Use of glycopeptide of medlar in preparation of medicine for preventing or treating noise-induced hearing loss

Abstract

The present application belongs to the technical field of biological medicine, and particularly relates to the use of glycopeptide of medlar in preparation of a medicament for preventing or treating noise-induced hearing loss. Experimental results show that the glycopeptide of medlar has a protective effect on hearing loss of a mouse induced by noise, and has an improvement effect on cochlear hair cell loss induced by noise. It can be seen that the glycopeptide of medlar, as a product extracted from traditional Chinese medicinal materials, can be effectively used for prevention and treatment of noise-induced hearing loss, has a prospect of being developed into a medicament for preventing and treating noise-induced hearing loss, provides more treatment options for preventing or treating noise-induced hearing loss in clinic, and has important social benefits and economic value.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to the use of a glycopeptide of wolfberry in the preparation of a medicament for preventing or treating noise-induced hearing loss. BACKGROUND

[0002] Hearing impairment is the most common ear disease in the world. According to the statistics of the World Health Organization (WHO), at present, about 466 million people worldwide suffer from hearing impairment, and by 2050, the number will rise to more than 900 million. With the aging of the population, the abuse of ototoxic drugs, noise and environmental pollution, the population of people with hearing loss and hearing impairment is increasing year by year, and hearing loss has become a global health problem that affects social politics and economy. Among them, about 63% of patients with hearing loss are sensorineural hearing loss, and cochlear hair cell damage is one of the main causes of sensorineural hearing loss. At present, the commonly used hearing aids and cochlear implantation in clinic can improve the hearing of patients to a certain extent, but there are certain limitations, and cannot fundamentally solve the problem. Moreover, its effect completely depends on the number and quality of hair cells, so it is very important to find the cause and mechanism of hair cell damage for the prevention and treatment of sensorineural hearing loss.

[0003] Noise-induced hearing loss, also known as noise-induced hearing loss, is a kind of progressive sensorineural hearing loss caused by exposure to harmful noise environment. Noise exposure can cause damage to the auditory system. The hearing loss caused by short-term exposure to noise environment can be naturally recovered within a few weeks after leaving the noise environment, which is called temporary hearing loss. The phenomenon that cannot be naturally recovered after leaving the noise environment is called permanent threshold shift. The damage of noise to the auditory organ is multifaceted, and the main damage site is the cochlea, which causes hypoxia, ischemia and excessive accumulation of free radicals in the inner ear. Studies have found that when exposed to noise, cochlear hair cells are in a state of stress, the calcium homeostasis in the cells is destroyed, the mitochondrial membrane potential is reduced, and reactive oxygen species (ROS) are released into the cytoplasm through the respiratory chain, which breaks the balance between oxidation and antioxidant in the cells, causing hair cell death and forming permanent hearing threshold shift. In the face of the serious situation of noise-induced hearing loss, exploring effective protective drugs is one of the focuses of research in the field of hearing in recent years. However, there is no effective treatment for noise-induced hearing loss in clinical treatment and drugs.

[0004] Gouqi is the dried mature fruit of Lycium barbarum L. in Solanaceae, which has the effects of tonifying liver and kidney, nourishing essence and improving eyesight, and is used for treating deficiency, essence deficiency, soreness of waist and knees, dizziness and tinnitus. Gouqi polysaccharide is a natural macromolecular water-soluble polysaccharide extracted from Gouqi. Studies have found that Gouqi polysaccharide has the pharmacological effects of immunomodulation, hypoglycemic and hypolipidemic, anti-aging, antioxidant and anti-tumor. Studies have also shown that Gouqi polysaccharide can protect neuron cells from damage and has neuroprotective effects such as anti-depression. Gouqi glycopeptide (Lycium barbarum Glycopeptide, LBGP) extracted from Gouqi polysaccharide has attracted attention in recent years. Compared with Gouqi polysaccharide, Gouqi glycopeptide removes inorganic salts and various monosaccharides. Studies have shown that Gouqi glycopeptide has strong immunological activity and anti-aging effects.

[0005] At present, there is no report on Gouqi glycopeptide for preventing or treating noise-induced hearing loss. SUMMARY

[0006] In order to solve the problem that there is no drug for effectively preventing or treating noise-induced hearing loss for clinical use at present, the inventors of the present application have carried out in-depth development and utilization of natural products by using modern drug research methods, combined with a large number of pharmacodynamic experimental researches, and provided the use of Gouqi glycopeptide in the preparation of a drug for preventing or treating noise-induced hearing loss.

[0007] Specifically, the present application is realized through the following technical solutions:

[0008] The present application provides the use of Gouqi glycopeptide in the preparation of a drug for preventing or treating noise-induced hearing loss.

[0009] Preferably, the present application provides the use of Gouqi glycopeptide as the only active ingredient in the preparation of a drug for preventing or treating noise-induced hearing loss.

[0010] As an optional mode, in the above use, the noise refers to noise with a frequency of 4-60 kHz and an intensity of 90-150 dB.

[0011] As an optional mode, in the above use, the noise-induced hearing loss also includes blast-induced hearing loss.

[0012] As an optional mode, in the above use, the symptoms of noise-induced hearing loss include one or more of the following: tinnitus, hearing reduction or hearing loss.

[0013] As an optional mode, in the above use, the Gouqi glycopeptide reduces the hearing threshold shift caused by noise and improves the cochlear hair cell survival rate after noise exposure.

[0014] As an optional mode, in the above use, the sugar peptide glycan content of the wolfberry sugar peptide is ≥60% by weight, the uronic acid content is ≥5%, and the protein content is 20-33%, and the wolfberry sugar peptide is in the form of a powder with a purity ≥90%.

[0015] As an optional mode, in the above use, the molecular weight of the wolfberry sugar peptide is 88kD as determined by SDS-PAGE, the sugar content is 70% by weight, and the sugar composition is ArA:GAl:GlC=2.5:1.0:1.0 by molar ratio.

[0016] As an optional mode, in the above use, the medicine comprises a prophylactically or therapeutically effective amount of wolfberry sugar peptide and a pharmaceutically acceptable carrier.

[0017] As an optional mode, in the above use, the medicine is an oral preparation, an injection, or a topical preparation.

[0018] Preferably, the oral preparation is selected from an oral liquid, a tablet, a powder, a capsule, or a granule.

[0019] Further preferably, the injection is selected from an aqueous injection or a sterile powder for injection.

[0020] Further preferably, the topical preparation is selected from a drop, a spray, or a gel.

[0021] As an optional mode, in the above use, the medicine is suitable for various administration modes, which can be oral administration or parenteral administration or intra-aural administration.

[0022] Preferably, the parenteral administration includes intravenous injection, intramuscular injection, subcutaneous injection, and the like.

[0023] The present application has the following beneficial effects relative to the prior art:

[0024] The present application, in combination with the advantages of China in the research of natural products, first discovers a new use of the natural product wolfberry sugar peptide in the prevention or treatment of noise-induced hearing loss. Experimental results show that wolfberry sugar peptide has a protective effect on noise-induced hearing loss in mice and an improving effect on noise-induced loss of cochlear hair cells. As can be seen, wolfberry sugar peptide, as a product extracted from traditional Chinese medicinal materials, can be effectively used for the prevention and treatment of noise-induced hearing loss and has the prospect of being developed into a medicine for preventing and treating noise-induced hearing loss, providing more treatment options for preventing or treating noise-induced hearing loss in the clinic and having important social and economic values. BRIEF DESCRIPTION OF DRAWINGS

[0025] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 Lycium barbarum glycopeptides protect against noise-induced hearing loss in mice (x±SD, n=6). <0.0001 vs Control group; **P<0.01, ***P<0.0001 vs Noise group. Among them, Figure 1 A represents the flowchart for in vivo animal experiments. Figure 1 B indicates that ABR testing was used to assess the hearing of different groups of mice.

[0027] Figure 2 Lycium barbarum glycopeptides protected against noise-induced cochlear hair cell loss (x±SD, n=3). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Figure 2 A represents an immunofluorescence image of mouse cochlear hair cells, magnified 100x. Myosin 7a is used to label hair cells, Phalloidin is used to label cilia, and DAPI is used to label cell nuclei. Figure 2 B represents a statistical graph of cochlear hair cell counts. Detailed Implementation

[0028] The inventors employed modern pharmaceutical research methods to deeply develop and utilize natural products. Through extensive screening, they discovered for the first time that wolfberry glycopeptides can prevent or treat noise-induced hearing loss. Based on this discovery, the present invention was completed.

[0029] Alternatively, in the above-described uses, the drug may be an oral preparation, an injection, or a topical preparation.

[0030] Preferably, the oral preparation is selected from oral liquid, tablets, powder, capsules or granules.

[0031] Preferably, the injection is selected from aqueous injections or sterile powders for injection.

[0032] Preferably, the topical preparation is selected from drops, sprays, or gels.

[0033] Alternatively, in the above-described uses, the drug is suitable for various administration methods, including oral administration, parenteral administration, or intraocular administration.

[0034] Preferably, the parenteral administration includes intravenous injection, intramuscular injection, and subcutaneous injection.

[0035] The pharmaceutically acceptable carrier refers to the pharmaceutical carrier conventional in the field of pharmaceutical preparation, which is selected from one or more of fillers, binders, disintegrants, lubricants, suspending agents, wetting agents, pigments, flavoring agents, solvents, and surfactants.

[0036] The fillers include, but are not limited to, starch, microcrystalline cellulose, sucrose, dextrin, lactose, sugar powder, glucose, etc.; the lubricants include, but are not limited to, magnesium stearate, stearic acid, sodium chloride, sodium oleate, sodium lauryl sulfate, poloxamer, etc.; the binders include, but are not limited to, water, ethanol, starch paste, sugar syrup, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, sodium alginate, polyvinylpyrrolidone, etc.; the disintegrants include, but are not limited to, starch effervescent mixture, i.e., sodium bicarbonate and citric acid, tartaric acid, low-substituted hydroxypropyl cellulose, etc.; the suspending agents include, but are not limited to, polysaccharides such as acacia, agar, alginic acid, cellulose ether, and carboxymethyl chitin, etc.; the solvents include, but are not limited to, water, balanced salt solution, etc.

[0037] The above various dosage forms can be prepared according to the conventional process in the field of pharmaceutical preparation.

[0038] The “gouqi glycopeptide” can be extracted and separated from plants containing the active ingredient, such as wolfberry fruit, by using the conventional natural product extraction method in the art, or can be purchased from commercially available products. In particular, the wolfberry glycopeptide can be prepared by using the preparation method described in CN107021995B. As the most effective active ingredient in wolfberry pulp, wolfberry glycopeptide is the essence of wolfberry. Modern pharmacological tests have fully confirmed that wolfberry glycopeptide can significantly improve the immune function of the body, participate in the immune regulation of the organism, participate in various activities of life cells, and has certain effects of lowering blood pressure, lowering blood lipids, lowering blood sugar, anti-inflammatory, antioxidant, anti-aging, anti-tumor, etc. The high biological activity of wolfberry glycopeptide makes it several hundred times more immunologically active than the currently clinically used oral polysaccharides. Further experiments have also proved that wolfberry glycopeptide has multiple pharmacological activities such as antioxidant, anti-aging immune regulation, and inhibition of tumor cell replication.

[0039] In the medical uses described herein, the administration time, administration frequency, and administration frequency of wolfberry glycopeptide, etc. need to be determined according to the specific diagnosis of the disease, which is within the technical scope mastered by those skilled in the art.

[0040] The treatment regimen for animals can be applied to humans, and the effective dose of all drugs for humans can be calculated by the effective dose of the drug for animals, which is also easy for ordinary skilled persons in the art to achieve.

[0041] In order to better understand the essence of the present application, in the following specific embodiments part with pharmacodynamics experiment and its results to further illustrate the new use of wolfberry glycopeptide in the field of pharmacy for preventing or treating noise-induced hearing loss.

[0042] The present application is further illustrated below with reference to specific examples. It should be understood that the specific examples described herein are merely intended to explain the present application and are not intended to limit the scope of the present application.

[0043] Unless otherwise specified in the examples, the techniques or conditions are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. Unless otherwise specified, the reagents or instruments used are conventional products that can be purchased through regular channels.

[0044] Unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, the test materials used in the following examples are commercially available products.

[0045] Example: Protective effect of wolfberry glycopeptide on noise-induced hearing loss in mice 1. Animal grouping and experiment Select 4-week-old C57BL / 6J mice (purchased from Nanjing Qinglongshan Animal Breeding Farm) and randomly divide them into 5 groups: blank control group, model group (noise damage group), wolfberry glycopeptide low-dose group (5 mg / kg), wolfberry glycopeptide medium-dose group (10 mg / kg) and wolfberry glycopeptide high-dose group (20 mg / kg). The wolfberry glycopeptide administration group of mice was administered intragastrically for 14 days, and a 110 dB single white noise exposure for 2 hours was used to establish a noise damage model. Two days later, the mice were subjected to auditory brainstem response (ABR) audiometry and cochlear hair cell immunofluorescence counting analysis.

[0046] 2. Auditory brainstem response (ABR)

[0047] The mice were anesthetized by intraperitoneal injection of pentobarbital sodium according to their body weight. The anesthetized mice were placed in a shielded room with a constant temperature pad at 37°C. The detection electrode was inserted into the subcutaneous position at the center of the mouse's head, the reference electrode was inserted into the subcutaneous position under the mouse's left pinna, and the ground electrode was inserted into the mouse's thigh. The sound wave device was placed at the mouse's left ear, and then 4 kHz, 8 kHz, 12 kHz, 16 kHz, 24 kHz and 32 kHz pure tones were used for stimulation, with the sound wave intensity gradually decreasing from 90 dB to 10 dB, until no repeatable waveform was recorded. The last recorded stimulation intensity was the hearing threshold of the mouse.

[0048] 3. Mouse cochlear hair cell immunofluorescence counting

[0049] Mice were euthanized and decapitated quickly. The auditory bulla was opened, and the obtained cochlear tissue was fixed in 4% paraformaldehyde (PFA) for 24 hours, followed by EDTA decalcification. After complete decalcification, the cochlea was placed in PBS, and the basement membrane was dissected under a dissecting microscope. Immunofluorescence staining was performed using myosin 7a (a hair cell marker) and phalloidin (a ciliary marker). The cochlear hair cells were then counted and statistically analyzed under a ZEISS 900 laser confocal microscope.

[0050] Statistical analysis methods: Graphpad Prism 9.0 software was used to calculate statistically significant differences through one-way ANOVA and Dunnett's test. A p-value <0.05 was considered statistically significant.

[0051] 4. Experimental Results

[0052] 4.1 Protective effect of Lycium barbarum glycopeptides on noise-induced hearing loss in mice

[0053] As shown in Figure 1A, four-week-old mice were given oral administration of Lycium barbarum glycopeptide for two weeks before noise damage (110dB) was induced. Two days later, ABR was used to detect the hearing level of the mice and hair cells were counted by immunofluorescence.

[0054] like Figure 1 As shown in B, the ABR thresholds of mice at 12kHz, 16kHz, 24kHz, and 32kHz were significantly increased after noise injury. Oral administration of Lycium barbarum glycopeptide significantly improved noise-induced hearing impairment, with the high-dose group (20mg / kg) showing the best effect.

[0055] 4.2 The effect of Lycium barbarum glycopeptides on improving noise-induced cochlear hair cell loss

[0056] like Figure 2 A and Figure 2 As shown in Figure B, immunofluorescence experiments after dissecting the cochlear basilar membrane of mice revealed that the number of hair cells in the middle and basal circles of the cochlea was significantly reduced in mice after noise damage compared with the blank control group. Lycium barbarum glycopeptide (10 mg / kg and 20 mg / kg) had a protective effect against noise-induced cochlear hair cell loss (middle circle); all three dose groups of Lycium barbarum glycopeptide (high, medium and low) had a protective effect against noise-induced cochlear hair cell loss (basal circle).

[0057] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. Use of a wolfberry glycopeptide in the preparation of a medicament for preventing or treating noise-induced hearing loss.

2. Use according to claim 1, characterized in that: The noise refers to noise with a frequency of 4-60 kHz and an intensity of 90-150 dB.

3. Use according to claim 1, characterized in that: The noise-induced hearing loss also includes blast-induced hearing loss.

4. Use according to any one of claims 1 to 3, characterized in that: The symptoms of the noise-induced hearing loss include one or more of the following: tinnitus, hearing reduction, or hearing loss.

5. Use according to claim 1, characterized in that: The wolfberry glycopeptide reduces the hearing threshold shift caused by noise and improves the cochlear hair cell survival rate after noise exposure.

6. Use according to claim 1, characterized in that: The wolfberry glycopeptide has a sugar content of ≥60%, a uronic acid content of ≥5%, and a protein content of 20-33% by weight; and the wolfberry glycopeptide is in the form of a powder with a purity of ≥90%.

7. Use according to claim 1, characterized in that: The medicament comprises a prophylactically or therapeutically effective amount of the wolfberry glycopeptide and a pharmaceutically acceptable carrier.

8. Use according to claim 7, characterized in that: The medicament is in the form of an oral preparation, an injection, or a topical preparation.

9. Use according to claim 8, characterized in that: The oral preparation is selected from the group consisting of an oral liquid, a tablet, a powder, a capsule, or a granule; the injection is selected from the group consisting of an aqueous injection or a sterile powder for injection; and the topical preparation is selected from the group consisting of a drop, a spray, or a gel.

10. Use according to any one of claims 7 to 9, characterized in that: The medicament is suitable for oral administration, parenteral administration, or intra-auricular administration.

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