Application of rehmannia glutinosa libosch D in preparation of medicine for protecting spermatogonium

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of rehmannia glutinosa libosch D in preparation of a spermatogonial cell protection medicine. According to the present invention, the protection experiment of the rehmannia glutinosa libosch D on the spermatogonium cells proves that the rehmannia glutinosa libosch D has significant protection effect on the spermatogonium cells, such that the new idea and the new scheme are provided for the preparation and the research of the spermatogonium cell protection drug, and the certain basis is provided for the subsequent male infertility drug development of the rehmannia glutinosa libosch D.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology. Specifically, it relates to the application of rehmannioside D in the preparation of drugs for protecting spermatogonia cells. Background Art

[0002] Rehmannia glutinosa has a long history of medicinal use. Its fresh or dried roots are used as medicine. Different processing methods result in different medicinal effects. For example, CN102716256A provides a traditional Chinese medicine preparation of prepared rehmannia root for treating headache, and CN103055023B discloses the effect of fresh rehmannia root in treating alopecia. Prepared rehmannia root is obtained by steaming and processing raw rehmannia root with yellow rice wine. It is slightly warm and sweet in taste, and has the effects of nourishing yin and blood, and replenishing essence and marrow. Traditional Chinese medicine believes that insufficient kidney yin and deficiency of kidney essence can lead to few sperm and small semen volume. Therefore, prepared rehmannia root is widely used in traditional Chinese medicine prescriptions for infertility due to deficiency of kidney essence (oligozoospermia and asthenospermia). Common classic traditional Chinese medicine prescriptions include Liuwei Dihuang Pills, Zhibai Dihuang Pills, Bawei Dihuang Pills, Wuzi Dihuang Oral Liquid, Dihuang Yijing Formula, etc.

[0003] However, the chemical composition of prepared rehmannia root is complex, and the main medicinal components for treating oligozoospermia and asthenospermia are still unknown. In addition, the complex chemical composition of prepared rehmannia root is not conducive to quality control and the development of related drugs, and the complex chemical composition is prone to cause adverse reactions.

[0004] Based on the above background, this application is specifically proposed. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide the application of rehmannioside D in the preparation of drugs for protecting spermatogonia cells.

[0006] To solve the above problems, the technical solutions adopted by the present invention are as follows: Technical Theme 1 The application of rehmannioside D and its medicinal salts or isomers in the preparation of drugs for protecting spermatogonia cells.

[0007] As a further improvement of the present invention, the drug can be made into one of the following dosage forms: tablets, capsules, granules, powders, suspensions, emulsions, powders, solutions, gels, syrups, pills, tinctures, wines, decoction extracts, lozenges, mixtures, suppositories, injections, inhalants or sprays.

[0008] Technical Theme 2 The application of rehmannioside D and its medicinal salts or isomers as the sole active ingredient in the preparation of drugs for treating oligozoospermia and asthenospermia.

[0009] The beneficial effects produced by adopting the above technical solutions are as follows: Through the experiment on the protection of spermatogonial cells by rehmannioside D, the present invention proves that rehmannioside D has an obvious protective effect on GC-1 spermatogonial cells. It provides new ideas and new schemes for the preparation and research of drugs for protecting spermatogonial cells, and provides a certain basis for the subsequent development of drugs for male infertility with rehmannioside D. Brief Description of the Drawings

[0010] Figure 1 It is the result diagram of the protection of spermatogonial cells by rehmannioside D in Example 1 of the present invention. In the figure, 15 µM represents the group of spermatogonial cells + culture medium + D-galactose + 15 µM rehmannioside D in the drug group, 20 µM represents the group of spermatogonial cells + culture medium + D-galactose + 20 µM rehmannioside D in the drug group, 25 µM represents the group of spermatogonial cells + culture medium + D-galactose + 25 µM rehmannioside D in the drug group, and 30 µM represents the group of spermatogonial cells + culture medium + D-galactose + 30 µM rehmannioside D in the drug group. Detailed Embodiments

[0011] As used herein, "pharmaceutically acceptable salts" refer to salts that retain the desired biological activity of the target compound and exhibit minimal undesired toxicological effects. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of the compounds of the present invention with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable bases include salts prepared from inorganic bases and organic bases. Examples of salts of inorganic bases include aluminum salts, ammonium salts, calcium salts, copper salts, iron salts, ferrous salts, lithium salts, magnesium salts, manganese salts, manganous salts, potassium salts, sodium salts, zinc salts, etc. Examples of salts of organic non-toxic bases include salts of primary amines, secondary amines, and tertiary amines, including substituted amines and cyclic amines. For example: N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, aminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucosamine, glucosamine hydrochloride, histidine, hydroxocobalamin, isopropylamine, lysine, methylglucosamine, morpholine, piperazine, piperidine, pyrrolidine, polyamine resin, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, etc. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts, where the inorganic acids include, for example, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, or nitric acid, etc.; organic acids such as formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, hexanoic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2-(4-hydroxybenzoyl)-benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, digluconic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, pamonic acid, pectinic acid, 3-phenylpropionic acid, picric acid, pivalic acid, 2-hydroxyethanesulfonic acid, itaconic acid, aminosulfonic acid, trifluoromethanesulfonic acid, dodecylsulfuric acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, mandelic acid, ascorbic acid, glucoheptonic acid, glycerophosphoric acid, aspartic acid, sulfosalicylic acid, etc. including salts formed with sodium, potassium, magnesium, lithium, aluminum, calcium, zinc, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucosamine, and procaine, etc.

[0012] As used herein, "isomer" means that in the presence of one or more asymmetric centers and / or double bonds, the compounds of the present invention can exist in the form of racemates, racemic mixtures, single enantiomers, diastereomer mixtures, single diastereomers, geometric isomers, etc. These compounds can be represented by the symbols "R" or "S", depending on the configuration of the substituents around the stereogenic carbon atom, and may also be represented by the symbols "Z" or "E", depending on the arrangement of the substituents around the carbon-carbon double bond, or the substituents around the carbon-carbon double bond can be referred to as "cis" or "trans". The compounds disclosed herein can exist as tautomers, and both tautomeric forms are intended to be included within the scope of the present invention, even if only one tautomeric structure is depicted, such as keto-enol tautomers, phenol-keto tautomers, nitroso-oxime tautomers, imine-enamine tautomers, etc. In the present invention, the described compounds and their stereoisomers are all within the scope of protection, including but not limited to enantiomers, diastereomers, and cis-trans isomers. Therefore, the compounds protected by the present invention include not only all possible single stereoisomers, but also their optically active mixtures and racemates. In addition, those skilled in the art should understand that different stereoisomers may have significant differences in biological activity, pharmacodynamic effects, and toxicity, so the selection of specific stereoisomers or their combinations may optimize the therapeutic effect. Stereoisomers can be obtained by known chemical or physical methods, such as chiral catalysis, chiral synthesis, or chiral resolution by chromatography or chemical methods. The present invention also includes the stereoisomers obtained by these methods and their pharmaceutically acceptable salts.

[0013] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be clearly and completely described below in conjunction with specific embodiments.

[0014] Rehmannioside D used in this application was purchased from the National Institutes for Food and Drug Control, with the batch number 112063-202304.

[0015] Example 1 Study on the protective effect of rehmannioside D on GC-1 spermatogonial cells The GC-1 spermatogonial cells were cultured in an incubator at 37°C and 5% CO2, and the culture medium was high-glucose DMEM medium containing 10% fetal bovine serum and double antibiotics (100 U / mL penicillin and 100 mg / L streptomycin).

[0016] Take GC-1 spermatogonial cells and prepare a single-cell suspension with high-glucose DMEM solution containing 10% fetal bovine serum and double antibiotics (100 U / mL penicillin and 100 mg / L streptomycin) as the culture medium, at a concentration of 5×10 4Cells were seeded at a density of

[0017] into 96-well plates at a volume of 100 μL per well. At the same time, a blank group, a control group, a model group, and a drug group were set up, with 6 replicates in each group. Only medium without GC-1 spermatogonial cells was added to the blank group. The blank group was medium; 4 The control group was GC-1 spermatogonial cells at a density of 5×10 4 cells / mL + medium; The model group was 4 GC-1 spermatogonial cells at a density of 5×10 cells / mL + medium + 150 mM D-galactose (D-Gal); −1 The drug group was −1 GC-1 spermatogonial cells at a density of 5×10

[0018] cells / mL + medium + 150 mM D-Gal + (15 μM, 20 μM, 25 μM, or 30 μM) rehmannioside D. −1 The medium in each of the above groups was DMEM high-glucose medium containing 10% fetal bovine serum, −1 100 U∙mL

[0019] penicillin, and 100 mg∙L

[0020] streptomycin.

[0021] After the above groups were prepared, they were cultured in an incubator at 37 °C with 5% CO2 for 24 hours, rinsed twice with PBS solution, and then Figure 1 100 μL of DMEM high-glucose medium containing 10% fetal bovine serum,

[0022] −1 100 U∙mL −1 penicillin, 100 mg∙L

[0019] streptomycin, and 10 μL of CCK-8 were added and incubated for 1 hour. The OD value at a wavelength of 450 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader.

[0020] Cell viability (%) = 100% × (OD value of each group - OD value of the blank group) / (OD value of the control group - OD value of the blank group).

[0021] Cell viability improvement rate (%) = 100% × (cell viability of the drug group / cell viability of the model group - 1). Figure 1 As shown in the results,

[0022] rehmannioside D had an obvious protective effect on GC-1 spermatogonial cells (Note: *p < 0.05; **p < 0.01; ***p < 0.001; ns: P ≥ 0.05, no statistical significance, sample size n = 6). Compared with the model group, the average cell viability of GC-1 spermatogonial cells treated with the 20 μM rehmannioside D drug group was 84.82%, and the cell viability improvement rate of the 20 μM rehmannioside D drug group compared with the model group was 21.66%.

[0022] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of rehmannia glycoside D and its pharmaceutically acceptable salts or isomers in the preparation of drugs for protecting spermatogonia.

2. The use according to claim 1, characterized in that: The drug can be prepared into one of the following dosage forms: tablets, capsules, granules, powders, suspensions, emulsions, powders, solutions, gels, syrups, pills, tinctures, wine preparations, decoctions, lozenges, mixtures, suppositories, injections, inhalants or sprays.

3. Use of rehmannoside D and its pharmaceutically acceptable salts or isomers as the sole active ingredient in the preparation of drugs for the treatment of oligoasthenospermia.

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