for improving male sexual function according to claim 1, characterized in that: The proanthocyanidin microemulsion is made of the following ingredients by weight percentage: 2% to 10% proanthocyanidin, 40% to 90% aqueous phase, 5% to 40% oil phase, 10% to 40% surfactant, 2% to 10% co-surfactant and 1% to 4% auxiliary stabilizer; the sum of the mass percentages of the above ingredients is 100%.

5. A preparation for improving male sexual function according to claim 4, characterized in that: The proanthocyanidin is an oligomeric proanthocyanidin with a polymerization degree of 2 to 5.

6. A preparation for improving male sexual function according to claim 4, characterized in that: The aqueous phase is water or physiological saline; The oil phase is a mixture of any one of isopropyl myristate, isopropyl palmitate, isopropyl octanoate, trioctanoin, soybean oil, peanut oil and olive oil and any one of nitrooleic acid and nitrolinoleic acid.

7. A preparation for improving male sexual function according to claim 4, characterized in that: The surfactant is made of any two materials selected from polyoxyethylene 40 castor oil, polyoxyethylene 35 castor oil, Tween 80, Tween 20 and Span 80; the mass ratio of any two materials is 0.15-5:1; The co-surfactant is any one of ethanol, propanol, 1,2-propylene glycol and glycerol; The mass ratio of the surfactant to the co-surfactant is 1 to 4:1; The mass ratio of the total mass of the surfactant and the co-surfactant to the mass of the oil phase is 0.4-5:

1.

8. A preparation for improving male sexual function according to claim 4, characterized in that: The auxiliary stabilizers are PEG-100 and vitamin E; The mass ratio of the vitamin E to the proanthocyanidins is 1:5-10; The PEG-100 accounts for 0.5% to 5% of the total mass of the proanthocyanidin microemulsion.

9. The method for preparing a preparation for improving male sexual function according to claim 1, characterized in that: The steps include: At a rotation speed of 200 rpm to 500 rpm, the lactobacillus extracellular vesicle solution is added to the proanthocyanidin microemulsion at a rate of 0.5 mL / min to 2 mL / min to obtain the preparation for improving male sexual function.

10. Use of a preparation for improving male sexual function in preparing a product for improving male sexual function, characterized in that: The product is an oral capsule or liquid health product prepared by using the preparation for improving male sexual function as claimed in claim 1 as a raw material and supplemented with pharmaceutically acceptable excipients.

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