CN116173112B - Traditional Chinese medicine formula probiotic composition, preparation method and application thereof in improving male sexual function - Google Patents
Traditional Chinese medicine formula probiotic composition, preparation method and application thereof in improving male sexual function Download PDFInfo
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Abstract
The invention belongs to the field of medicines, in particular to the field of traditional Chinese medicines and microorganisms, and more particularly relates to a traditional Chinese medicine formula probiotic composition, a preparation method and application thereof in improving male sexual functions. The probiotic composition comprises traditional Chinese medicine extract powder and raw fungus freeze-dried powder; wherein the traditional Chinese medicine extract powder comprises, by weight, 6-15 parts of cistanche deserticola, 10-25 parts of oyster, 6-15 parts of mulberry, 1-8 parts of cinnamon and 4-15 parts of fingered citron; the raw bacteria freeze-dried powder consists of freeze-dried powder of combined bacteria of lactobacillus reuteri, lactobacillus plantarum, lactobacillus rhamnosus, lactobacillus paracasei and lactobacillus salivarius. The composition can be prepared into medicine for improving male sexual function.
Description
Technical Field
The invention belongs to the field of medicines, in particular to the field of traditional Chinese medicines and microorganisms, and more particularly relates to a traditional Chinese medicine formula probiotic composition, a preparation method and application thereof in improving male sexual functions.
Background
As one of the most common high-rise diseases of adult males, the population mainly concentrates on males between 40 and 70 years old, and researches show that about 50 to 60 percent of males in the age group have male sexual dysfunction with different severity degrees, and patients with sexual dysfunction show a younger trend along with various factors such as the acceleration of life rhythm and irregular diet, and if the patients cannot be treated efficiently in time, the conditions of delayed illness and even aggravation are extremely easily caused, so that the optimal treatment time is missed.
Male sexual dysfunction is a serious disease of the male family, and the patients have insufficient sexual function and sexual satisfaction, and are particularly manifested by symptoms such as sexual desire disorder, impotence, premature ejaculation, spermatorrhea, non-ejaculation, retrograde ejaculation and the like. Male sexual activity is a physiological process involving a variety of conditioned and unconditioned reflexes, including multiple links such as libido, penile erection, intercourse, ejaculation, and orgasm. Sexual function is an organic lesion such as a physiological process, a reproductive system disease and a systemic disease which occurs due to the synergistic effect of multiple aspects such as a nervous system, an endocrine system and a sexual organ, and the occurrence of a disorder in any link can be regarded as a performance disorder, and the disease can affect the normal life and even mental health of a patient. Therefore, it is extremely important to study the risk factor of male sexual dysfunction and further to take corresponding effective therapeutic measures to improve the quality of sexual life of patients.
Currently, male sexual dysfunction is mostly treated by western medicines, surgery and the like. However, since surgery requires a certain recovery period and treatment period, and the price of surgery treatment is high, most patients do not want to choose a surgical method for treatment. However, most of the traditional Chinese medicines for treating male sexual dysfunction have the defects of long treatment time and insignificant effect. Based on the research, the action mechanism of the probiotics in the traditional Chinese medicine formula on improving the sexual dysfunction is discussed, and clinical guidance and basis are provided for more effectively treating the sexual dysfunction.
The probiotics are used for treating male sexual dysfunction and hyposexuality, so far, no related literature reports exist, but the effect of the probiotics on intestinal tracts is confirmed, in recent years, along with the occurrence of intestinal-testicular axis and intestinal-renal axis theory, the inventor has a certain inspired effect on male sexual dysfunction and hyposexuality, the inventor combines five probiotic strains according to the characteristics of the probiotic strains, and fuses effective traditional Chinese medicine components with the combined probiotics, so that the probiotics not only have the effects of enhancing immunity, maintaining intestinal flora balance and participating in overall metabolism improvement, but also have good probiotic effects, and the effect of improving the sexual dysfunction and hyposexuality is obviously better than that of pure traditional Chinese medicine treatment through animal observation.
Disclosure of Invention
The inventor obtains a traditional Chinese medicine formula probiotic composition and a preparation method through research, and the composition can be used for improving male sexual function and is verified by experiments, thereby completing the invention.
The invention provides a probiotic composition with a traditional Chinese medicine formula, which comprises traditional Chinese medicine extract powder and raw fungus freeze-dried powder. The traditional Chinese medicine extract powder and the probiotics freeze-dried powder are uniformly mixed according to the volume ratio of 1:0.8-1.2 (preferably 1:0.9-1.1, more preferably 1:1), and the traditional Chinese medicine extract powder and the probiotics freeze-dried powder are particularly prepared by uniformly stirring and mixing by a micro-stirrer, and are stored in the environment of 4 ℃ after being prepared.
The preparation method of the traditional Chinese medicine extract powder comprises the following steps: 6-15 parts of cistanche, 10-25 parts of oyster, 6-15 parts of mulberry, 2-8 parts of cinnamon and 6-15 parts of fingered citron. Cutting or pulverizing the above materials in proportion, soaking in water for 30 min, heating to boil, leaching, separating decoction, decocting residues for 2-3 times, and collecting decoction; extracting the decoction with ethanol; drying the filtrate, and pulverizing into superfine powder smaller than 10 μm.
The invention adopts the traditional Chinese medicine formula as the experience formula of the inventor, combines the dosage range of various traditional Chinese medicines in Chinese pharmacopoeia, is selected through clinical experience, has better curative effect, and has the key effects of tonifying qi and yin, tonifying spleen and kidney, strengthening reproduction, improving medicine species essence and special curative effect in the prescription composition, and is the key for exerting the effect of improving sexual function.
The lyophilized powder of raw bacteria comprises Lactobacillus reuteri, lactobacillus plantarum, lactobacillus rhamnosus, lactobacillus paracasei and Lactobacillus salivarius. The probiotics have no literature report on treating male sexual dysfunction and hyposexuality, but the effect of the probiotics on intestinal tracts has been confirmed, and in recent years, the occurrence of intestinal-testicular axis and intestinal-renal axis theories has inspired the inventors to treat male sexual dysfunction and hyposexuality, the inventors have combined strains according to the characteristics of the probiotics strains, and the combined strains have a certain help to improve the sexual dysfunction and hyposexuality. Wherein, lactobacillus reuteri: has good probiotic properties. Lactobacillus plantarum: and (3) increasing mucin yield, and participating in the whole metabolism improvement. Lactobacillus rhamnosus: has the functions of regulating immunity, promoting growth of bifidobacterium and playing a role. Lactobacillus paracasei: has the effects of enhancing immunity, maintaining intestinal flora balance, promoting digestion, etc. Lactobacillus salivarius: the external interaction with dendritic cells has a certain adsorption effect, and can well colonise and grow on the surface of intestinal tracts so as to play a role in immunity.
The preparation process of the raw fungus freeze-dried powder is completed through the following steps: firstly, storing the thalli in a refrigerator at the temperature of minus 80 ℃ for fermentation and amplification, then freezing/granulating the thalli by strain, freeze-drying the thalli, and finally grinding the thalli into fine powder. The 5 probiotics freeze-dried powders can be purchased from commercial approaches, and then are stored in a low-temperature refrigerator for standby after being uniformly mixed according to the proportion. Specifically, one part of lactobacillus paracasei, lactobacillus plantarum and lactobacillus rhamnosus dry powder, two parts of lactobacillus reuteri and lactobacillus salivarius dry powder are uniformly mixed according to a proportion for standby.
The invention also provides a preparation method of the traditional Chinese medicine formula probiotic composition, which is characterized in that: the method comprises the following steps:
cutting or pulverizing the above materials in proportion, soaking in water in a decoction vessel, heating to boil, leaching, separating decoction, decocting residues for 2-3 times, and collecting decoction; extracting the decoction with ethanol; drying the filtrate extracted by alcohol, and then crushing the dried filtrate by using a superfine pulverizer, wherein the superfine powder is preferably processed into superfine powder smaller than 10 mu m;
the traditional Chinese medicine extract powder and the probiotics freeze-dried powder are uniformly mixed according to the volume ratio of 1:0.8-1.2 (preferably 1:1), and the traditional Chinese medicine extract powder and the probiotics freeze-dried powder are particularly uniformly mixed by a micro-mixer.
The invention further provides application of the traditional Chinese medicine formula probiotic composition in preparing medicines for treating or improving male sexual functions. The improvement of male sexual function refers to modification of male hyposexuality, improvement of sexual dysfunction and the like. Specifically, the improvement of male sexual function refers to the following sexual function problems: sexual dysfunction, impotence, premature ejaculation, spermatorrhea, non-ejaculation and retrograde ejaculation.
The medicine is suitable for injection and oral administration.
According to the invention, by means of the method of extracting the traditional Chinese medicine and adding probiotics, the two processes are deeply fused to prepare the probiotics with the traditional Chinese medicine formula, so that the treatment effect is improved, and the treatment expectation is realized. Specifically, the invention combines the traditional Chinese medicine extraction method, and deeply fuses the traditional Chinese medicine extraction method with probiotics to prepare the probiotics in the traditional Chinese medicine formula, thereby improving the treatment effect and realizing the treatment expectation. Animal experiments show that the traditional Chinese medicine formula probiotics prepared by combining the traditional Chinese medicine formula with probiotics has obvious treatment effect, and the combination of the probiotics and the traditional Chinese medicine decoction has complementary effects, so that the defects of decoction and inconvenient administration of traditional Chinese medicine decoction are overcome, the effect of pure traditional Chinese medicine treatment is enhanced, and the treatment effects of traditional Chinese medicine + probiotics treatment on improvement of immune indexes such as sexual hyposexuality animal model sexual function recovery, blood lipid level, anti-sperm antibodies and the like, oxidative stress, sperm activity, testis tissue morphology, semen viable bacteria, microorganism subgroups and the like are better than those of pure traditional Chinese medicine treatment of a control group, so that the traditional Chinese medicine composition can improve sexual dysfunction through a plurality of targets. The invention has obvious technical advantages compared with the prior art through strict experimental verification.
Animal experiments show that the treatment effect of probiotics and traditional Chinese medicine treatment of the treatment group on improvement of immune indexes such as sexual hyposexuality animal models, blood lipid level, anti-sperm antibodies and the like, oxidative stress, sperm motility, testis tissue morphology, semen viable bacteria, microorganism subpopulations and the like is superior to that of the control group pure traditional Chinese medicine treatment, and the treatment effect is superior to that of the control group pure traditional Chinese medicine treatment, so that the treatment group can improve sexual dysfunction through a plurality of targets. The invention has obvious technical advantages compared with the prior art through strict experimental verification.
Drawings
FIG. 1Westernblot detection results. And (3) injection: the left image is a Western blot exposure image, and the right image is the calculated relative protein expression quantity (expressed by gray values) of each group. * P <0.01, P <0.0001.AsAb: sperm antibodies; control: a negative control group; model: positive control group of rats with hyposexuality; a: a traditional Chinese medicine treatment group for rats with hyposexuality; b: traditional Chinese medicine + probiotic treatment group for hyposexuality rats.
Figure 2 serological assay. And (3) injection: levels of A.sperm antibodies, B.IgA antibodies, C.IgG antibodies, D.IgM antibodies, E.complement protein 3, F.complement protein 4, G.pro-inflammatory factor IL-2, and H.IL-6 in the serum of each group of rats. * P <0.01, P <0.0001.
FIG. 3 oxidative stress detection. And (3) injection: concentration of a, malondialdehyde, b, glutathione peroxidase, c, superoxide dismutase in the serum of each group of rats. * P <0.05, < P <0.01, < P <0.001, < P <0.0001.
FIG. 4 immunohistochemical detection. And (3) injection: the left panel is an immunohistochemical light microscope image (x 100), and polyclonal antibodies against the rat pigment epithelium elongation factor (pigment epithelium derived factor, PEDF) are expressed as brown yellow strips or granular materials positive for immune response; the right panel shows the relative levels of positive immune response calculated for each group. * P <0.05, < P <0.01, < P <0.001.
FIG. 5 blood lipid level detection. And (3) injection: serum of rats of each group contains A. Testosterone, B. Follicle stimulating hormone, C. Luteinizing hormone, D. Estradiol, and E. Testosterone to estradiol ratio. * P <0.01, P <0.001, P <0.0001.
FIG. 6 semen viable bacteria and microorganism subpopulation analysis and detection. And (3) injection: NGS sequencing detects the number of a. Pseudomonas, b. Prasuvorexa, c. Lactobacillus in each group of rat semen. * P <0.05, < P <0.01, < P <0.0001.
FIG. 7 animal physiological function test. And (3) injection: A. electrophysiological experiments on sexual activity the ejaculation latency of rats of each group was examined and b. mating experiments were carried out to determine the insertion latency of rats of different treatment groups. * P <0.05, P <0.001, P <0.0001.
FIG. 8 sperm motility assay. And (3) injection: the amount of a. Sperm, the number of b. Sperm, the motility of c. Sperm, and the motility of d. Sperm in each group of rats. * P <0.01, P <0.001, P <0.0001.
Detailed Description
The following examples further illustrate the invention but are not to be construed as limiting the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.
The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated. The data result is analyzed by SPSS20.0 and Prism graphic pad 8.0 software, and the analysis result and report are provided.
1. Preparation of probiotic composition with traditional Chinese medicine formula
Preparation of traditional Chinese medicine extraction powder: 6-15 parts of cistanche, 10-25 parts of oyster, 6-15 parts of mulberry, 2-8 parts of cinnamon and 6-15 parts of fingered citron (in the embodiment, 10 parts of cistanche, 15 parts of oyster, 10 parts of mulberry, 2 parts of cinnamon and 6 parts of fingered citron are all parts by weight). Cutting or pulverizing the above materials in proportion, soaking in water for 30 min, heating to boil, leaching, separating decoction, decocting residues for 2-3 times, and collecting decoction; extracting the decoction with ethanol; drying the filtrate, and pulverizing into superfine powder smaller than 10 μm.
Preparation of probiotic freeze-dried powder: consists of lactobacillus reuteri, lactobacillus plantarum, lactobacillus rhamnosus, lactobacillus paracasei and lactobacillus salivarius. In the embodiment, the lactobacillus paracasei strain is composed of lactobacillus paracasei LC86, lactobacillus plantarum LP90, lactobacillus rhamnosus Lra05, lactobacillus reuteri LE16 and lactobacillus salivarius LS 33. The preparation method comprises the following steps: mixing lactobacillus paracasei, lactobacillus plantarum and lactobacillus rhamnosus dry powder respectively in one portion, lactobacillus reuteri and lactobacillus salivarius dry powder respectively in two portions according to a proportion for later use.
The probiotics of the traditional Chinese medicine formula: the preparation method comprises weighing Chinese medicinal extract powder and probiotic lyophilized powder at a volume ratio of 1:1, mixing, stirring with a micro stirrer, and storing in 4deg.C environment (such as low temperature refrigerator).
2. Experimental design and grouping:
(1) SPF-grade Sprague Dawley (SD) male rats at 8 weeks of age purchased 30 rats, weighing approximately 200-300 g/rat, and remained ready for use.
(2) The grouping is as follows:
wherein, 10 XPCR buffer is self-made buffer, and the formula is: 500mmol/L KCl,100mmol/L Tris-HCl (pH 8.3), 5mmol/L MgCl 2 。MgCl 2 The concentration of MgCl is significantly lower than that in conventional commercial PCR reagents 2 Concentration. 5mmol/L MgCl 2 To more accurately amplify a target template (bacteroides fragilis in human intestinal tract) in a mixed sample (human fecal dilution).
3. Building an animal model:
the experiment mainly discusses the improvement effect of probiotics in the traditional Chinese medicine formula on sexual dysfunction, and structural damage to the sexual function of rats can influence the healing and improvement of the sexual dysfunction, so that a sexual dysfunction model is not necessarily constructed by a testis extraction method, and the sexual dysfunction model is constructed by stimulating the cavernous nerve of the penis by adopting electrophysiology.
1. The experimental method comprises the following steps:
(1) After the experimental grouping, except the control group, 6 rats in each of the three groups are anesthetized by intraperitoneal injection of pentobarbital sodium according to the dosage of 50mg/kg, and the anesthesia is maintained by supplementing an appropriate amount of pentobarbital sodium every 45-60min during the experimental period.
(2) The experimental rats were anesthetized and fixed in the supine position, and then were cut through the skin and muscle in the middle of their lower abdomen in order to find out the prostate, and the fat was carefully and slowly peeled off on both sides of the prostate, freeing the rear of the lobes on both sides of the prostate, leaving the pelvic stellate ganglion (Major Pelvic ganglion, MPG) clearly exposed in the field of vision of the experimenter.
(3) Three identifiable nerve-pelvic, infrontal and corpora cavernosa nerves were then carefully dissociated along the MPG to the periphery using a glass minute hand. Among them, the most medial one, the most output branch running along the lateral side of the urethra is Cavernous Nerve (CN).
(4) After the corpora cavernosa nerve is found, the glans tissue of the rat is pulled out, the prepuce around the penis is carefully cut, the skin and the white film on the surface of the prepuce are carefully peeled off, and the corpora cavernosa is fully exposed.
(5) Then lightly holding the corpora cavernosa with the forceps to keep the cavernosa in a horizontal state, then puncturing the 25G needle head into the corpora cavernosa at one side in parallel and slightly vertically, and drawing back blood to ensure that the needle head punctures into the corpora cavernosa, and then carefully fixing the punctured needle head.
(6) Then, after skin and muscle are cut in turn at the median incision of the neck, the left common carotid artery is exposed by a glass needle, and arterial catheterization is used to move a arterial catheterization for continuously monitoring arterial pressure.
(7) The needles of the corpora cavernosa and carotid artery were then connected to a multichannel physiological recording system via a catheter, and the catheter was filled with heparin sodium solution (200U/ml physiological saline) and then the air bubbles in the catheter were purged.
(8) The CN is hooked by a refined stainless steel bipolar electrode, and the electric stimulation is prepared. After all readies, the corpora cavernosa nerve was electrically stimulated and Mean Arterial Pressure (MAP) and intracavernosal pressure (ICP) were recorded.
(9) The stimulation parameters of the cavernous nerve are: continuous square wave stimulation, 10ms wave width, 20HZ frequency, 3mA current, and 90s duration for each electrical stimulation. The cavernous nerve on each side was stimulated 3 times, and then the ratio of ICP to MAP was recorded and calculated for evaluation of erectile function in rats.
(10) And (5) wrapping the wound after the experiment is finished, and placing the wound back into a rearing cage for rearing.
2. Model verification: electrophysiological determination of sexual activity
After feeding for 1-2 weeks, the back of the model rat was fixed after the rat was observed for recovery of physiological functions such as diet, the program of the biological function test system was set to an electric stimulation mode, stimulation electrodes were placed at the penis part and the lower abdomen of the rat near the anus, electric stimulation was given, and the erection latency and ejaculation latency (the time required from the start of stimulation to the erection of the penis; the time from the erection of the penis to the start of ejaculation; the ejaculation latency) of the rat were measured. After one week of continuous testing, test data were recorded to see if the sexual dysfunction model was constructed successfully.
4. Dose and related function detection
1. Different doses and optimal dose verification:
the dosage of the traditional Chinese medicine for adults is 9.8g/70kg of adults/day, and the human is dosed according to the literature: murine = 1:0.018, rat dosing was about 0.5mg/200 g/rat/day.
According to the administration experience of experimental animals, the human being is administrated: murine = 1:0.1, rat dosing was about 2.8mg/200 g/rat/day.
The study discusses simultaneous feeding of 2 doses to investigate the optimal dose.
2. Detecting the physiological functions of animals;
the sexual dysfunction model is the same as the sexual dysfunction model in verification method, and the sexual function recovery condition of the rat is observed through electrophysiological measurement of sexual activity.
After 4 weeks of feeding, the model rat was fixed at the back, the program of the bioelectrical function test system was set to an electric stimulation mode, stimulation electrodes were placed at the penis and lower abdomen of the rat near the anus, electric stimulation was given, and the erection latency and ejaculation latency (the time required from the start of stimulation to the erection of the penis; the time from the erection of the penis to the start of ejaculation; the ejaculation latency) of the rat were measured. The test was continued for one week and the test data was recorded to observe the restoration of sexual dysfunction.
3. Detecting animal serum indexes;
the WB method detects anti-sperm antibody (AsAb) expression.
ELISA method for detecting immune function index [ complement protein 3 (C3), complement protein 4 (C4), interleukin 2 (IL-2), interleukin 6 (IL-6), immunoglobulin A (IGA), immunoglobulin G (IGG), immunoglobulin M (IGM) ].
The specific experimental steps are as follows:
(1) WB Experimental procedure
A. 0.1ml of rat serum was taken in a 10ml EP tube, 1ml of tissue lysate (50 ml of Tris-HCl150 mM NaCl, 0.1% SDS, 1% NP-40 and 0.1% PMSF) was added for sufficient homogenization, and the mixture was allowed to stand under ice bath conditions for 10min;
Centrifuging at 4deg.C for 20min at 12,000Xg, and collecting supernatant;
detecting the protein concentration of each histone sample by using a BCA kit;
D. the concentration of each group of proteins is adjusted to 2mg/ml by the lysate;
E. dripping 1/4 volume of 5×loading Buffer and 1/20 volume of beta-mercaptoethanol into each group of protein samples, and fully and uniformly mixing;
F. each set of samples was denatured by placing in boiling water for 5 min.
G. Sucking 20 μl into each hole for protein electrophoresis, wherein the voltage is constant at 80V, and adjusting the voltage to 130V after the sample enters the separation gel until the gel running is finished;
H. cutting the separating gel of the anti-sperm antibody (AsAb) protein according to the molecular weight of the protein and transferring the membrane;
I. after the transfer, the PVDF membrane is incubated in 5% skim milk powder for 1h, and then incubated with anti-sperm antibody (AsAb) (1:1000) primary antibody at 4deg.C overnight;
J. the PVDF membrane was washed 6 times with 1 XTBE buffer every day for 8min;
K. then placing PVDF into a diluted secondary antibody solution, and incubating the PVDF film on a rotary shaking table at room temperature for 2h;
washing PVDF membrane with 1 XTBST buffer for 8 times each for 8min;
and M, finally, performing western blot imaging on the PVDF film by using a Tanno full-automatic digital gel/chemiluminescence Image analysis system, and analyzing the gray value of each strip by using Image J software.
(2) ELISA procedure
A. 0.1ml of rat serum was centrifuged at 10000 Xg for 1 minute, and 1.0ml of standard & sample diluent was added to the lyophilized standard, the tube cap was screwed, left standing for 10 minutes, inverted upside down several times, and after it was sufficiently dissolved, it was gently mixed with a pipette (concentration: 200 ng/ml). The ratios were then diluted to 200, 100, 50, 25, 12.5, 6.25ng/mL standards.
B. Sample adding: and a blank hole, a standard hole and a sample hole to be tested are respectively arranged. Blank Kong Jiabiao standard substance and sample diluent 100 μl, standard Kong Jiabiao standard substance 100 μl, sample hole adding sample to be detected 20 μl and sample diluent 80 μl, and adding sample to the bottom of the ELISA plate during sample adding, and mixing by gently shaking. The ELISA plate was covered and incubated at 37℃for 90 minutes.
C. Discard the liquid, spin-dry, and do not need washing. 100 μl of biotinylated antibody working solution (formulated within 15 minutes prior to use) was added to each well, and the elisa plate was covered with a membrane and incubated for 1 hour at 37 ℃.
D. The liquid in the wells was discarded, dried, and the plate was washed 3 times, each time soaking for 1-2 minutes, about 350 μl/well, dried and tapped on absorbent paper to dry the liquid in the wells.
E. Mu.l of enzyme conjugate working solution (prepared within 15 minutes immediately before use) was added to each well, and the mixture was covered with a membrane and incubated at 37℃for 30 minutes.
F. Discarding the liquid in the holes, spin-drying, washing the plate for 5 times, and the method is the same as the step 5.
G. Mu.l of primer solution (TMB) was added to each well, and the ELISA plate was incubated at 37℃for 15 minutes in the absence of light.
H. The reaction was stopped by adding 50. Mu.l of stop solution to each well, and the blue color turned yellow immediately. The order of addition of the stop solution is as similar as possible to the order of addition of the substrate solution.
I. The optical density (OD value) of each well was measured immediately with an enzyme-labeled instrument at a wavelength of 450 nm.
J. And drawing a standard curve by taking the concentration of the standard substance as an abscissa and the OD value as an ordinate. The OD value of the sample carried into the well to be measured is used for calculating the concentration.
4. Effects of the drug on animal serum oxidative stress index and the like:
ELISA method for detecting serum oxidative stress related indexes [ Malondialdehyde (MDA), glutathione peroxidase (GSH-PX) and superoxide dismutase (SOD) ].
ELISA test procedure:
A. 0.1ml of rat serum was centrifuged at 10000 Xg for 1 minute, and 1.0ml of standard & sample diluent was added to the lyophilized standard, the tube cap was screwed, left standing for 10 minutes, inverted upside down several times, and after it was sufficiently dissolved, it was gently mixed with a pipette (concentration: 200 ng/ml). The ratios were then diluted to 200, 100, 50, 25, 12.5, 6.25ng/mL standards.
B. Sample adding: and a blank hole, a standard hole and a sample hole to be tested are respectively arranged. Blank Kong Jiabiao standard substance and sample diluent 100 μl, standard Kong Jiabiao standard substance 100 μl, sample hole adding sample to be detected 20 μl and sample diluent 80 μl, and adding sample to the bottom of the ELISA plate during sample adding, and mixing by gently shaking. The ELISA plate was covered and incubated at 37℃for 90 minutes.
C. Discard the liquid, spin-dry, and do not need washing. 100 μl of biotinylated antibody working solution (formulated within 15 minutes prior to use) was added to each well, and the elisa plate was covered with a membrane and incubated for 1 hour at 37 ℃.
D. The liquid in the wells was discarded, dried, and the plate was washed 3 times, each time soaking for 1-2 minutes, about 350 μl/well, dried and tapped on absorbent paper to dry the liquid in the wells.
E. Mu.l of enzyme conjugate working solution (prepared within 15 minutes immediately before use) was added to each well, and the mixture was covered with a membrane and incubated at 37℃for 30 minutes.
F. Discarding the liquid in the holes, spin-drying, washing the plate for 5 times, and the method is the same as the step 5.
G. Mu.l of primer solution (TMB) was added to each well, and the ELISA plate was incubated at 37℃for 15 minutes in the absence of light.
H. The reaction was stopped by adding 50. Mu.l of stop solution to each well, and the blue color turned yellow immediately. The order of addition of the stop solution is as similar as possible to the order of addition of the substrate solution.
I. The optical density (OD value) of each well was measured immediately with an enzyme-labeled instrument at a wavelength of 450 nm.
J. And drawing a standard curve by taking the concentration of the standard substance as an abscissa and the OD value as an ordinate. The OD value of the sample carried into the well to be measured is used for calculating the concentration.
5. Animal sperm motility detection:
(1) Visual inspection of sperm morphology
A. Amount of essential liquid: the semen is poured into a graduated test tube or cup and the semen volume is measured.
B. Observe the semen color and smell. The color is as follows: normal semen is generally milky or white, and the higher the density, the more milky the degree of whiteness and the lower the transparency. The other colors are abnormal colors. Smell: generally odorless or slightly fishy.
C. The seminal fluid turns and rolls in a cloud shape. Apparent cloud indicated by "+++", the cloud is indicated by "++", the cloudiness is not apparent and is indicated by "+".
(2) Sperm motility test
Sperm motility is of three types: advancing, rotating and oscillating movements, the motility of sperm is evaluated according to the percentage of sperm count in sperm in straight advancing movement to total sperm count. Sperm motility is an important index for evaluating semen quality, and is closely related to sperm fertility.
A. Visual assessment method: viability was judged using microscope magnification at 100 x. The sperm motility rating was evaluated by a ten-level rating method, i.e., by an estimated percentage of the number of sperm moving in a straight line in the visual field to the total number of sperm, with 100% of the straight line moving being rated as 1.0, 90% as 0.9, 80% as 0.8, and so on. During examination, a drop of semen is taken on a glass slide to prepare a tabletting specimen, the tabletting specimen is observed under a microscope of 100 times, and the semen with large concentration is firstly diluted with normal saline or isotonic diluent and then is examined. The proportion of the sperm in the advancing movement to the total sperm is not less than 70 percent, namely the vitality is not less than 0.7 percent, and the sperm can be further processed.
B. Cytometer counting method: the scrubbed blood cell count plate is placed on the stage of the microscope and covered with a cover slip. The diluted semen in the test tube is dropped on the edge of the cover glass of the counting plate to automatically infiltrate the semen into the counting chamber, and the phenomenon that the semen overflows out of the cover glass and cannot be insufficient to cause bubbles or dryness in the counting chamber is needed to be reworked if the phenomenon exists. Find counting chamber and first middle square: the counting plate was fixed in the pusher of the microscope and the counting chamber was found with 100 x magnification and then the first middle square of the counting chamber was found with 400 x magnification. Counting total sperms in five middle lattices from the upper left corner to the lower right corner, counting the sperms on the lattice lines according to the principle that the number is not counted down and the number is not counted right or left or right by taking the head of the sperms as the standard, and counting the sperms in the first row of lattices from the middle lattice to the right, to the left of the second row, to the right of the third row and to the left of the fourth row. Counting the number X of nonlinearly (including dead sperm and swinging and rotating sperm) motile sperm in five large squares, namely 80 small squares at four corners and the center, and obtaining the number of nonlinearly motile sperm in each milliliter of semen. The number of sperm counted by five large squares (80 small squares) is substituted into the following formula to obtain the number of sperm per milliliter, (X/80) ×400×dilution×10000=the number of sperm contained in per milliliter.
Sperm motility = (total sperm count-number of non-progressive motile sperm count)/total sperm count.
(3) Sperm density examination
A small drop of semen was placed on a clean slide, and a cover slip was added to disperse the semen into a uniform thin layer, but no bubbles were present, nor could the semen flow out or spill onto the cover slip, and the density was assessed as follows under a microscope with 400 x magnification.
The density-the precision in the whole visual field is very high, the gaps between the two are very small, and the movement condition of each sperm is not clear, which belongs to the field of 'density', and the sperm content per milliliter is about more than 10 hundred million.
The space between the sperm is obvious, and the distance between the sperm is about one sperm length. Some sperm motility can be clearly seen. The density of this semen was rated "medium" and contained between about 2-10 billion sperm per milliliter.
Thin sperm-the existence of dispersed sperm, the interval between sperm exceeds the length of one sperm, and the sperm contained in each milliliter of semen is less than 2 hundred million.
6. Animal semen viable bacteria and microorganism subgroup analysis and detection:
the number and proportion of three species of bacteria Pseudomonas, prevoltella and Lactobacillus in the semen were determined. And performing NGS sequencing detection based on free nucleic acid in the semen sample, analyzing the nucleic acid sequence of the microorganism in the sample, and identifying the microorganism by comparing the nucleic acid sequence with the nucleic acid sequence of the microorganism in the database. The detection process comprises the following steps: nucleic acid extraction, library construction, sequencing, information analysis, report interpretation, and the like. The minimum detection limit of the detection technology for the microbial nucleic acid in the sample is 100-1000copies/mL, the detection specificity for the microorganism with the copy number larger than the minimum detection limit is larger than 99%, and the repeatability is larger than 99%.
(note: live bacteria detection will send semen back to the party for self-detection).
7. Animal testis histomorphology detection:
(1) Penis tissue material
A. Anesthesia: all rats were anesthetized with 3% sodium pentobarbital intraperitoneally at a dose of 50mg/kg;
B. fixing: placing the deeply anesthetized mice in a supine position on an experimental animal operating table, fixing limbs and exposing penile tissues thereof;
C. drawing materials: the head of the penis is clamped by flat tooth forceps, the penis is naturally straightened, the skin of the external layer of the penis above the root below the head of the penis is carefully sheared by using an ophthalmic scissors, fascia is carefully peeled off, and then the penis tissue is sheared off from the root. The whole operation process needs to be careful as much as possible so as not to damage the blood vessels and nerves of the penis;
D. and (3) preserving: according to a preset experimental scheme, the head of the extracted penile tissue is cut off, residual blood in the tissue is gently squeezed out by using a pair of curved forceps, and the penile tissue is frozen in liquid nitrogen and subjected to paraffin embedding treatment.
(2) Paraffin embedding
A. Fixing: the penile tissue to be paraffin-embedded was fixed in 50ml of a solution of 4% PFA freshly prepared and pre-chilled at 4 ℃. After 24h fixation, the tissue was removed and rinsed overnight with tap water to rinse out residual PFA from the tissue;
B. Dehydrating: the washed tissue is dehydrated by ethanol gradient after the water is absorbed by filter paper, the ethanol concentration gradients are 70%, 80%, 90%, 95%, 100% (I) and 100% (II) in sequence, and each concentration gradient is dehydrated for 1h;
C. and (3) transparency: sequentially placing the dehydrated tissue in xylene (I) and xylene (II) for transparent treatment, and separating out residual ethanol in the tissue, wherein each transparent treatment is 5min (if the dehydrated tissue is placed in xylene (I), white water mist appears on the surface of the tissue, which indicates that the tissue is not dehydrated completely, the tissue can be placed in 100 percent ethanol (I) and 100 percent ethanol (II) for respectively and again dehydrating for 0.5 h);
D. wax dipping: sequentially placing the transparent treated tissue into three-cylinder paraffin of paraffin (I) and paraffin (II) dissolved at 60 ℃ to carry out paraffin dipping treatment and separating out dimethylbenzene in the tissue, wherein the time of each paraffin dipping is 1h; 5) Embedding: the penis tissue after wax dipping treatment is longitudinally erected in a stainless steel embedding box filled with molten paraffin, a plastic embedding frame is covered, the embedding box is filled with the molten paraffin, and after the paraffin is solidified at room temperature, the penis tissue is horizontally placed at 4 ℃ to be quickly solidified. After the paraffin is completely solidified, the metal embedding box is carefully glass, and the embedded tissue is preserved in a refrigerator at 4 ℃ for preparing paraffin sections.
(3) Paraffin section
A. Slide cleaning: the unused glass slide is placed in an acid jar for soaking overnight (> 16 h), then acid-resistant gloves are worn, protective clothing is worn, the glass slide is carefully fished out and placed under tap water to fully wash the residual liquid, and then ddH2O is used for three times to fully remove the residual tap water, and then the glass slide is placed in a ventilation place for natural airing or oven drying.
B. Preparing APES working solution: a certain amount of APES concentrate was sucked up with a pipette and placed in a plastic beaker, followed by 1:49 (i.e. 1 part concentrate +49 parts acetone) and finally thoroughly mixing.
C. Coating: the well-washed and dried slide glass was immersed in the diluted APES working solution for several tens of seconds or ten times in an ice bath, and the solution was drained slightly for about 10 seconds, and then the solution was put into distilled water to wash the residual APES. Naturally airing or baking at the temperature lower than 60 ℃ after being placed for 12-24 hours at room temperature.
(4) Immunohistochemistry
A. Immunohistochemical sections, spreads, patches and bakeware: taking out a pre-chilled penile tissue paraffin embedded block at-20 ℃, vertically placing the paraffin embedded block in a bayonet on a paraffin slicer, carefully adjusting the embedded block to be parallel to a blade, fixing the embedded block, adjusting the embedded block to be 5 mu m, slicing, and continuously cutting 3 slices for each sample, wherein each group of 3 samples; spreading the slices in a water bath at 37 ℃ and placing the slices in an oven at 60 ℃ for baking for 1h.
B. Dewaxing and rehydrating: sequentially placing the baked slices into xylene (II) and xylene (I) for dewaxing for 10min each time; sequentially placing the dewaxed tissue slices in 100% (I), 100% (II), 95%, 85% and 75% ethanol for dehydration for 3min each time; placing the dehydrated tissue slice in ddH2O for hydration for 2min multiplied by 3 times;
C. antigen retrieval: and performing antigen thermal remediation by adopting a microwave heating method. 200ml of freshly prepared citric acid antigen retrieval liquid (0.01M, pH6.0) is firstly placed in a microwave oven and heated for 3min at medium and high grade, at this time, the temperature of the antigen retrieval liquid reaches 92-95 ℃, then the slices are placed in the retrieval liquid, heated for 20s and stopped for 2min, repeatedly, accumulated for 15min, taken out and naturally cooled to room temperature.
D. Blocking and blocking: after antigen retrieval, ddH2O was washed 2min×3 times, incubated with 3% H2O2 for 10-15min at room temperature to block endogenous peroxidase in the tissue, washed 3min×3 times with PBS, and sliced tissue blocked with 5% BSA for 15min to reduce nonspecific staining.
E. Incubating primary antibodies: after blocking, 20. Mu.l/piece of rabbit anti-rat PEDF polyclonal antibody (1:100) was added dropwise and incubated overnight at 4 ℃. The negative control was replaced with an antibody dilution.
F. Warming and incubation of secondary antibodies: the primary antibody is incubated overnight (> 15 hours), the tissue sections are taken out, after rewarming for 30min at room temperature, PBS is used for 3min×3 times, 20 μl/piece of polymer auxiliary reagent is dripped, incubation is carried out for 20min at room temperature, PBS is used for 3min×3 times, 20 μl/piece of horseradish peroxide-labeled goat anti-rabbit IgG polymer (secondary antibody) is dripped, and after incubation for 30min at room temperature, PBS is used for 3min×3 times.
G. Color development and counterstain: DAB color development, observation under a microscope, brown particles appear, immediately stop the color development reaction with ddH2O, and wash with ddH2O for 2min; hematoxylin counterstain for 5s, and washing with tap water for 15min to return to blue;
H. dewatering, transparentizing and sealing: dehydrating the tissue slice by ethanol with the concentration of 95 percent (I), 95 percent (II), 100 percent (I) and 100 percent (II) in sequence for 5 minutes each time; and (3) sequentially passing through xylene (I) and xylene (II) for transparency after dehydration, wherein each time is 5min, and then sealing the neutral resin.
I. Photographing: the results were observed under an optical microscope (x 100) and photographed, and immunoreaction-positive expression was brown-yellow streak-like or granular material.
8. Detecting the blood lipid level of animals:
ELISA method for detecting rat blood lipid level [ FSH (follicle stimulating hormone), LH (luteinizing hormone), T (testosterone) and E2 (estradiol), and T/E2 ratio ].
ELISA test procedure:
A. 0.1ml of rat serum was centrifuged at 10000 Xg for 1 minute, and 1.0ml of standard & sample diluent was added to the lyophilized standard, the tube cap was screwed, left standing for 10 minutes, inverted upside down several times, and after it was sufficiently dissolved, it was gently mixed with a pipette (concentration: 200 ng/ml). The ratios were then diluted to 200, 100, 50, 25, 12.5, 6.25ng/mL standards.
B. Sample adding: and a blank hole, a standard hole and a sample hole to be tested are respectively arranged. Blank Kong Jiabiao standard substance and sample diluent 100 μl, standard Kong Jiabiao standard substance 100 μl, sample hole adding sample to be detected 20 μl and sample diluent 80 μl, and adding sample to the bottom of the ELISA plate during sample adding, and mixing by gently shaking. The ELISA plate was covered and incubated at 37℃for 90 minutes.
C. Discard the liquid, spin-dry, and do not need washing. 100 μl of biotinylated antibody working solution (formulated within 15 minutes prior to use) was added to each well, and the elisa plate was covered with a membrane and incubated for 1 hour at 37 ℃.
D. The liquid in the wells was discarded, dried, and the plate was washed 3 times, each time soaking for 1-2 minutes, about 350 μl/well, dried and tapped on absorbent paper to dry the liquid in the wells.
E. Mu.l of enzyme conjugate working solution (prepared within 15 minutes immediately before use) was added to each well, and the mixture was covered with a membrane and incubated at 37℃for 30 minutes.
F. Discarding the liquid in the holes, spin-drying, washing the plate for 5 times, and the method is the same as the step 5.
G. Mu.l of primer solution (TMB) was added to each well, and the ELISA plate was incubated at 37℃for 15 minutes in the absence of light.
H. The reaction was stopped by adding 50. Mu.l of stop solution to each well, and the blue color turned yellow immediately. The order of addition of the stop solution is as similar as possible to the order of addition of the substrate solution.
I. The optical density (OD value) of each well was measured immediately with an enzyme-labeled instrument at a wavelength of 450 nm.
J. And drawing a standard curve by taking the concentration of the standard substance as an abscissa and the OD value as an ordinate. The OD value of the sample carried into the well to be measured is used for calculating the concentration.
4. Results of the study
1. Sperm antibody protein Westernblot detection results: WB detection of expression of sperm antibody (AsAb) proteins in serum of rats from different treatment groups, see fig. 1. The results show that the protein expression Model of AsAb in rat serum is greater than A > B > control; compared with normal rats in the control group, the serum AsAb protein expression of the rats in the model group is obviously increased, the increase of the AsAb protein expression is inhibited by the treatment of the treatment group A and the treatment group B, and the treatment effect of the treatment group B is better than that of the treatment group A.
2. ELISA serological test results: ELISA detection of sperm antibody (AsAb) expression in serum from rats of different treatment groups (FIG. 2-A): the results show that the expression Model of AsAb in rat serum is greater than A > B > control; compared with normal rats in the control group, the serum AsAb expression of the rats in the model group is obviously increased, the increase of the AsAb expression is inhibited by the treatment of the treatment group A and the treatment group B, and the treatment effect of the treatment group B is better than that of the treatment group A.
ELISA detection of IgA expression in serum from rats of different treatment groups (FIG. 2-B): the results show that IgA expression in rat serum is Model > A > B > control; compared with normal rats in the control group, the serum IgA expression of rats in the model group is obviously increased, the increase of IgA expression is inhibited by the treatment of the treatment group A and the treatment group B, and the treatment effect of the treatment group B is better than that of the treatment group A.
ELISA detection of IgG expression in serum from rats of different treatment groups (FIG. 2-C): the results show that the expression Model of IgG in rat serum is greater than A > B > control; compared with normal rats in the control group, the serum IgG expression of the rats in the model group is obviously increased, the increase of the IgG expression is inhibited by the treatment of the treatment group A and the treatment group B, and the treatment effect of the treatment group B is better than that of the treatment group A.
ELISA to detect IgM expression in serum from rats of different treatment groups (FIG. 2-D): the results show that the expression Model of IgM in rat serum is greater than A > B > control; compared with normal rats in the control group, the IgM expression of the serum of the rats in the model group is obviously increased, the IgM expression is inhibited from being increased by the treatment of the treatment group A and the treatment group B, and the treatment effect of the treatment group B is better than that of the treatment group A.
ELISA detects the expression of C3 in the serum of rats of different treatment groups (FIG. 2-E): the results show that the expression Model of C3 > A > B > control in rat serum; compared with the normal rats in the control group, the serum C3 expression of the rats in the model group is obviously increased, the treatment of the treatment group A and the treatment group B inhibits the increase of the C3 expression, and the treatment effect of the treatment group B is better than that of the treatment group A.
ELISA to detect the expression of C4 in serum from rats of different treatment groups (FIG. 2-F): the results show that the expression Model of C4 > A > B > control in rat serum; compared with the normal rats in the control group, the serum C4 expression of the rats in the model group is obviously increased, the treatment of the treatment group A and the treatment group B inhibits the increase of the C4 expression, and the treatment effect of the treatment group B is better than that of the treatment group A.
ELISA detection of IL-2 expression in serum from rats of different treatment groups (FIG. 2-G): the results show that the expression Model of IL-2 in rat serum is greater than A > B > control; compared with normal rats in the control group, the serum IL-2 expression of rats in the model group is obviously increased, the IL-2 expression is inhibited from being increased by the treatment of the treatment group A and the treatment group B, and the treatment effect of the treatment group B is better than that of the treatment group A.
ELISA detection of IL-6 expression in serum from rats of different treatment groups (FIG. 2-H): the results show that the expression Model of IL-6 in rat serum is greater than A > B > control; compared with normal rats in the control group, the serum IL-6 expression of rats in the model group is obviously increased, the IL-6 expression is inhibited from being increased by the treatment of the treatment group A and the treatment group B, and the treatment effect of the treatment group B is better than that of the treatment group A.
3. Oxidative stress detection results: malondialdehyde (MDA) kit detects the content of MDA in serum from rats of different treatment groups (a in fig. 3): the results show that the MDA content in the rat serum is Model > A > B > control; compared with normal rats in the control group, the MDA content of the serum of the rats in the model group is obviously increased, the MDA content is inhibited from being increased by the treatment of the treatment group A and the treatment group B, and the treatment effect of the treatment group B is better than that of the treatment group A.
Glutathione peroxidase (GSH-PX) kit detects GSH-PX content in serum of rats of different treatment groups (B in FIG. 3): the results show that the content of GSH-PX in the rat serum is Model < A < B < control; compared with normal rats in the control group, the serum GSH-PX content of rats in the model group is obviously reduced, the GSH-PX content is reduced by the treatment of the treatment group A and the treatment group B, and the treatment effect of the treatment group B is better than that of the treatment group A.
Superoxide dismutase (SOD) kit to detect the content of SOD in serum of rats of different treatment groups (C in fig. 3): the result shows that the SOD content in the serum of the rat is Model < A < B < control; compared with normal rats in the control group, the serum SOD content of the rats in the model group is obviously reduced, the SOD content is inhibited from being reduced by the treatment of the treatment group A and the treatment group B, and the treatment effect of the treatment group B is better than that of the treatment group A.
4. Immunohistochemical detection assay results: immunohistochemistry detection of expression of the penile tissue pigment epithelium derived factor PEDF in rats from different treatment groups see fig. 4: the results show that expression of PEDF in penile tissue of rats Model > a > B > control; compared with the normal rats in the control group, the expression of PEDF in the penile tissue of the rats in the model group is obviously increased, the treatment of the treatment group A and the treatment group B inhibits the increase of the expression of PEDF, and the treatment effect of the treatment group B is better than that of the treatment group A.
5. Blood lipid level detection experimental results: t (testosterone) kit the content of testosterone in serum of rats of different treatment groups (a in fig. 5): the result shows that the testosterone content in the serum of the rat is Model < A < B < control; compared with normal rats in the control group, the serum testosterone content of rats in the model group is obviously reduced, the testosterone content is inhibited from being reduced by the treatment of the treatment group A and the treatment group B, and the treatment effect of the treatment group B is better than that of the treatment group A.
SH (follicle stimulating hormone) kit the content of follicle stimulating hormone in the serum of rats of different treatment groups (B in fig. 5): the results show that the content of follicle stimulating hormone in the serum of the rat is Model < A < B < control; compared with normal rats in a control group, the content of the follicle-stimulating hormone in the serum of rats in a model group is obviously reduced, the reduction of the content of the follicle-stimulating hormone is inhibited by the treatment of the treatment group A and the treatment group B, and the treatment effect of the treatment group B is better than that of the treatment group A.
LH (luteinizing hormone) kit assay for the content of luteinizing hormone in serum of rats of different treatment groups (fig. 5-C): the result shows that the content of luteinizing hormone in the serum of the rat is Model < A < B < control; compared with normal rats in the control group, the serum luteinizing hormone content of rats in the model group is obviously reduced, the luteinizing hormone content is reduced by the treatment of the treatment group A and the treatment group B, and the treatment effect of the treatment group B is better than that of the treatment group A.
E2 (estradiol) kit detection of the amount of estradiol in serum from rats of different treatment groups (fig. 5-D): the results show that the content of estradiol in the serum of the rat is Model & gtA & gtcontrol & gtB; compared with normal rats in the control group, the serum estradiol content of rats in the model group is obviously increased, the increase of the luteinizing hormone content is inhibited by the treatment of the treatment group A and the treatment group B, and the treatment effect of the treatment group B is better than that of the treatment group A.
Ratios of testosterone to estradiol in serum from rats of different treatment groups (fig. 5-E): the results show that the ratio Model < A < control < B of testosterone to estradiol in the serum of the rat; compared with normal rats in the control group, the ratio of testosterone to estradiol in serum of rats in the model group is obviously reduced, the reduction of the ratio of testosterone to estradiol is inhibited by the treatment of the treatment group A and the treatment group B, and the treatment effect of the treatment group B is better than that of the treatment group A.
6. Semen viable bacteria and microorganism subgroup analysis and detection results: NGS sequencing the number of Pseudomonas (Pseudomonas) in the semen of rats from different treatment groups (a in fig. 6): the results show that the number of Pseudomonas in the semen of rats Model > A > control > B; compared with normal rats in the control group, the number of pseudoomas in the semen of the rats in the model group is obviously increased, the number of pseudoomas is inhibited from being increased by the treatment of the treatment group A and the treatment group B, and the treatment effect of the treatment group B is better than that of the treatment group A.
NGS sequencing the number of Prevolvulella (Prevoltella) in semen from rats of different treatment groups (B in fig. 6): the results show that the number of Prevoltella > A > B > control in rat semen; the number of Prevotella in the semen of the rats in the model group is significantly increased compared with that of the normal rats in the control group, the number of Prevotella is inhibited from being increased by the treatment of the treatment group A and the treatment group B, and the treatment effect of the treatment group B is better than that of the treatment group A.
NGS sequencing the number of Lactobacillus (C in fig. 6) in semen of rats of different treatment groups was examined: the results show that the quantity of Lactobacillus in the semen of the rat is Model < A < B < control; compared with normal rats in the control group, the number of Lactobacillus in semen of the rats in the model group is obviously reduced, the treatment of the treatment group A and the treatment group B inhibits the reduction of the number of the Prevotella, and the treatment effect of the treatment group B is better than that of the treatment group A.
7. Animal physiological function detection results: electrophysiological determination of viability ejaculation latency in rats of different treatment groups (a in fig. 7): the results show that the ejaculation latency Model > A > control > B in rats; compared with normal rats in the control group, the ejaculation latency of rats in the model group is obviously increased, the ejaculation latency is inhibited from being increased by the treatment of the treatment group A and the treatment group B, and the treatment effect of the treatment group B is better than that of the treatment group A.
Mating test the insertion latency of rats of different treatment groups was determined (B in fig. 7): the results show that the insertion latency Model > A > B > control for rats; compared with the normal rats in the control group, the insertion latency of the rats in the model group is obviously increased, the treatment of the treatment group A and the treatment group B inhibits the increase of the insertion latency, and the treatment effect of the treatment group B is better than that of the treatment group A.
8. Sperm motility test results: results of test tube testing of semen volume (A in FIG. 8) of rats in different treatment groups showed that the semen volume Model < A < B < control; compared with normal rats in the control group, the semen volume of rats in the model group is obviously reduced, the treatment of the treatment group A and the treatment group B inhibits the reduction of the semen volume of rats, and the treatment effect of the treatment group B is better than that of the treatment group A.
Results of cytometry detection of sperm count (B in FIG. 8) in rats of different treatment groups showed that the sperm count Model < A < B < control; compared with normal rats in the control group, the number of sperms of rats in the model group is obviously reduced, the reduction of the number of sperms of rats is inhibited by the treatment of the treatment group A and the treatment group B, and the treatment effect of the treatment group B is better than that of the treatment group A.
Results of cytometry detection of sperm motility (C in FIG. 8) of rats of different treatment groups showed that sperm motility Model < A < B < control of rats; compared with normal rats in the control group, the sperm motility of rats in the model group is obviously reduced, the sperm motility of rats is inhibited by the treatment of the treatment group A and the treatment group B, and the treatment effect of the treatment group B is better than that of the treatment group A.
Results of cytometry detection of motility (D in FIG. 8) of sperm from rats of different treatment groups showed that the motility Model < A < B < control of sperm from rats; compared with normal rats in the control group, the motility of the sperms of the rats in the model group is obviously reduced, the motility of the sperms of the rats is inhibited by the treatment of the treatment group A and the treatment group B, and the treatment effect of the treatment group B is better than that of the treatment group A.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that it will be apparent to those skilled in the art that several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the scope of the invention.
Claims (9)
1. A traditional Chinese medicine formula probiotic composition for improving male sexual function is characterized in that: the preparation method comprises the steps of extracting traditional Chinese medicine powder and probiotics freeze-dried powder; wherein the traditional Chinese medicine extract powder comprises, by weight, 6-15 parts of cistanche deserticola, 10-25 parts of oyster, 6-15 parts of mulberry, 1-8 parts of cinnamon and 4-15 parts of fingered citron; the probiotics freeze-dried powder consists of freeze-dried powder of combined bacteria consisting of lactobacillus reuteri, lactobacillus plantarum, lactobacillus rhamnosus, lactobacillus paracasei and lactobacillus salivarius;
Wherein the probiotic freeze-dried powder comprises one part of lactobacillus paracasei, lactobacillus plantarum and lactobacillus rhamnosus dry powder, and two parts of lactobacillus reuteri and lactobacillus salivarius dry powder;
mixing the Chinese medicine extract powder and the probiotics freeze-dried powder according to the volume ratio of 1:1.
2. The traditional Chinese medicine formula probiotic composition according to claim 1, wherein: the traditional Chinese medicine extract powder and the probiotics freeze-dried powder are stirred and mixed uniformly by a micro stirrer.
3. The traditional Chinese medicine formula probiotic composition according to claim 1, wherein: the preparation method of the traditional Chinese medicine extract powder comprises the following steps: cutting or pulverizing the above materials in proportion, soaking in water in a decoction vessel, heating to boil, leaching, separating decoction, decocting residues for 2-3 times, and collecting decoction; extracting the decoction with ethanol; drying the filtrate, pulverizing with superfine pulverizer, and making into superfine powder smaller than 10 μm.
4. The traditional Chinese medicine formula probiotic composition according to claim 1, wherein: the traditional Chinese medicine extract powder comprises 10 parts of cistanche, 15 parts of oyster, 10 parts of mulberry, 2 parts of cinnamon and 6 parts of fingered citron by weight.
5. A method for preparing a probiotic composition according to a traditional Chinese medicine formula for improving male sexual function according to any one of claims 1 to 4, characterized in that: the method comprises the following steps:
Cutting or pulverizing the above materials in proportion, soaking in water in a decoction vessel, heating to boil, leaching, separating decoction, decocting residues for 2-3 times, and collecting decoction; extracting the decoction with ethanol; drying the filtrate obtained by alcohol extraction, and pulverizing with a superfine pulverizer to obtain superfine powder smaller than 10 μm to obtain Chinese medicinal extract powder;
uniformly mixing lactobacillus paracasei, lactobacillus plantarum, lactobacillus rhamnosus, lactobacillus reuteri and lactobacillus salivarius dry powder according to a proportion to obtain probiotic freeze-dried powder;
the traditional Chinese medicine extraction powder and the probiotics freeze-dried powder are prepared according to the volume ratio of 1:1, mixing evenly.
6. Use of a probiotic composition of a traditional Chinese medicine formulation for improving male sexual function according to any one of claims 1 to 4 or a probiotic composition of a traditional Chinese medicine formulation obtained by the preparation method of claim 5 for the preparation of a medicament for treating or improving male sexual function.
7. The use according to claim 6, wherein: the improvement of male sexual function means improvement of male hyposexuality and improvement of sexual dysfunction.
8. The use according to claim 6, wherein: the improvement of male sexual function means improvement of the following sexual function problems: sexual dysfunction, impotence, premature ejaculation, spermatorrhea, non-ejaculation and retrograde ejaculation.
9. The use according to any one of claims 6 to 8, wherein the medicament is adapted for oral administration.
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| CN104524024A (en) * | 2014-12-15 | 2015-04-22 | 江瀚生物科技（上海）有限公司 | Compound microorganism preparation for improving male sperm quality and preparation method thereof |
| CN106174485A (en) * | 2016-07-11 | 2016-12-07 | 佛山市朗达信息科技有限公司 | There is the enzyme composition of kidney-reinforcing Yang strengthening effect, effervescent tablet and preparation method |
| CN110301633A (en) * | 2019-07-31 | 2019-10-08 | 大连山海物华科技开发有限公司 | A kind of healthy food and preparation method thereof improving male's sex-health |
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| CN104524024A (en) * | 2014-12-15 | 2015-04-22 | 江瀚生物科技（上海）有限公司 | Compound microorganism preparation for improving male sperm quality and preparation method thereof |
| CN106174485A (en) * | 2016-07-11 | 2016-12-07 | 佛山市朗达信息科技有限公司 | There is the enzyme composition of kidney-reinforcing Yang strengthening effect, effervescent tablet and preparation method |
| CN110301633A (en) * | 2019-07-31 | 2019-10-08 | 大连山海物华科技开发有限公司 | A kind of healthy food and preparation method thereof improving male's sex-health |
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