CN114209717A - Deer glue and its medicinal composition and use - Google Patents
Deer glue and its medicinal composition and use Download PDFInfo
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- CN114209717A CN114209717A CN202111634623.0A CN202111634623A CN114209717A CN 114209717 A CN114209717 A CN 114209717A CN 202111634623 A CN202111634623 A CN 202111634623A CN 114209717 A CN114209717 A CN 114209717A
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Abstract
The invention discloses deer glue and a pharmaceutical composition and application thereof. Wherein, the pharmaceutical composition comprises deer glue with effective treatment amount. According to the invention, the deer glue can increase the copy number of mitochondrial mtDNA by obviously improving the expression of Bcl-2 protein, so that the dibutyl phthalate mediated apoptosis can be effectively inhibited. The invention further discloses a method for inhibiting apoptosis comprising the step of contacting a cell with a pharmaceutical composition of the invention.
Description
Technical Field
The invention relates to the field of medicines, in particular to deer-horn glue, a pharmaceutical composition and application thereof.
Background
Apoptosis generally refers to a kind of programmed cell death (programmed cell death) of body cells in the development process or under the action of some factors through the regulation and control of intracellular genes and their products. Apoptosis has become a hot spot in cell biology. The apoptosis can attract attention, and the root of the apoptosis is that the apoptosis is closely related to a plurality of human diseases, and the apoptosis is expected to bring a new treatment method for certain diseases, such as tumors, cardiovascular diseases, degenerative diseases, inflammations, immune system diseases and the like without the phenomenon of not including apoptosis. Many of the drugs currently under investigation are also being developed for apoptosis.
The traditional Chinese medicine or the effective components thereof can regulate apoptosis, but the molecular mechanism of the traditional Chinese medicine still needs to be further researched. For example, chinese patent application CN107496512A discloses a traditional Chinese medicine composition for treating cerebral ischemia, which is prepared from the following raw material medicines in parts by weight: 500 parts of rhodiola rosea, 500 parts of corydalis tuber, 300 parts of ligusticum wallichii and 7 parts of borneol. The Chinese medicinal composition can be used for preventing and treating cerebral ischemia diseases, and reducing cerebral cortex apoptosis rate.
For another example, chinese patent application CN104940326A discloses an application of a Chinese medicinal composition in preparing a medicament for reducing apoptosis of mesenchymal stem cells, which is prepared from the following raw material medicines in parts by weight: 5-9 parts of ginseng, 5-11 parts of leech, 6-9 parts of ground beeltle, 2-4 parts of prepared frankincense, 4-7 parts of red paeony root, 2-4 parts of dalbergia wood, 2-4 parts of sandalwood, 3-7 parts of scorpion, 6-10 parts of cicada slough, 1-3 parts of centipede, 2-6 parts of borneol and 4-9 parts of fried spina date seed. However, there is still a need for a pharmaceutical composition that is simple in composition and can effectively inhibit apoptosis.
Disclosure of Invention
In order to solve at least part of technical problems in the prior art, the inventor discovers that deer glue can increase the copy number of mitochondrial mtDNA by improving the expression of Bcl-2 protein, so that dibutyl phthalate mediated apoptosis can be effectively inhibited. The present invention includes the following.
In a first aspect of the invention, the invention provides an application of deer glue in preparing a medicament for inhibiting apoptosis.
According to the use of the present invention, preferably, the deer-horn glue comprises a solid glue aqueous extract prepared by decocting and concentrating the skin and/or horn of a cervidae animal.
According to the use of the present invention, preferably, it is capable of increasing the expression of the Bcl-2 protein.
According to the use of the invention, it is preferably capable of up-regulating mitochondrial mtDNA copy number.
According to the use of the present invention, preferably, the apoptosis is associated with dibutyl phthalate mediated apoptosis.
According to the use of the present invention, preferably, the deer-horn glue is 2-200 mg.
In a second aspect of the present invention, there is provided a pharmaceutical composition for inhibiting apoptosis, comprising a therapeutically effective amount of the deer glue according to the first aspect.
In a third aspect of the invention, there is provided a method for inhibiting apoptosis comprising the step of contacting a cell with a pharmaceutical composition according to the second aspect.
The method according to the present invention preferably further comprises the step of combining the pharmaceutical composition with other therapeutic agents.
Preferably, according to the method of the present invention, the therapeutic agent comprises an apoptosis signaling pathway-related inhibitor.
Drawings
The result of figure 1 shows that the deer-horn glue can remarkably improve the dibutyl phthalate-induced apoptosis of eye cells of the zebra fish juvenile fish
FIG. 2 is a DBP-induced zebra fish aging model Bcl-2 protein expression electrophoretogram of deer glue
FIG. 3 shows the effect of deer glue on the expression of DBP-induced anti-apoptotic factor Bcl-2 in zebra fish aging model
Wherein, compared to the blank control group,##p is less than 0.01; in comparison to the set of models,**P＜0.01，***P＜0.001。
FIG. 4 is a graph showing the effect of deer glue on DBP-induced mitochondrial DNA expression in zebra fish senescence model
Wherein, compared to the blank control group,##p is less than 0.01; in comparison to the set of models,**P＜0.01，***P＜0.001。
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that the upper and lower limits of the range, and each intervening value therebetween, is specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
Application of deer glue in preparation of medicine for inhibiting apoptosis
In the present invention, the deer glue is a known product or an extract containing an effective ingredient prepared by a known method. Preferably, the deer-horn glue of the present invention does not contain any additional additives. Examples of known products of deer glue include, but are not limited to, deer glue produced by Guangxiang health pharmaceutical Co., Ltd. Examples of the preparation by the known method include, but are not limited to, solid gum aqueous extracts prepared by, for example, decocting and concentrating the skin and/or horn of cervidae animals.
In the present invention, the process conditions for preparing the deer-horn glue are not particularly limited as long as the desired deer-horn glue can be obtained.
In the present invention, the Cervidae animal includes Cervus elaphus Linnaeus or Cervus nippon Temminck.
In the present invention, the amount of deer-horn glue is 2-200mg, preferably 5-200mg, and more preferably 10-150 mg. The concentration of deer glue is 0.01-5mg/mL, preferably 0.05-2mg/mL, and more preferably 0.1-0.5 mg/mL.
The inventor finds that the deer glue can obviously relieve the apoptosis in a dose-dependent mode in an experimental model, and particularly, the deer glue inhibits the apoptosis by regulating Bcl-2 protein. Preferably, the deer glue of the present invention can up-regulate the expression of Bcl-2 protein. Methods for measuring the expression level of Bcl-2 protein are known in the art, and qualitative/quantitative analysis can be performed using, for example, Westernblot, enzyme-linked immunosorbent assay (ELISA), immunofluorescence staining, immunohistochemical staining, and the like.
The inventor also surprisingly found that deer glue can inhibit apoptosis by regulating mitochondrial mtDNA copy number in experimental models. Preferably, the deer glue is capable of up-regulating mitochondrial mtDNA copy number. This is probably due to the protective effect of deer glue on damaged mitochondria, thereby effectively inhibiting apoptosis. The method for detecting the number of mitochondrial mtDNA copies is not particularly limited, and detection can be performed by, for example, the second-generation sequencing technique or fluorescent quantitative PCR.
Pharmaceutical composition
The invention further provides a pharmaceutical composition for inhibiting apoptosis, which comprises deer glue. Optionally, the pharmaceutical composition of the present invention comprises a pharmaceutically acceptable carrier or adjuvant. It is understood that the pharmaceutical compositions of the present invention may also include deer glue in combination with other therapeutic agents. Preferably, the therapeutic agent comprises an apoptosis signaling pathway-related inhibitor.
In the present invention, the pharmaceutically acceptable carrier includes a diluent or excipient or other additives, examples of which include, but are not limited to, for example, wetting agents, disintegrants, lubricants, binders, surfactants, and the like. Examples of other additives include, but are not limited to, for example, shellac, gum arabic, talc, titanium oxide, sugars (e.g., sucrose), gelatin, water, polysaccharides such as lactose or glucose, paraffin (e.g., petroleum fractions), vegetable oils (e.g., peanut or sesame oil), and pharmaceutically acceptable organic solvents such as alcohols (e.g., ethanol or glycerol), natural mineral powders (e.g., kaolin, clay, talc, and chalk), synthetic mineral powders (e.g., highly dispersed silicic acid and silicates), emulsifiers (e.g., lignin, sulfite solutions, methylcellulose, starch, and polyvinylpyrrolidone), magnesium stearate, stearic acid, sodium lauryl sulfate, and the like.
The pharmaceutical composition of the present invention may be prepared in various dosage forms including, but not limited to, for example, pharmaceutical preparations suitable for oral administration, such as solid oral preparations including tablets, coatings, capsules, granules, powders, pills, powders, and the like, or liquid oral preparations including solutions, syrups, suspensions, emulsions, and the like; pharmaceutical preparations suitable for parenteral administration, for example in the form of suppositories for rectal administration or else transdermal patches for topical administration.
Methods for inhibiting apoptosis
The present invention also provides a method for inhibiting apoptosis, the method for modulating expression of Bcl-2 protein and/or mitochondrial mtDNA copy number, comprising the step of contacting a cell with a pharmaceutical composition, wherein the pharmaceutical composition comprises deer glue.
Preferably, the cell is an in vitro cell. The detection of changes in protein expression levels and copy number is performed by exposing animal cells to deer-gelatin in an environment in which the cells are cultured in vitro, to highly mimic the effects of deer-gelatin on the regulation of Bcl-2 protein expression and/or mitochondrial mtDNA copy number in vivo. The culture conditions are not particularly limited as long as normal growth of cells in vitro and normal expression of proteins can be achieved.
It will be appreciated that the method of the invention for inhibiting apoptosis further comprises administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition of the invention, wherein the pharmaceutical composition comprises or consists of deer-horn glue.
The term "subject" as used herein includes mammals. The mammal can be, for example, any mammal, e.g., a human, primate, bird, mouse, rat, poultry, dog, cat, cow, horse, goat, camel, sheep, or pig. Preferably, the mammal is a human.
In the present invention, the "therapeutically effective amount" means an amount of the pharmaceutical composition of the present invention sufficient to significantly inhibit apoptosis without causing serious side effects. Generally, the pharmaceutical composition contains 0.1-2000mg of deer-gelatin, more preferably, 0.1-200mg of deer-gelatin. For a person with a body weight of 60kg, the daily dose administered is generally 1-2000mg, preferably 20-500 mg. Of course, the particular dosage regimen will take into account factors such as the route of administration, the health of the patient, and the like, which are within the skill of the skilled practitioner.
It will be appreciated by those skilled in the art that other steps or operations may be included in the method of the invention, for example to further optimize and/or improve the method of the invention, as long as the objects of the invention are achieved.
Examples
This example is a pharmacological test against apoptosis, as follows.
1. Basis of molding
Dibutyl phthalate (DBP) can enter animals or human bodies through the ways of respiratory tract, digestive tract, venous transfusion, skin contact or biological enrichment of animals and plants, enter food chains and the like, interfere with the hormone level in blood, improve the oxidative stress level or influence the metabolism of substances in the bodies, and cause the abnormalities in aspects of reproduction, development, behavior and the like. Research has shown that DBP can cause oxidative stress damage to ocular cells.
The zebra fish eyes have the characteristics of short experimental period, large head eyeball proportion and the like similar to other vertebrates in the aspects of structure, tissue components and biological signals, and the protective effect of the medicine on damaged cells can be directly observed by observing the change of eye cells.
The dibutyl phthalate is used for inducing apoptosis of eye cells of the zebra fish, a zebra fish eye cell apoptosis model is established, and the influence of the deer glue on the eye cell apoptosis is observed.
2. Experimental Material
2.1 Experimental animals: zebra fish.
2.2 test substance
The colla Cornus Cervi is solid gelatin prepared by decocting and concentrating dried or fresh skin of Cervus elaphus Linnaeus or Cervus nippon Temminck of Cervidae.
The deer glue sample for the experiment is provided for the production of Guangji Tang health pharmaceutical industry Co.
2.3 reagent: dibutyl phthalate (DBP)
2.4 Instrument: m205FA fluorescence microscope (Leica corporation), SPX-150 biochemical incubator (Shanghai Yuan-Chi medical apparatus Co., Ltd.)
3. Method of producing a composite material
3.1 preparation of the drug solution
3.1.1 embryo culture fluid:
measuring 1000ml of pure water, adding saline to adjust the electric conductivity to 550-.
3.1.2DBP stock:
precisely weighing 21mg DBP, adding embryo culture solution to 7.54ml to obtain 10mmol/L DBP stock solution, placing in refrigerator at 4 deg.C, and diluting with embryo culture solution to desired concentration when in use.
3.1.3 deer-horn glue stock solution:
precisely weighing 16.27mg colla Cornus Cervi, adding embryo culture solution to 1.994ml to obtain 500mmol/L colla Cornus Cervi stock solution, placing in refrigerator at 4 deg.C, and diluting with embryo culture solution to desired concentration when in use.
3.1.4 donkey-hide gelatin stock solution:
precisely weighing colla Corii Asini, adding embryo culture solution, preparing to obtain colla Corii Asini stock solution with concentration of 0.5mg/mL, placing in refrigerator at 4 deg.C, and diluting with embryo culture solution to desired concentration when in use.
3.1.5 Strand protease E solution:
precisely weighing 25mg of pronase E powder, placing the powder in 50ml of embryo culture solution, and completely dissolving to obtain 0.5g/L pronase E solution.
3.1.6 acridine orange staining (AO) solution:
mu.l of AO staining solution (1g/L) was diluted to 5mg/L with 9.95ml of embryo culture medium.
3.1.7 anesthetic:
precisely weighing 400mg of tricaine powder, adding Tris buffer solution (adjusted to pH 7), adding ultrapure water to 100ml, mixing uniformly to obtain a tricaine storage solution, storing in a refrigerator at 4 ℃, measuring 4.2ml of the tricaine storage solution by time, adding ultrapure water to 100ml, and mixing uniformly to obtain the tricaine.
4. Effect of DBP on Zebra Fish survival
The selected normal embryos are placed in a 6-well plate, 20 embryos are placed in each well, 3ml of embryo culture medium is added, DBP (0.1, 10, 50 and 100 mu mol/L) with various concentrations is added from 30h (hpf) after fertilization, the normal environment is cultured for 42h, the number of dead embryos is observed at 18h and 42h, the experiments are repeated in parallel for 3 times, and the survival rate is counted after data are summarized.
5. Model for DBP induction of eye apoptosis
The selected normal embryos are placed in a 6-well plate, 12 embryos per well are added, 3ml of embryo culture medium is added, the embryo culture medium is completely sucked from 30h or 54h after fertilization, DBP (0.1, 1, 5, 10 and 50 mu mol/L) with each concentration is added, the culture is continued for 18h under the condition of direct light (54-324lux) or non-direct light, and the experiment is repeated for 3 times in parallel. AO staining, observing under a body type microscope, displaying eye green fluorescence as apoptotic cells, collecting pictures and performing data processing.
6. Mode of administration
Placing normal zebra fish embryos in 6-well plates with 12 fish eggs per well, feeding 3ml of embryo culture medium to 30hpf, and completely sucking out the culture medium; grouping, adding 3mL embryo culture medium into blank control group, adding 3mL 10 μmol/L DBP into model group, adding 1.5mL 20 μmol/L DBP into each well of 6 administration groups, respectively adding colla Cornus Cervi to final concentration of 0.02 mg/mL-1、0.1mg·mL-1And 0.5mg/mL-1The culture was carried out for 18h, and the above experiment was repeated 3 times in parallel. AO staining and observing eye apoptosis cells under a body type microscopeAnd collecting pictures and processing data.
7. Picture acquisition and data processing
Placing the embryo in a culture dish containing 0.5g/L protease E solution, and standing at room temperature for 8 min; when the embryo individually begins to be stripped, the embryo culture solution is immediately added for dilution, the stripping is stopped, the stripped embryo is quickly moved into a clean culture dish, after 2 times of rinsing, 5mg/L AO solution is used for staining for 1 hour in a dark room, then the embryo culture solution is used for rinsing for 3 times, 5 minutes each time, then 2ml of 0.04g/L MS-222 is used for anaesthetizing for 5 minutes, the embryo culture solution is placed on a glass slide and is subjected to microscopic examination and photo taking under a body type fluorescence microscope, and ImageJ software is used for counting the number of eye apoptotic cells of the zebra fish.
8. Statistical analysis
The indexes of each group are as follows
Statistical analysis was performed using GraphPad Prism 5.0 software. Differences between groups were compared using the one-way ANOVA method, and the Dunnett's T3 test was used for comparison between groups.
9. Results
9.1 as shown in figure 1, compared with the blank group, the embryo eyes of the model group have a large amount of apoptosis, the eye apoptosis of the zebra fish is obvious, and the model modeling of the eye apoptosis model is successful (P is less than 0.001). Compared with the model group, deer glue 0.02, 0.1, 0.5mg/mL-1The dose group significantly reduced apoptosis of ocular cells in a dose-dependent manner (P < 0.001). Experiments show that: the deer glue can obviously inhibit apoptosis of zebra fish eye cells caused by DBP.
9.2 As shown in FIGS. 2 and 3, the expression level of model group Bcl-2 protein was significantly decreased compared to the blank group, indicating successful modeling. 0.1 and 0.5mg/mL compared to model group-1The deer glue can obviously up-regulate the expression of Bcl-2 protein (P is less than 0.01 and P is less than 0.001). The experimental results show that: the deer glue can effectively improve the expression of apoptosis-related Bcl-2 protein and effectively inhibit the apoptosis of aging model animals. This indicates that deer glue has the function of resisting apoptosis of anti-aging cells.
9.3 comparison with blank set, as shown in FIG. 4The mitochondrial mtDNA copy number of the model group is obviously reduced (P is less than 0.05); after the treatment of administration, the mitochondrial mtDNA copy number shows a rising trend, wherein 0.1 mg/mL-1The deer glue can obviously up-regulate the copy number of mitochondrial mtDNA (P is less than 0.01 and P is less than 0.001), and the expression level of the mitochondrial DNA is obviously improved. The results suggest that deer glue has protective effect on damaged mitochondria, thereby inhibiting cell damage and reducing apoptosis.
Therefore, the deer glue of the invention can observe obvious protection effect on DBP-induced apoptosis.
While the present invention has been described with reference to exemplary embodiments, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. Various modifications or changes may be made to the exemplary embodiments without departing from the scope or spirit of the present invention. The scope of the invention should be accorded the broadest interpretation so as to encompass all modifications and equivalent structures and functions.
Claims (10)
1. The application of deer glue in preparing medicine for inhibiting apoptosis is provided.
2. The use as claimed in claim 1, wherein said deer-horn gelatin comprises a solid gelatin aqueous extract prepared by decocting and concentrating the skin and/or horn of a cervidae animal.
3. Use according to claim 2, characterized in that it is capable of increasing the expression of the Bcl-2 protein.
4. Use according to claim 3, characterized in that it is capable of up-regulating the mitochondrial mtDNA copy number.
5. Use according to claim 4, wherein said apoptosis is associated with dibutyl phthalate mediated apoptosis.
6. The use according to claim 5, wherein the deer-horn glue is 2-200 mg.
7. A pharmaceutical composition for inhibiting apoptosis, comprising a therapeutically effective amount of deer-horn glue according to any one of claims 1-6.
8. A method for inhibiting apoptosis comprising the step of contacting a cell with the pharmaceutical composition of claim 7.
9. The method of claim 8, further comprising the step of combining the pharmaceutical composition with an additional therapeutic agent.
10. The method of claim 9, wherein the therapeutic agent comprises an apoptosis signaling pathway-related inhibitor.
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| 杜江等: "龟鹿二仙胶及其拆方对去势骨关节炎大鼠关节软骨中Bax和Bcl-2的影响", 《中国中医骨科伤杂志》, vol. 21, no. 3, pages 4 - 7 * |
| 林胜友等: "龟鹿二仙胶抵抗化疗小鼠脾T淋巴细胞凋亡的实验研究", 《中国中西医结合杂质》, vol. 28, no. 4, pages 339 - 341 * |
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