CN108392487B - Application of icariside II or pharmaceutically acceptable carrier thereof in erectile dysfunction - Google Patents
Application of icariside II or pharmaceutically acceptable carrier thereof in erectile dysfunction Download PDFInfo
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Abstract
The invention provides an application of icariside II or a pharmaceutically acceptable carrier thereof in erectile dysfunction, which comprises the icariside II or the pharmaceutically acceptable carrier thereof, and particularly but not exclusively is applied to preventing and/or treating the erectile dysfunction of mammals.
Description
Technical Field
The invention belongs to the field of medicines, and relates to application of icariside II or a pharmaceutically acceptable carrier thereof in erectile dysfunction and application thereof, in particular, but not limited to application in preventing and/or treating erectile dysfunction of mammals.
Background
In 1995, about 1.52 million ED patients had worldwide, and it was expected that 2025 would increase the number to 3.22 million [1] . According to statistics, the ED prevalence rate of middle-aged men (40.2+/-5.8 years old) in Beijing and other areas of China is as high as 40.2 percent [2] . Currently, oral PDE5 inhibitors (e.g. sildenafil, tadalafil) are the first therapy for clinical treatment of ED, but the therapeutic response of ED patients of different etiologies to drugs varies greatly. For example, ED patients with hypogonadism are best treated with PDE5 inhibitors after receiving testosterone replacement therapy (effective rate: 85%), while ED patients with diabetes (effective rate: 44%) or after radical prostate cancer therapy (effective rate: 43%) are poorly treated [3] . In recent years, partial scholars have considered that continuous administration of small doses of PDE5 inhibitors improves vascular endothelial function and increases penile blood flow in ED patients [4,5] . However, it has been found that ED treatment efficiency is only 24-33% [6-8] . Furthermore, it was found that [9] The continuous administration of small doses of sildenafil did not significantly improve the pathology of the neurogenic ED animal model.
Reference is made to:
1.Ayta I A,Mckinlay J B,and Krane R J.The likely worldwide increase in erectile dysfunction between 1995and 2025and some possible policy consequences.BJU Int,1999.84(1):50-6.
2. zhang Qingjiang three cities 2226 epidemiological surveys of male erectile function, J.Chi Men's science 2003,17 (3): 191-193.
3.Mccullough A R,Barada J H,Fawzy A,Guay A T,and Hatzichristou D.Achieving treatment optimization with sildenafil citrate(Viagra)in patients with erectile dysfunction.Urology,2002.60(2Suppl2):28-38.
4.Nakano Y,Miyake H,Chiba K,and Fujisawa M.Impact of penile rehabilitation with low-dose vardenafil on recovery of erectile function in Japanese men following nerve-sparing radical prostatectomy.Asian J Androl,2014.16(6):892-6.
5.Vardi Y,Appel B,Ofer Y,Greunwald I,Dayan L,and Jacob G.Effect of chronic sildenafil treatment on penile endothelial function:a randomized,double-blind,placebo controlled study.J Urol,2009.182(6):2850-5.
6.Brock G,Ni X,Oelke M,Mulhall J,Rosenberg M,Seftel A,D'souza D,and Barry J.Efficacy of Continuous Dosing of Tadalafil Once Daily vs Tadalafil On Demand in Clinical Subgroups of Men With Erectile Dysfunction:A Descriptive Comparison Using the Integrated Tadalafil Databases.J Sex Med,2016.13(5):860-75.
7.Mccullough A R,Levine L A,and Padma-Nathan H.Return of nocturnal erections and erectile function after bilateral nerve-sparing radical prostatectomy in men treated nightly with sildenafil citrate:subanalysis of a longitudinal randomized double-blind placebo-controlled trial.J Sex Med,2008.5(2):476-84.
8.Padma-Nathan H,Mccullough A R,Levine L A,Lipshultz L I,Siegel R,Montorsi F,Giuliano F,and Brock G.Randomized,double-blind,placebo-controlled study of postoperative nightly sildenafil citrate for the prevention of erectile dysfunction after bilateral nerve-sparing radical prostatectomy.Int J Impot Res,2008.20(5):479-86.
Disclosure of Invention
The invention aims to: the invention aims to provide an application of icariside II or a pharmaceutically acceptable carrier thereof in erectile dysfunction.
The invention adopts the technical scheme that: one aspect of the present invention relates to icariside II or a pharmaceutically acceptable carrier thereof for use in the prevention and/or treatment of erectile dysfunction in a mammal. Particularly for the prevention and/or treatment of erectile dysfunction in mammals insensitive to PDE5 inhibitors.
Another aspect of the present invention relates to icariside ii obtained by biological enrichment comprising the steps of: culturing trichoderma in a trichoderma culture medium, inoculating the cultured trichoderma into a liquid culture medium containing icariside for fermentation culture, finally obtaining trichoderma bodies enriched with icariside II, and extracting and purifying to obtain a pure icariside II product.
As a preferred embodiment of the present invention, trichoderma is Trichoderma viride. The trichoderma viride used in the method has the advantages of convenient source, easy propagation and no need of extracting hydrolase; the fermentation equipment is common, the subsequent purification method is simple, the low boiling point solvent extraction is carried out, the concentration is easy, and the high-purity product can be obtained by only extracting and soaking, filtering, adsorption chromatography and recrystallization, thus being applicable to industrial production and having low production cost. Can transfer icariside into cells, and is hydrolyzed by hydrolase in the cells, and hydrolysate is enriched, so that the industrialized preparation of icariside II is possible, the content of the prepared icariside II is more than 99%, and the yield is more than 91%. The method has the advantages of simple operation, easy implementation, low cost, high yield and no pollution, is suitable for industrialized mass production, and has extremely high application prospect.
Furthermore, the application of icariside II or a pharmaceutically acceptable carrier thereof in erectile dysfunction is that firstly, a PDA solid culture medium is prepared to cultivate trichoderma viride, and then the cultivated trichoderma viride is inoculated into a liquid culture medium containing icariside for fermentation culture. The trichoderma viride can transport icariside into cells, and hydrolysis is completed by hydrolytic enzymes in the cells, and hydrolysate is enriched, so that the industrialized preparation of icariside II is possible.
The method specifically comprises the following steps: a. trichoderma viride is cultured in a small test tube culture medium in a slant way; b. inoculating the cultivated Trichoderma viride into a liquid culture medium containing epimedium for fermentation culture; c. when icariin is completely converted and hydrolyzed, filtering the fermentation liquor to obtain filter residue containing Trichoderma viride and icariside II (and a small amount of icariside); d. soaking the residue in methanol, filtering again to obtain filtrate containing icariside II (and small amount of icariside), and repeating the process until all icariside II and small amount of icariside are dissolved in methanol solution; e. high performance liquid detection, if the conversion rate of icariside II is more than 50% and less than 85%, purifying by silica gel adsorption column chromatography; f. and (3) further purifying the secondary glycoside II with the conversion rate being more than 85% and passing through a silica gel column in the step (e) by a recrystallization method to obtain a pure icariside II product.
Further, the preparation of the culture medium for culturing Trichoderma viride comprises the following steps: firstly, preparing a PDA solid culture medium by using potatoes and glucose, and culturing the trichoderma viride in the PDA solid culture medium for 3-5 days at the temperature of 26-29 ℃ to obtain the trichoderma viride which is a high-activity thallus.
Further, the culture of Trichoderma viride in the PDA solid culture medium is specifically performed by slant culture in a small tube culture medium. The preparation method of the PDA solid culture medium comprises the following steps: weighing 200g of potato and 1L of distilled water, boiling the potato in distilled water until the potato is soft, filtering, heating the filtrate, adding 20g of glucose and 15-20g of agar, subpackaging into small test tubes after the potato is dissolved, sterilizing for 15-20 minutes at the temperature of 121 ℃, taking out, shaking uniformly, obliquely placing at an angle of 45 degrees, and cooling to obtain the PDA solid culture medium.
Further, the preparation method of the icariin-containing liquid culture medium comprises the following steps: adding crushed potato 0.5-10g and icariin 0.8-16g into 0.1-3L triangular conical flask, adding distilled water to 0.05-1L, fastening the bottle mouth with sealing film, sterilizing in 121 deg.C sterilizing box for 15-20 min, and cooling to 20-30deg.C to obtain icariin-containing liquid culture medium.
Preferably, the liquid culture medium containing icariin is prepared by crushing and sterilizing fresh potatoes, wherein the ratio of the fresh potatoes to the distilled water is 10g, and the ratio of the distilled water is 1L, and the ratio of the icariin to the fresh potatoes is 16 g.
Further, the fermentation culture process comprises the following steps: inoculating the cultured Trichoderma viride to a liquid culture medium containing icariin on an ultra-clean workbench, placing the liquid culture medium into a shaking table at 28 ℃ and at a rotating speed of 165r/min for 9 days, filtering a fermentation broth, soaking filter residues obtained by filtering with methanol, filtering again, and repeating the process for 2-5 times until the Trichoderma viride is soaked and whitened, and the icariside II is completely dissolved in the methanol; and concentrating the methanol soaking solution to obtain icariside II-containing powder, and purifying the powder to obtain icariside II pure product. Icariside II and icariside have the best solubility in methanol, and Trichoderma viride is insoluble in methanol, from which Trichoderma viride can be separated.
Further, the inoculation, i.e., 16g of icariin, requires 4 small tubes (1.2 cm in diameter) of Trichoderma to be cultured for 9 days, where the conversion rate is highest.
Further, the method for purifying the icariside II-containing powder is as follows: firstly, detecting the conversion rate of icariside II by using a liquid phase of powder containing the icariside II, filling mixed powder with the conversion rate of the icariside II being more than 50% and less than 85% into a column by using 200-300 mesh industrial chromatographic silica gel of which the size is 6-8 times that of the powder, and filling the column with dichloromethane by using 1.5-2 times of macroporous resin DM301 for dry sample stirring: the volume ratio of methanol is 15:1 and pure methanol, mixing the solutions, concentrating at 60deg.C under reduced pressure to obtain powder, dissolving with methanol at 65-70deg.C by heating reflux method, removing methanol solution containing icariside II, standing at normal temperature, separating out crystals, filtering, and vacuum drying below 45deg.C to constant weight to obtain icariside II pure product with a content of above 99%. When the conversion rate is between 50% and 85%, the icariside II is subjected to silica gel adsorption column chromatography purification and then recrystallization purification, and when the conversion rate is more than 85%, the icariside II is directly recrystallized and purified, so that the quality of the prepared icariside II can be effectively ensured.
The mammal according to the invention is a male mammal. In the present invention, the mammal is, for example, a human, a dog, a monkey, a cow, a horse, a cat, a bear, a tiger, a sheep, a mouse, or the like.
Wherein the icariside II is used in a unit dose amount of, for example, 0.1 to 100mg/kg body weight/day.
The use according to any of the preceding claims, wherein the product is for enteral or parenteral use.
The invention also relates to a product, which contains effective dose of icariside II and pharmaceutically acceptable carrier or excipient; a pharmaceutical pack or other form in separate packaging containers is obtained.
The icariside II-containing product of the present invention may be administered to a desired host such as a human by an enteral or parenteral route. The product of the present invention administered enterally can be administered in the form of an oral formulation, exemplified by: tablets, capsules, granules, suspending agents, sustained release agents and the like. The product of the present invention may be in the form of an injection, a topical formulation such as a skin patch, or a spray, etc. by parenteral administration.
The product of the present invention generally contains 0.1 to 90% by weight of the active ingredient (icariside II). Icariside ii may be prepared according to methods known in the art or by bioaugmentation methods described herein. For this purpose, the active ingredient may be combined, if desired, with one or more solid or liquid pharmaceutical excipients and/or adjuvants, into a suitable form of administration or dosage form for human use.
The pharmaceutical compositions of the present invention may be administered in unit dosage form by the enteral or parenteral route, such as oral, intramuscular, subcutaneous, nasal, oral mucosal, dermal, peritoneal or rectal. For example, tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories, freeze-dried powder injection and the like. Can be common preparation, slow release preparation, controlled release preparation and various microparticle administration systems. For the purpose of shaping the unit dosage form into a tablet, various carriers known in the art can be widely used. Examples of carriers are, for example, diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; humectants and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, dextrose solution, acacia slurry, gelatin slurry, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone, and the like; disintegrants such as dry starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid ester, sodium dodecyl sulfonate, methylcellulose, ethylcellulose, etc.; disintegration inhibitors such as sucrose, glyceryl tristearate, cocoa butter, hydrogenated oils and the like; absorption promoters such as quaternary ammonium salts, sodium lauryl sulfate, and the like; lubricants such as talc, silica, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, and the like. The tablets may be further formulated into coated tablets, such as sugar coated tablets, film coated tablets, enteric coated tablets, or bilayer and multilayer tablets. For the preparation of the dosage unit into a pill, various carriers well known in the art can be widely used. Examples of carriers are, for example, diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oils, polyvinylpyrrolidone, gelucire, kaolin, talc, etc.; binders such as acacia, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or batter, and the like; disintegrants such as agar powder, dry starch, alginate, sodium dodecyl sulfate, methylcellulose, ethylcellulose, etc. For preparing the dosage unit into suppositories, various carriers well known in the art can be widely used. Examples of carriers include polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semisynthetic glycerides, and the like. In order to make the administration unit into a capsule, the active ingredient is mixed with the above-mentioned various carriers, and the thus-obtained mixture is placed in a hard Ming capsule or a soft capsule. The active ingredients can also be made into microcapsule, suspension in aqueous medium to form suspension, or hard capsule or injection. For preparing the administration unit into an injectable preparation such as a solution, emulsion, lyophilized powder for injection and suspension, all diluents commonly used in the art, for example, water, ethanol, polyethylene glycol, 1, 3-propanediol, ethoxylated isostearyl alcohol, polyoxyisostearyl alcohol, polyoxyethylene sorbitol fatty acid ester, etc. may be used. In addition, in order to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose or glycerin may be added to the preparation for injection, and further, a conventional cosolvent, a buffer, a pH adjuster, and the like may be added.
In addition, colorants, preservatives, flavors, flavoring agents, sweeteners, or other materials may also be added to the pharmaceutical formulation, if desired.
The dosage of the drug of the present invention to be administered depends on many factors such as the nature and severity of the disease to be prevented or treated, the sex, age, weight and individual response of the patient or animal, the administration route and the administration frequency, etc. The above-mentioned doses may be administered in a single dosage form or divided into several, for example two, three or four dosage forms. The dosage level will be selected based on the particular route of administration, the severity of the condition being treated, the condition and past medical history of the patient to be treated, and the like. However, it is practiced in the art to administer doses that begin at levels below that required to achieve the desired therapeutic effect and gradually increase until the desired effect is achieved.
The term "effective amount" refers to the amount that achieves treatment, prevention, alleviation and/or relief of a disease or condition of the present invention in a subject.
It will be appreciated that the total daily amount of the medicament of the present invention must be determined by the physician within the scope of sound medical judgment. For any particular patient, the particular therapeutically effective dose level will depend on a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; age, weight, general health, sex and diet of the patient; time of administration, route of administration, and rate of excretion; duration of treatment; a drug used in combination or simultaneously; and similar factors well known in the medical arts. For example, it is common in the art to administer doses that begin at levels below that required to achieve the desired therapeutic effect and gradually increase until the desired effect is achieved. In general, the pharmaceutical composition of the present invention may be used in a dosage of 0.001 to 1000mg/kg body weight/day, for example, 0.01 to 100mg/kg body weight/day, for example, 0.01 to 10mg/kg body weight/day, calculated as the active ingredient (icariside II) for mammals, particularly humans.
The beneficial effects of the invention are as follows: the application of icariside II or the pharmaceutically acceptable carrier thereof in erectile dysfunction has remarkable effect. On the other hand, since nnos+ nerve fibers are damaged after nerve injury, cCMP is inhibited from being synthesized, and cGMP cannot be accumulated even after administration of PDE5 inhibitors such as sildenafil, such patients generally have poor efficacy of PDE5 inhibitors. Icariside II restores cGMP synthesis by repairing nNOS+ nerve fibers, so that PDE5 inhibitors such as sildenafil and the like play a role basis. Icariside II can effectively compensate for synthetic cCMP disorder caused by nNOS+nerve fiber damage after nerve injury, so that cGMP cannot be accumulated further after PDE5 inhibitors such as sildenafil and the like are taken, and the problem of poor curative effect of the common PDE5 inhibitors of patients is solved.
On the other hand, the problem of impaired structure leading to penile erection, which is caused by smooth muscle disuse atrophy after denervation or by smooth muscle decrease due to failure to erect hypoxia, etc., is solved. Icariside II promotes differentiation of endogenous stem cells of the penis of EdU+ to smooth muscle on the one hand, and restores effector cells of penile erection. In addition, after the two are combined, on the basis of the pathological change repair of the tissues related to penile erection, the medicine is again effective for patients insensitive to the original oral PDE5 inhibitor.
Therefore, the application of icariside II or the pharmaceutically acceptable carrier thereof in erectile dysfunction can repair pathological changes of penile tissue, and has important clinical significance for preventing and treating erectile dysfunction, in particular for curing PDE5 inhibitor insensitive erectile dysfunction.
Drawings
FIG. 1 is a graph showing the effect of ICAII and sildenafil at various doses on penile erectile function in rats according to example 2 of the present invention (< 0.05vsBCNI-3wgroup, # p <0.05vsICAII-1.25 group);
FIG. 2 is a diagram of evaluation of penile erection function according to example 4 of the invention (< 0.05 vsVehicclejujuup, #p <0.05vsSilgroup, @ p <0.05vsICAII group & p <0.05 vsICAII+Silgroup);
FIG. 3 is a statistical plot of cGMP content in tissue (< 0.05 vsVehicclergep, #p <0.05vsSilgroup, & p <0.05vsICA II group) for example 5 of the present invention;
FIG. 4 is a graph of the measurement of the level of Masson trichromatic smooth muscle in example 6 of the present invention (p <0.05 vsVehicclegarop, # p <0.05 vsSilgroup);
FIG. 5 is a graph of the measurement of smooth muscle content of an immunofluorescent-stained for 7. Alpha. -SMA according to the present invention (p <0.05 vsVehicclectrosup, # p <0.05 vsSilgroup);
FIG. 6 is a graph of the amount of nNOS+ nerve fibers measured in accordance with example 8 of the present invention (p <0.05 vsVehicclergep, #p <0.05 vsSilgroup);
FIG. 7 is a graph showing the measurement of the number of EdU+ cells in example 9 of the present invention; the right side is an enlarged view of the left square frame;
FIG. 8 is a graph of smooth muscle cell differentiation assay of example 9 of the present invention (< 0.05 vsShamsroup, #p <0.05 vsVehicclegaroup, & p <0.05vsSilgroup p)
FIG. 9 shows the result of the HPLC detection in step e of example 10 according to the present invention;
FIG. 10 shows the result of the HPLC detection in step f of example 10 according to the present invention;
FIG. 11 shows the result of the HPLC detection in step e of example 11 according to the present invention;
FIG. 12 shows the result of the HPLC detection in step f of example 11 according to the present invention.
Detailed Description
The invention will now be further illustrated by means of several specific examples, which are given for illustrative purposes only and are not intended to be limiting.
Example 1:
the experimental method comprises the following steps:
(1) Laboratory animals and groups
96 male SD rats were born 1 day.
After 12 weeks, 12 Sham groups (Sham, open abdomen but not damage cavernous nerve, n=12) were randomly selected. The two side cavernous nerves (see 2) are damaged by the rest 84 SD rats to establish a neurogenic ED model; randomly selecting 12 sildenafil drenches from the above medicines, wherein the concentration is 1.25mg/kg/d; randomly selecting 12 sildenafil drenches, and 2.5mg/kg/d; randomly selecting 12 sildenafil drenches, 5mg/kg/d; randomly selecting 12 icariside II to be drenched, wherein the dose is 2.5mg/kg/d; randomly selecting 12 icariside II to be drenched, wherein the dose is 2.5mg/kg/d; randomly selecting 12 icariside II to be infused, and 5mg/kg/d; the remaining 12 were used as model sets (BCNI-3 w).
(2) Establishment of a neurogenic ED rat model:
after the SD rat is born at 12w, the corpus cavernosum of the penis is damaged to establish an ED animal model, and the process is as follows:
1) SD rats were anesthetized by intraperitoneal injection of 5% sodium pentobarbital, at a dose of 30mg/kg;
2) After SD rats are anesthetized, the rats are fixed on an operating table, and the abdomen is shaved and disinfected;
3) A median incision of the lower abdomen is carried out, the pelvic ganglion is searched outside the dorsal aspect of the double-sided prostate under an operation microscope, and branches such as cavernous nerve, abdominal nerve, pelvic nerve and the like are distinguished; after the cavernous nerve is emitted from the pelvic ganglion, the cavernous nerve extends to the urethra direction on the surface of the prostate and enters the penis foot behind the pubic symphysis;
4) Clamping the cavernous nerve at a position 5mm away from the cavernous nerve and from the pelvic ganglion for 2min left and right;
5) After injury to the cavernous nerve, the abdominal cavity is closed layer by layer and the incision site of the abdomen is disinfected by iodophor. The control group did not clamp the cavernous nerve, and the other operations were the same as the experimental group.
(3) Determination of intracavernosal and mean arterial pressures
1) SD rats were anesthetized by 5% sodium pentobarbital (30 mg/kg) intraperitoneal injection, and were placed in supine position, and were cut off the abdominal hair, and the lower abdomen was cut off in the middle, exposing the bladder and prostate, and finding the pelvic ganglion and cavernous nerve (located posterolateral from the prostate);
2) Taking 2 24G puncture needles, filling 200IU/ml heparin, and connecting the other end with a Biopac multi-guide physiological recorder (MP 150) through a PE-50 pipe;
3) Cutting the skin of the penis to expose the corpora cavernosa of the rat penis, and inserting a puncture needle into the corpora cavernosa sinus of the penis; freeing the left common carotid artery and inserting another puncture needle;
4) The cavernous nerve of penis was stimulated by a Biopac multi-lead physiological recorder stimulator for 50 seconds with parameters of 1.5mA and 20Hz. Intracavernosal pressure (ICP) and Mean Arterial Pressure (MAP) were synchronously recorded and changes in penile erectile function before and after treatment were assessed in animal models by ICP/MAP ratio.
(4) Drug infusion:
small dose ica ii group: after 7 days of successful construction of ED model, ICAII (1.25 mg/kg) was administered for 4 weeks of gastric lavage;
medium dose ica ii group: after 7 days of successful construction of ED model, ICAII (2.5 mg/kg) was administered for 4 weeks of gastric lavage;
high dose ica ii group: after 7 days of successful construction of ED model, ICAII (5 mg/kg) was administered for 4 weeks of gastric lavage treatment
Small dose Sildenafil group (Sildenafil): after 7 days of successful construction of the ED model, sildenafil (1.25 mg/kg) was administered for 4 weeks; and (3) injection: after the drug treatment is finished, the subsequent experiment is carried out after the elution period is set to 2 weeks.
Sildenafil group (Sildenafil) at medium dose: after 7 days of successful construction of the ED model, sildenafil (2.5 mg/kg) was administered for 4 weeks; and (3) injection: after the drug treatment is finished, the subsequent experiment is carried out after the elution period is set to 2 weeks.
High dose Sildenafil group (Sildenafil): after 7 days of successful construction of the ED model, sildenafil (5 mg/kg) was administered for 4 weeks in a gastric lavage treatment; and (3) injection: after the drug treatment is finished, the subsequent experiment is carried out after the elution period is set to 2 weeks.
Example 2:
penis erection assessment
As shown in FIG. 1, sildenafil was infused daily, with no significant improvement in penile erectile function in the neuropathic ED rat model (VSBCNI-3 w group); the penis erection function (VSBCNI-3 w group) of the neuropathic ED rat model can be obviously improved by drenching ICAII 1.25mg/kg, 2.5mg/kg and 5 mg/kg; and ICAII 2.5mg/kg and 5mg/kg groups have significant differences compared to ICAII 1.25mg/kg group; and compared with the ICAII group of 2.5mg/kg and 5mg/kg, the ICAII group of 5mg/kg has no obvious improvement on the penile erection kinetic energy of rats.
Example 3:
the experimental method comprises the following steps:
(1) Labelling of systemic endogenous stem cells/precursor cells using labelling retention cell technology
50mgEdU was dissolved in 2.5ml DMSO, sterilized deionized water (2.5 ml) was added, and the mixture was homogenized and incubated in a 37℃water bath. Within 12 hours after the suckling mice are born, 50 mu l of EdU solution is injected into the abdominal cavity, and the dosage is 50mg/kg.
(2) Laboratory animals and groups
48 neonatal 1 day male SD rats were intraperitoneally injected with EdU labeled endogenous stem cells/precursor cells.
After 12 weeks, 12 Sham groups (Sham, open abdomen but not damage cavernous nerve, n=12) were randomly selected. The two side cavernous nerves (see 4) are damaged by the rest 48 SD rats to establish a neurogenic ED model; randomly selecting 12 sildenafil drenches, 2.5mg/kg/d; the rest 12 are drenched with icariside II, 2.5 mg/kg/d).
Continuously drenching for 4 weeks, detecting erectile function and obtaining the medicament eluting period before the specimen for 7 days.
(3) Establishment of a neurogenic ED rat model: same as in example 1
(4) The intracavernosal pressure and mean arterial pressure were determined: same as in example 1
(5) Effects of cavernous nerve injury and treatment of groups on differentiation of endogenous stem cells/precursor cells of penile tissue
And obtaining each group of penile tissue specimens, and freezing and slicing after OCT embedding. The co-expression of the labeled cells (EdU+) in the corpora cavernosa and the smooth muscle cell marker alpha-SMA is detected by immunofluorescence staining, and the superposition indicates differentiation.
(6) Statistical analysis
All experimental groups adopt a random principle; after collecting the data, applying statistical software SPSS to carry out statistical analysis on the data; p <0.05 was considered to have significant statistical differences, and p <0.01 was considered to have very significant statistical differences.
Example 4:
1. penis erection assessment
As shown in table 1 and fig. 2, sildenafil was infused daily, with no significant improvement in penile erectile function in the neuropathic ED rat model (VS Vehicle group, ICP/map=0.24±0.08, p=0.867); the penis erection function (VS Vehicle group, ICP/MAP=0.50+ -0.05, p=0.047) of the neuropathic ED rat model can be remarkably improved by drenching ICAII 2.5mg/kg, but has a remarkable difference from the penis erection kinetic energy of normal rats.
Table 1: penis erection assessment
Example 5:
2 effects on cGMP content in tissues
As shown in table 2 and fig. 3, after nerve injury, the cGMP content in the fresh corpora cavernosa was significantly reduced, and cGMP could not be further accumulated after simple administration. After ICAII is taken, the cGMP content is improved, so that the cGMP in the corpora cavernosa is further and obviously improved, and the defect of poor effect of the PDE5 inhibitor can be overcome by combining with the PDE5 inhibitor.
Table 2: influence of cGMP content in tissue
Since PDE5 inhibitors exert therapeutic effects primarily by reducing cyclic guanosine monophosphate (cGMP) hydrolysis. So that the number of the parts to be processed,sexual life The administration of PDE5 inhibitors "one-time" before life only temporarily alleviates EDSymptoms, not completely removing the etiology and improving the histopathology And (3) a change.Furthermore, in the case of ED caused by vascular/neuropathy (diabetes mellitus and prostate cancer radical surgery), the reduction of Nitric Oxide (NO) release from cavernous nerve endings and/or endothelial cells results in insufficient synthesis of cyclic guanosine monophosphate (cGMP) in smooth muscle cells, and PDE5 inhibitors have poor therapeutic effects due to lack of molecular basis for exerting therapeutic effects.
Icariside II restores cGMP synthesis by repairing nNOS+ nerve fibers, so that PDE5 inhibitors such as sildenafil and the like play a role basis. Icariside II can effectively compensate for synthetic cCMP disorder caused by nNOS+nerve fiber damage after nerve injury, so that cGMP cannot be accumulated further after PDE5 inhibitors such as sildenafil and the like are taken, and the problem of poor curative effect of the common PDE5 inhibitors of patients is solved.
Example 6:
3.2.1 Effect on smooth muscle content (Masson trichromatic dyeing)
As shown in table 3 and fig. 4, the degree of fibrosis in the cavernous body was increased after nerve injury, and oral sildenafil could not significantly improve the degree of fibrosis in the cavernous body; the degree of cavernous body fibrosis is remarkably improved after ICAII administration.
Table 3: influence on smooth muscle content (Masson trichromatic dyeing)
Example 7:
3.2.2 effects on smooth muscle content (alpha-SMA immunofluorescence staining)
As shown in table 4 and fig. 5, after nerve injury, the content of smooth muscle in the corpora cavernosa was significantly reduced, and oral sildenafil could not significantly increase the content of smooth muscle in the corpora cavernosa; the content of smooth muscle in the cavernous body is increased after ICAII administration.
Table 4: influence on smooth muscle content (alpha-SMA immunofluorescence staining)
Example 8:
influence on the number of nNOS+ nerve fibers
As shown in table 5 and fig. 6, after nerve injury, the amount of nnos+ nerve fibers in the corpora cavernosa was significantly reduced, and oral sildenafil could not significantly increase the amount of nnos+ nerve fibers in the corpora cavernosa; the increase in the number of nNOS+ nerve fibers in the corpora cavernosa after ICAII administration suggests that the increase in the number of nNOS+ nerve fibers in the corpora cavernosa may be mainly related to ICAII drenching.
Table 5: influence on the number of nNOS+ nerve fibers
Example 9:
effect on EdU+ cell count
As shown in table 6 and fig. 7-8, the three groups, except the sham-operated group, all used the cavernous nerve injury ED animal model, immunofluorescent staining found a significant increase in the number of edu+ cells in these groups over the sham-operated group, but no statistical differences between the three groups. Further counting of the number of cells of edu+ with sma+ revealed that the number of cells of edu+ with sma+ was significantly increased in the group to which ICA ii was administered compared to the other groups, but there was no statistical difference in the number of cells between the two groups. The data suggests that EdU+ cells differentiate into smooth muscle cells after ICAII administration.
Table 6: effects on EdU+ cell number and EdU+ cell number of SMA+ at the same time
Example 10:
the method for obtaining icariside II through bioconversion and enrichment of 8g of icariside comprises the following steps:
a. 4 ℃ cryopreserved Trichoderma viride is adopted to be propagated in a potato culture medium for 4 days, the propagation culture temperature is 28 ℃, and when the inclined surface of a test tube grows to be full of thalli, the trichoderma viride is placed in a 4 ℃ refrigerator to be cryopreserved with high activity; the potato culture medium is prepared from fresh potatoes, anhydrous glucose and agar by adding distilled water, namely 200g of fresh potatoes are firstly boiled to be soft by using an iron pot and distilled water, filtered, the filtrate is heated again, 20g of anhydrous glucose and 18g of agar are added, the mixture is packaged into small test tubes with the diameter of 1.2cm after being dissolved, the height of the culture medium is not more than 1/3 of the total height of the test tubes, the test tubes are plugged, each 7 test tubes are bundled, the test tubes are wrapped by kraft paper, sterilized for 20 minutes at the temperature of 121 ℃, the test tubes are uniformly shaken after being taken out, the test tubes are obliquely placed at an angle of 45 degrees, and the test tubes are cooled and stored for standby;
b. adding 0.5g of potatoes (broken) and 0.8g of icariin into a 100mL triangular conical flask, adding distilled water to 50mL, tying the bottle mouth tightly by using a sealing film, placing 10 vials in a sterilizing box at 121 ℃ for sterilization for 20 minutes, cooling to about 25 ℃, inoculating the trichoderma viride of 1 test tube cultured in the step a into a liquid culture medium of the triangular conical flask on an ultra-clean workbench, and placing the trichoderma viride into a shaking table at 28 ℃ and a rotating speed of 165r/min for 9 days;
c. after 9 days of culture, filtering out icariside II and icariside (small amount) which contain trichoderma viride and are insoluble in water by using a suction filtration device;
d. dissolving icariside II and icariside filtered in the step c by using pure methanol until the trichoderma viride is soaked to be whitened, and dissolving the icariside II and the icariside in the methanol;
the Trichoderma viride was filtered off by soaking with methanol 3 times in amounts of 1000ml, 500ml, respectively.
e. Concentrating the methanol soaking solution in the step d to obtain mixture powder mainly containing icariside II, and weighing 6.2430g; the conversion was measured by liquid phase and the measured result was calculated to be 91.56% (fig. 9);
f. dissolving the powder in the step e with minimum methanol at 65-70deg.C by heating reflux, removing methanol solution containing icariside II, standing at normal temperature, standing to precipitate crystals, filtering to obtain crystals, standing at normal temperature, standing to precipitate crystals for the second time, filtering to obtain crystals, mixing the crystals obtained by the first filtering, and vacuum drying below 45deg.C to constant weight to obtain 5.5371g of icariside II pure product with 99% content, and yield 91.07% (figure 10).
Example 11:
a method for bioconverting and enriching icariside II from 192g of icariside, comprising the following steps:
a. 4 ℃ cryopreserved Trichoderma viride is adopted to be propagated in a potato culture medium for 4 days, the propagation culture temperature is 28 ℃, and when the inclined surface of a test tube grows to be full of thalli, the trichoderma viride is placed in a 4 ℃ refrigerator to be cryopreserved with high activity; the potato culture medium is prepared from fresh potatoes, anhydrous glucose and agar by adding distilled water, namely 200g of fresh potatoes are firstly boiled to be soft by using an iron pot and distilled water, filtered, the filtrate is heated again, 20g of anhydrous glucose and 18g of agar are added, the mixture is packaged into small test tubes with the diameter of 1.2cm after being dissolved, the height of the culture medium is not more than 1/3 of the total height of the test tubes, the test tubes are plugged, each 7 test tubes are bundled, the test tubes are wrapped by kraft paper, sterilized for 20 minutes at the temperature of 121 ℃, the test tubes are uniformly shaken after being taken out, the test tubes are obliquely placed at an angle of 45 degrees, and the test tubes are cooled and stored for standby;
b. adding 10g of potatoes (broken), 16g of icariin and distilled water into a 3L triangular conical flask, adding distilled water into the 3L triangular conical flask until the bottle mouth is 1L, tightly tying the bottle mouth by using a sealing film, adding 12 bottles, placing the bottle into a sterilizing box at 121 ℃, sterilizing for 20 minutes, cooling the bottle until the bottle is cooled to about 25 ℃, inoculating the trichoderma viride with 6 test tubes cultured in the step a into a liquid culture medium of the triangular conical flask on an ultra-clean workbench, and placing the bottle into a shaking table at 28 ℃ and culturing for 9 days at a rotating speed of 165 r/min;
c. after 9 days of culture, filtering out icariside II and icariside (small amount) which contain trichoderma viride and are insoluble in water by using a suction filtration device;
d. dissolving icariside II and icariside filtered in the step c by using pure methanol until the trichoderma viride is soaked to be whitened, and dissolving the icariside II and the icariside in the methanol;
the Trichoderma viride was filtered off by soaking 3 times with methanol in amounts of 36L, 18L, respectively.
e. Concentrating the methanol soaking solution in the step d to obtain mixture powder mainly containing icariside II, and weighing 143.5265g; the conversion was measured by liquid phase and the measured result was calculated as 93.12% (fig. 11);
f. dissolving the powder in the step e with minimum methanol at 65-70deg.C by heating reflux method, removing methanol solution containing icariside II, standing at normal temperature, standing to precipitate crystals, filtering to obtain crystals, standing at normal temperature, standing to precipitate crystals for the second time, filtering to obtain crystals, mixing the crystals obtained by the first filtering, and vacuum drying below 45deg.C to constant weight to obtain 133.4286g of icariside II with product content of 99.12% and yield of 91.44% (figure 12).
The application of icariside II or a pharmaceutically acceptable carrier thereof in erectile dysfunction is shown in the invention, and the action mechanism of the icariside II or the pharmaceutically acceptable carrier thereof is as follows: on the one hand, nnos+ nerve fibers are damaged after nerve injury, and the synthesis of cCMP is impaired, so that cGMP cannot be accumulated further after sildenafil administration, and thus such patients generally have poor efficacy of PDE5 inhibitors. On the other hand, after denervation, the smooth muscle is disabled, or the smooth muscle is reduced due to the failure to erect and lack of oxygen, etc., so that the most important structure of penile erection is damaged. Icariside II promotes differentiation of endogenous stem cells of the penis of EdU+ to smooth muscle on the one hand, and restores effector cells of penile erection. On the other hand, restoration of nnos+ nerve fibers, restoration of cGMP synthesis, and the like have been developed as a basis for the action of PDE5 inhibitors such as sildenafil. Therefore, after the two are combined, on the basis of the pathological change repair of the tissues related to penile erection, the patients insensitive to the original oral PDE5 inhibitor can take effect on the medicine again.
The foregoing is merely a preferred embodiment of the invention, and it should be noted that modifications could be made by those skilled in the art without departing from the principles of the invention, which modifications would also be considered to be within the scope of the invention.
Claims (2)
1. A method for obtaining icariside II through biotransformation and enrichment of icariside is characterized in that: the method comprises the following steps:
a. 4 ℃ cryopreserved Trichoderma viride is adopted to be propagated in a potato culture medium for 4 days, the propagation culture temperature is 28 ℃, and when the inclined surface of a test tube grows to be full of thalli, the trichoderma viride is placed in a 4 ℃ refrigerator to be cryopreserved with high activity; the potato culture medium is prepared from fresh potatoes, anhydrous glucose and agar by adding distilled water, namely 200g of fresh potatoes are firstly boiled to be soft by using an iron pot and distilled water, filtered, the filtrate is heated again, 20g of anhydrous glucose and 18g of agar are added, the mixture is packaged into small test tubes with the diameter of 1.2cm after being dissolved, the height of the culture medium is not more than 1/3 of the total height of the test tubes, the test tubes are plugged, each 7 test tubes are bundled, the test tubes are wrapped by kraft paper, sterilized for 20 minutes at the temperature of 121 ℃, the test tubes are uniformly shaken after being taken out, the test tubes are obliquely placed at an angle of 45 degrees, and the test tubes are cooled and stored for standby;
b. adding 0.5g of potatoes into a 100mL triangular conical flask, smashing, adding 0.8g of icariin, adding distilled water to 50mL, tying the bottle mouth tightly by using a sealing film, placing 10 vials in a sterilizing box at 121 ℃ for sterilization for 20 minutes, cooling to about 25 ℃, inoculating the trichoderma viride of 1 test tube cultured in the step a into a liquid culture medium of the triangular conical flask on an ultra-clean workbench, and culturing for 9 days in a shaking table at 28 ℃ and a rotating speed of 165 r/min;
c. after 9 days of culture, filtering out icariside II and icariside which contain trichoderma viride and are insoluble in water by using a suction filtration device;
d. dissolving icariside II and icariside filtered in the step c by using pure methanol until the trichoderma viride is soaked to be whitened, and dissolving the icariside II and the icariside in the methanol;
soaking in methanol for 3 times, and filtering out Trichoderma viride in amounts of 1000ml, 500ml and 500ml respectively;
e. concentrating the methanol soaking solution in the step d to obtain mixture powder mainly containing icariside II, and weighing 6.2430g; the conversion was measured by passing the liquid phase, and the measured result was calculated to be 91.56%;
f. dissolving the powder in the step e with minimum methanol at 65-70deg.C by heating reflux, removing methanol solution containing icariside II, standing at normal temperature, separating out crystals, filtering, standing at normal temperature, separating out crystals for the second time, filtering, mixing the crystals obtained by the first filtering, and vacuum drying below 45deg.C to constant weight to obtain 5.5371g of icariside II with 99% content and 91.07% yield.
2. A method for obtaining icariside II through biotransformation and enrichment of icariside is characterized in that: the method comprises the following steps:
a. 4 ℃ cryopreserved Trichoderma viride is adopted to be propagated in a potato culture medium for 4 days, the propagation culture temperature is 28 ℃, and when the inclined surface of a test tube grows to be full of thalli, the trichoderma viride is placed in a 4 ℃ refrigerator to be cryopreserved with high activity; the potato culture medium is prepared from fresh potatoes, anhydrous glucose and agar by adding distilled water, namely 200g of fresh potatoes are firstly boiled to be soft by using an iron pot and distilled water, filtered, the filtrate is heated again, 20g of anhydrous glucose and 18g of agar are added, the mixture is packaged into small test tubes with the diameter of 1.2cm after being dissolved, the height of the culture medium is not more than 1/3 of the total height of the test tubes, the test tubes are plugged, each 7 test tubes are bundled, the test tubes are wrapped by kraft paper, sterilized for 20 minutes at the temperature of 121 ℃, the test tubes are uniformly shaken after being taken out, the test tubes are obliquely placed at an angle of 45 degrees, and the test tubes are cooled and stored for standby;
b. adding 10g of potatoes into a 3L triangular conical flask, crushing, adding 16g of icariin, adding distilled water to 1L, tightly tying a bottle mouth by using a sealing film, adding 12 bottles, placing into a sterilizing box at 121 ℃, sterilizing for 20 minutes, cooling to about 25 ℃, inoculating the trichoderma viride of 6 test tubes cultured in the step a into a liquid culture medium of the triangular conical flask on an ultra-clean workbench, and culturing for 9 days in a shaking table at 28 ℃ and a rotating speed of 165 r/min;
c. after 9 days of culture, filtering out icariside II and icariside which contain trichoderma viride and are insoluble in water by using a suction filtration device;
d. dissolving icariside II and icariside filtered in the step c by using pure methanol until the trichoderma viride is soaked to be whitened, and dissolving the icariside II and the icariside in the methanol;
soaking in methanol for 3 times, and filtering out Trichoderma viride at 36L, 18L and 18L respectively;
e. concentrating the methanol soaking solution in the step d to obtain mixture powder mainly containing icariside II, and weighing 143.5265g; the conversion was measured by passing the liquid phase, and the measured result was calculated as 93.12%;
f. dissolving the powder in the step e with minimum methanol at 65-70deg.C by heating reflux, removing methanol solution containing icariside II, standing at normal temperature, separating out crystal, filtering to obtain crystal, standing at normal temperature, separating out crystal for the second time, filtering to obtain crystal, mixing the first filtered crystal, and vacuum drying below 45deg.C to constant weight to obtain 133.4286g of icariside II with product content of 99.12% and yield of 91.44%.
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| CN112870211A (en) * | 2021-02-05 | 2021-06-01 | 遵义医科大学 | Application of icariside II in preparation of medicine for preventing and treating acute liver injury |
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