Application of triterpenoid saponin and analogue thereof in preparation of medicine for treating senile deafness

Abstract

The invention discloses application of triterpenoid saponin and analogues thereof in preparation of a medicine for treating senile deafness, and belongs to the technical field of biological medicine. The triterpenoid saponin and the analogue of the triterpenoid saponin comprise Chiisanoside, Divaroside, Sessiloside A1 (Sessiloside A1) or Chiisanogenin (Sessiloside A1), Sessiloside A1 (Sessiloside A1) and Sessiloside A1 (Sessiloside A1). The triterpenoid saponin and the analogues thereof provided by the invention are derived from leaves and fruits of acanthopanax senticosus and acanthopanax sessiliflorus which are rich in resources and relatively renewable, are rich in original plants or can be obtained through simple conversion, and are safe to operate and low in production cost. The triterpenoid saponin and the analogues thereof provided by the invention achieve the effect of treating the senile deafness by inhibiting ear hair cell aging, can accurately act on targets related to the senile deafness, enable the drug effect to be more direct and effective, can effectively reduce the generation of toxic and side effects, and have a good application prospect. Reference data and a new research and development thought are provided for treatment of senile senile deafness.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to the use of triterpenoid saponin and analogs thereof in the preparation of a medicine for treating presbycusis. Background Art

[0002] Age-related hearing loss (ARHL), also known as presbycusis, is one of the most common sensory dysfunction diseases in the elderly. ARHL is characterized by the high-frequency area of ​​the auditory spectrum being damaged first, and then expanding to the low-frequency area with age. ARHL is a progressive, irreversible, and bilaterally symmetrical hearing loss, the main cause of which may be cell damage caused by apoptosis of hair cells and cell aging. The occurrence of ARHL seriously affects the language communication ability of the elderly, reduces the quality of life, and is the main cause of social isolation and cognitive decline in the elderly. With the development of aging in modern society, the incidence of presbycusis has also increased year by year. Studies have shown that among people over 65 years old, about half of them have varying degrees of hearing loss. At present, there are six main types of presbycusis: sensorineural, metabolic (vascular streak), central, mixed, cochlear conductive, and unformed. Among them, the most important and common is sensorineural presbycusis, which is mainly caused by damage to hair cells and manifests as high-frequency hearing loss and age-related hearing loss.

[0003] At present, the clinical treatment strategies for sensorineural presbycusis mainly include hearing aids, cochlear implants, drug therapy, auditory rehabilitation training, etc. A hearing aid is a device that improves hearing by amplifying sound. It can collect and amplify external sounds and transmit them into the ear, so that patients can hear clearer sounds. However, it also amplifies the surrounding noise, resulting in no obvious improvement in patients' hearing in noisy environments. Long-term wearing can cause ear discomfort, such as ear pain and itching. A cochlear implant is a method of restoring hearing by implanting an electronic device in the body. It converts external sounds into electrical signals, directly stimulates the auditory nerve, and enables patients to hear sounds. However, this operation has certain risks, such as infection and bleeding. It is also expensive, and certain rehabilitation training is required after implantation. The effect is not ideal for some patients, such as patients with severe auditory nerve degeneration. Some drugs may delay or improve sensorineural presbycusis by improving blood circulation in the inner ear, protecting auditory nerve cells, and inhibiting inflammatory responses. For example, vasodilators can increase blood supply to the inner ear, and neurotrophic drugs can promote the growth and repair of auditory nerve cells. However, there is currently no specific drug that can completely cure sensorineural presbycusis. The effect of drug treatment varies from person to person, and it needs to be taken for a long time, which may have side effects. In short, although the clinical treatment strategy for sensorineural presbycusis has certain effects, it still has many limitations. Therefore, it is necessary to further study its etiology and pathogenesis and develop more effective treatment methods. Summary of the invention

[0004] The purpose of the present invention is to provide an application of triterpenoid saponins and analogs thereof in the preparation of a drug for treating presbycusis to solve the problems existing in the above-mentioned prior art. The triterpenoid saponins and analogs thereof extracted from Acanthopanax senticosus or Acanthopanax brevis in the present invention can accurately act on targets related to presbycusis, making the drug efficacy more direct and effective, and can effectively reduce the generation of toxic and side effects, providing reference data and new research and development ideas for the treatment of sensorineural presbycusis.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides use of a triterpenoid saponin and an analog thereof in preparing a medicament for treating presbycusis. The triterpenoid saponin and an analog thereof have a general structure as shown in Formula I:

[0007]

[0008] wherein R1 is selected from -H, -β-D-Glc, -β-D-Glc-(6←1)-β-D-Glc or -β-D-Glc-(6←1)-β-D-Glc-(6←1)-α-L-Rha;

[0009] R2 is selected from -H, -OH or -O(CH2)2CH3;

[0010] R3 is selected from -CH3 or -CH2OH;

[0011] R4 is selected from -OH or -H;

[0012] R5 is selected from -CH(CH2)CH3, -C(O)CH3 or -CH(CH3)COOH.

[0013] Furthermore, the triterpenoid saponins and their analogs include Chiisanoside, Divaroside, Sessiloside A1 or Chiisanogenin.

[0014] Furthermore, the triterpenoid saponin and its analogs are Chiisanoside.

[0015] Furthermore, the dosage form of the drug includes oral preparation and injection.

[0016] Furthermore, the oral dosage form includes tablets.

[0017] Furthermore, the injection includes a suspension injection.

[0018] Furthermore, the presbycusis is sensorineural presbycusis.

[0019] Furthermore, the triterpenoid saponins and their analogs achieve the treatment of presbycusis by inhibiting the aging of ear hair cells.

[0020] Furthermore, the triterpenoid saponins and their analogs inhibit aging of hair cells in the ear by inhibiting apoptosis and cell cycle arrest of hair cells in the ear.

[0021] The present invention also provides the use of the triterpenoid saponins and analogs thereof in the preparation of inhibitors of TRAF2, PERK, Caspase-3, DAXX, p-MKK3 or p-p38 MAPK.

[0022] The present invention also provides the use of the triterpenoid saponin and its analogs in preparing an activator of CyclinE or CyclinE-CDK2.

[0023] The present invention discloses the following technical effects:

[0024] The triterpenoid saponins and analogs thereof provided by the present invention are all natural products, which are derived from the leaves and fruits of Acanthopanax senticosus and Acanthopanax brevis, which are abundant in resources and relatively renewable. The natural products involved in the present invention are rich in the original plants or can be obtained by simple transformation, and the operation is safe and the production cost is low.

[0025] The present invention proves through pharmacological experiments that the triterpenoid saponins extracted from Acanthopanax senticosus or Acanthopanax brevis and their analogs inhibit the expression of TRAF2 / PERK / Caspase-3 proteins through precise multi-target effects, thereby preventing the ear hair cells from ER stress, Ca 2+ Overload, mitochondrial dysfunction, damage, surge in ROS content, and then lead to cell apoptosis. At the same time, by inhibiting the expression of DAXX / MKK3 / p38 protein, activating the expression of CyclinE protein, activating the kinase activity of CyclinE-CDK2, preventing the ear hair cells from stagnating in the G1 phase instead of entering the S phase, thereby preventing the characteristic changes of ear hair cell aging. Its mechanism of action is clear, and it can accurately act on the target, making the drug effect more direct and effective, and can effectively reduce the generation of toxic and side effects. Compared with existing clinical treatment strategies, the triterpenoid saponins and analogs provided by the present invention have both precise target correspondence and embody the multi-target overall intervention effect on the disease. The present invention provides reference data and new research and development ideas for the treatment of sensorineural presbycusis. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1 This is a statistical chart showing the effects of CSS and its analogs on HEI-OC1 cell viability.

[0028] Figure 2 This is a statistical graph showing the effects of CSS and its analogs on the cell viability of D-Gal-induced senescent HEI-OC1 cells;

[0029] Figure 3 The crystal violet staining results of CSS on D-Gal-induced HEI-OC1 cells;

[0030] Figure 4 The results of FDA / PI staining of HEI-OC1 cells induced by D-Gal by CSS;

[0031] Figure 5 The results of flow cytometry detection of CSS on D-Gal-induced HEI-OC1 cells;

[0032] Figure 6 This is the KEGG analysis of differentially expressed genes in transcriptome sequencing of HEI-OC1 cells induced by D-Gal by CSS;

[0033] Figure 7 Statistical graph showing the effect of CSS on the expression of apoptosis and senescence-related genes TRAF2 (A), PERK (B), Caspase-3 (C), DAXX (D), MKK3 (E), p38 MARK (F) and CyclinE (G) in HEI-OC1 cells induced by D-Gal;

[0034] Figure 8 This is a statistical chart showing the effect of CSS on the ROS in HEI-OC1 cells induced by D-Gal;

[0035] Fig. 9 This is a statistical chart of the results of CSS detection of mitochondrial membrane potential of HEI-OC1 cells induced by D-Gal;

[0036] Fig.10 The biochemical index Ca2+ related to CSS in HEI-OC1 cells induced by D-Gal 2+ (A), statistical graphs of MDA (B), SOD (C), LDH (D), HMBG-1 (E) and GSH (F) test results;

[0037] Fig.11 The statistical graphs show the results of CSS detection of the expression of apoptosis-related proteins TRAF2 (A), PERK (B) and Caspase-3 (C) in HEI-OC1 cells induced by D-Gal;

[0038] Fig.12 This is a statistical chart of the results of CSS detection of the cell cycle of HEI-OC1 cells induced by D-Gal;

[0039] Fig.13 This is a statistical graph of the results of CSS staining of SA-β-gal in HEI-OC1 cells induced by D-Gal;

[0040] Fig.14 The statistical graphs show the results of CSS detection of the expression of senescence-related proteins DAXX (A), MKK3 (B), p38 MARK (C) and CyclinE (D) in HEI-OC1 cells induced by D-Gal.

[0041] Fig.15 This is a statistical graph of the ABR test results of CSS on D-Gal-induced aging mice;

[0042] Fig.16 This is a statistical graph of the results of CSS staining of basement membrane spreads of D-Gal-induced aging mice;

[0043] Fig.17The statistical graphs show the results of CSS detection of apoptosis-related proteins TRAF2 (A), PERK (B) and Caspase-3 (C) in D-Gal-induced aging mice;

[0044] Fig.18 Statistical graphs showing the results of CSS detection of the expression of cell cycle regulation-related proteins DAXX (A), MKK3 (B), p38MARK (C) and CyclinE (D) in D-Gal-induced aging mice. DETAILED DESCRIPTION

[0045] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0046] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0047] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0048] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0049] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0050] Terminology Note:

[0051] TRAF2 protein refers to tumor necrosis factor-related receptor factor 2;

[0052] PERK protein refers to protein kinase R-like endoplasmic reticulum kinase;

[0053] Caspase-3 protein refers to caspase 3;

[0054] DAXX protein refers to death domain-associated protein;

[0055] MKK3 protein refers to mitogen-activated protein kinase kinase 3;

[0056] p38 protein refers to mitogen-activated protein kinase;

[0057] CyclinE protein refers to cell cycle protein E.

[0058] The parent nucleus structure formula of Chiisanoside and its analogs referred to in the present invention is as follows:

[0059]

[0060] Wherein, R1 is selected from -H, -β-D-Glc, -β-D-Glc-(6←1)-β-D-Glc and -β-D-Glc-(6←1)-β-D-Glc-(6←1)-α-L-Rha;

[0061] R2 is selected from -H, -OH and -O(CH2)2CH3;

[0062] R3 is selected from -CH3 and -CH2OH;

[0063] R4 is selected from -OH and -H;

[0064] R5 is selected from -CH(CH2)CH3, -C(O)CH3 and -CH(CH3)COOH.

[0065] The monomer compounds used in the following examples were extracted from the leaves (or fruits) of Acanthopanax senticosus (or Acanthopanax brevis), and their structures and content information are shown in Table 1. The extraction and separation process is briefly described as follows:

[0066] 1. Short-stemmed Acanthopanax leaves as raw material

[0067] Extraction and enrichment of total saponins: 5 kg of Acanthopanax sphenanthera leaves (collected in mid-July, withered and dried) were ultrasonically extracted three times with 10 times the amount (volume-to-mass ratio) of 70% ethanol water. The extracts were combined, the ethanol was recovered until there was no alcohol taste, and the mixture was suspended on a D101 macroporous adsorption resin column. The mixture was washed with water until the effluent was colorless, eluted with 30% ethanol until the effluent was colorless, eluted with 50% ethanol water until the effluent was colorless, and the eluate was collected and dissolved to obtain a 50% ethanol elution component (for standby use), and eluted with 90% ethanol water until the effluent was colorless, and the eluate was collected and dissolved to obtain a 90% ethanol elution component (for standby use).

[0068] Separation of the components eluted with 50% ethanol: take the components eluted with 50% ethanol, dry-load them on a silica gel column (upflow-silica gel = 1:20), elute with chloroform-methanol (20:1), and monitor on silica gel thin layer chromatography until no fraction is eluted; elute with chloroform-methanol (5:1), monitor on silica gel thin layer chromatography until no fraction is eluted, and recover the solvent to obtain component Y-Fr-5-1; elute with chloroform-methanol (3:1), monitor on silica gel thin layer chromatography until no fraction is eluted, and recover the solvent to obtain component Y-Fr-3-1; elute with chloroform-methanol (1:1), monitor on silica gel thin layer chromatography until no fraction is eluted, and recover the solvent to obtain component Y-Fr-1-1.

[0069] Y-Fr-5-1 component separation: Y-Fr-5-1 components were dissolved in methanol, separated by C18 reverse phase high performance liquid chromatography, detected at 205 nm, and gradient eluted with acetonitrile-water (18:82-32:68). The fractions at the corresponding peaks were collected according to the chromatogram, the solvent was recovered, and the main component Chiisanoside was obtained by recrystallization with methanol.

[0070] Y-Fr-3-1 component separation: Y-Fr-3-1 components were dissolved in methanol, separated by C18 reverse phase high performance liquid chromatography, detected at 205 nm, and gradient eluted with acetonitrile-water (24:76-38:62). The fractions at the corresponding peaks were collected according to the chromatogram, and the solvents were recovered respectively. The main components, Divaroside and Sessiloside A1, were obtained by recrystallization with methanol.

[0071] Y-Fr-1-1 component separation: Y-Fr-1-1 component was dissolved in methanol, separated by C18 reverse phase HPLC, detected at 205 nm, and gradient eluted with acetonitrile-water (33:67-41:59). The fractions at the corresponding peaks were collected according to the chromatogram, the solvent was recovered, and the main component Chiisanogenin was obtained by recrystallization with methanol.

[0072] 2. Acanthopanax senticosus fruit as raw material

[0073] Extraction and enrichment of total saponins: 5 kg of Acanthopanax senticosus fruit (collected at the end of September and dried) was ultrasonically extracted three times with 10 times the amount (volume-to-mass ratio) of 70% ethanol water. The extracts were combined, the ethanol was recovered until there was no alcohol taste, and the mixture was suspended on a D101 macroporous adsorption resin column, washed with water until the effluent was colorless, eluted with 30% ethanol until the effluent was colorless, eluted with 50% ethanol water until the effluent was colorless and the eluate was collected and dissolved to obtain a 50% ethanol elution component (for standby use), and eluted with 90% ethanol water until the effluent was colorless and the eluate was collected and dissolved to obtain a 90% ethanol elution component (for standby use).

[0074] Separation of the components eluted with 50% ethanol: take the components eluted with 50% ethanol, dry-load them on a silica gel column (upflow-silica gel = 1:20), elute with chloroform-methanol (20:1), and monitor on silica gel thin layer chromatography until no fraction is eluted; elute with chloroform-methanol (5:1), monitor on silica gel thin layer chromatography until no fraction is eluted, and recover the solvent to obtain the G-Fr-5-1 component; elute with chloroform-methanol (3:1), monitor on silica gel thin layer chromatography until no fraction is eluted, and recover the solvent to obtain the G-Fr-3-1 component; elute with chloroform-methanol (1:1), monitor on silica gel thin layer chromatography until no fraction is eluted, and recover the solvent to obtain the G-Fr-1-1 component.

[0075] G-Fr-5-1 component separation: G-Fr-5-1 components were dissolved in methanol, separated by C18 reverse phase high performance liquid chromatography, detected at 205 nm, and gradient eluted with acetonitrile-water (18:82-32:68). The fractions at the corresponding peaks were collected according to the chromatogram, and the solvents were recovered respectively. The main components 24-Hydroxychiisanoside, 22α-Hydroxychiisanoside, Acanthosessilioside K, Chiisanoside, and 1-Deoxychiisanoside were obtained by recrystallization with methanol.

[0076] G-Fr-3-1 component separation: G-Fr-3-1 component was dissolved in methanol, separated by C18 reverse phase high performance liquid chromatography, detected at 205 nm, and gradient eluted with acetonitrile-water (24:76-38:62). The fractions at the corresponding peaks were collected according to the chromatogram, the solvent was recovered, and the main component Acanthosessilioside F was obtained by recrystallization with methanol.

[0077] G-Fr-1-1 component separation: G-Fr-1-1 component was dissolved in methanol, separated by C18 reverse phase high performance liquid chromatography, detected at 205 nm, and gradient eluted with acetonitrile-water (33:67-41:59). The fractions at the corresponding peaks were collected according to the chromatogram, and the solvents were recovered respectively. The main components, Elesesterpene F, 22α-Hydroxychiisanogenin, Elesesterpene G, and Chiisanogenin were obtained by recrystallization with methanol.

[0078] Table 1 Structure and content information of each monomer compound

[0079]

[0080] The structural data of each monomer compound separated above were determined by NMR and MS data analysis as follows:

[0081] Chiisanoside (English abbreviation CSS), white crystal, C48 H 74 O 19 .MS: [M] + 954.4815. 13 C NMR (100MHz, MeOD) δppm: 175.00, 173.84, 150.74, 147.92, 114.60, 111.56, 104.02, 101.72, 94 .89, 78.05, 77.81, 77.68, 76.71, 76.40, 75.35, 74.93, 73.59, 73.11, 71.83, 71.83, 71.12, 70. 67, 69.83, 69.15, 61.26, 57.00, 49.84, 49.57, 47.74, 44.48, 44.25, 42.54, 41.95, 39.03, 36.98, 35.39, 33.68, 32.73, 32.20, 31.07, 29.71, 25.64, 24.71, 19.85, 19.53, 18.87, 18.38, 14.46.

[0082] Divaroside (English abbreviation DVS), white crystal, C 42 H 64 O 15 .MS: [M] + 808.4268. 13 C NMR (100MHz, MeOD) δppm: 175.59, 175.56, 150.46, 147.53, 113.65, 110.23, 104.02, 94.89, 78.12, 77.81, 77.33, 76.71, 76.42, 73.58, 73.41, 73.11, 71.12, 70.63, 69.1 5, 62.26, 57.12, 50.20, 50.09, 47.76, 44.26, 44.17, 42.40, 42.20, 38.03, 36.73, 35.61, 33.56, 32.64, 32.11, 30.87, 29.63, 25.35, 22.60, 18.70, 18.51, 17.52, 13.37.

[0083] SessilosideA1 (English abbreviation SSA), white crystal, C 36 H 54 O 10 .MS: [M] + 646.3728. 13C NMR (100MHz, MeOD) δppm: 175.00, 173.05, 150.16, 147.75, 113.93, 110.76, 95.55, 79.56, 78.95, 75.30, 74.36, 71.11, 70.54, 62.20, 56.81, 49.7 8. 49.62, 47.65, 44.15, 44.12, 42.23, 41.78, 38.83, 36.73, 35.27, 33.53 , 32.39, 32.17, 30.82, 29.58, 25.22, 23.56, 19.05, 18.95, 17.91, 13.83.

[0084] Chiisanogenin (English abbreviation CSG), white crystals, C 30 H 44 O5. MS: [M] + 484.3169. 13 C NMR (100MHz, CDCl3) δppm: 180.14, 172.62, 148.53, 146.35, 113.71, 110.52, 75.20, 70.69, 55.70, 49.67, 48.74, 46.63, 43.4 2. 43.34, 41.47, 41.08, 37.10, 36.35, 34.67, 32.08, 31.64, 31.50, 29.98, 28.79, 24.16, 22.08, 18.53, 18.46, 17.47, 13.37.

[0085] 24-Hydroxychiisanoside (24-HCS), white crystals, C 48 H 74 O 20 .MS: [M] + 970.4753. 13C NMR (100MHz, Pyridine-d5) δppm: 175.11, 172.98, 152.71, 150.06, 111.35, 110.76, 105.02, 102. 62, 95.38, 78.81, 78.45, 77.98, 77.71, 76.40, 75.42, 75.23, 74.13, 73.91, 72.73, 72.53, 70.92, 70.34, 70.12, 69.35, 67.26, 61.26, 56.81, 49.62, 47.55, 45.68, 44.33, 44.31, 42.23, 41.76, 38.36, 36.78, 35.21, 33.45, 32.83, 32.15, 30.68, 29.61, 26.71, 18.92, 18.73, 18.47, 18.12, and 13.83.

[0086] 22α-Hydroxychiisanoside (22α-HCS), white powder, C 48 H 74 O 20 .MS: [M] + 970.4788. 13 C NMR (100MHz, Pyridine-d5) δppm: 175.11, 173.15, 150.06, 147.68, 113.51, 110.76, 105.02, 102. 62, 95.38, 78.81, 78.45, 77.98, 77.71, 76.40, 75.42, 75.42, 75.23, 74.13, 73.91, 72.73, 72.53, 70.92, 70.34, 70.12, 69.35, 62.51, 61.26, 49.65, 47.55, 44.33, 44.31, 44.12, 42.23, 41.65, 41.36, 38.02, 35.21, 33.45, 32.47, 29.61, 26.72, 25.24, 23.15, 19.27, 18.92, 18.47, 18.12, 13.83.

[0087] 22α-Hydroxychiisanogenin (22α-HCG), white powder, C 30 H 44 O6. MS: [M] + 500.3126. 13C NMR (100MHz, Pyridine-d5) δppm: 178.52, 173.08, 151.04, 147.75, 113.86, 110.93, 75.61, 75.33, 70.58, 62.53, 49.58, 48.21, 4 4.35, 44.18, 44.05, 42.17, 41.77, 41.35, 38.79, 35.06, 33.75, 32.59, 29.15, 27.12, 25.37, 23.51, 19.28, 18.82, 17.34, 13.71.

[0088] 1-Deoxychiisanoside (1-DCS), white powder, C 48 H 74 O 18 .MS: [M] + 938.4875. 13 C NMR (100MHz, Pyridine-d5) δppm: 175.45, 175.11, 150.06, 147.65, 114.32, 110.76, 105.02, 102. 62, 95.38, 78.81, 78.45, 77.98, 77.71, 76.40, 75.42, 75.23, 74.13, 73.91, 72.73, 72.53, 70.34, 70.12, 69.35, 61.26, 56.81, 56.81, 52.45, 49.62, 47.55, 42.23, 41.76, 39.98, 39.51, 36.78, 35.21, 33.45, 32.83, 32.15, 30.68, 30.28, 29.61, 25.33, 23.67, 18.92, 18.73, 18.47, 17.62, 13.83.

[0089] Elesesterpene F (ESP F), white powder, C 30 H 44 O7. MS: [M] + 532.3059. 13C NMR (100MHz, Pyridine-d5) δppm: 178.52, 178.02, 173.08, 147.75, 113.86, 75.33, 70.58, 56.88, 49.58, 48.21, 44.35, 44.18, 4 4.05, 42.91, 42.17, 41.77, 38.79, 37.12, 35.12, 35.06, 32.65, 32.59, 29.15, 25.78, 25.37, 23.51, 18.82, 18.75, 17.34, 13.71.

[0090] Acanthosessilioside F (ASS F), white powder, C 36 H 54 O 11 .MS: [M] + 662.3751. 13 C NMR (100MHz, Pyridine-d5) δppm: 174.28, 173.15, 150.06, 147.68, 113.51 ,111.08,95.38,79.52,78.82,75.42,74.82,74.21,71.12,70.52,62.51,6 2.12, 49.65, 47.55, 44.33, 44.31, 44.12, 42.23, 41.65, 41.36, 38.85, 35. 21, 33.45, 32.47, 28.91, 26.72, 25.24, 23.15, 19.27, 18.92, 17.79, 13.83.

[0091] Elesesterpene G (English abbreviation ESP G), white powder, C 29 H 42 O6. MS: [M] + 486.3058. 13 C NMR (100MHz, Pyridine-d5) δppm: 211.13, 178.52, 172.81, 147.75, 113.86, 75.33, 70.58, 56.88, 51.98, 49.58, 49.31, 44.1 8. 44.05, 42.17, 41.77, 38.79, 37.12, 34.85, 34.41, 32.65, 32.59, 29.21, 29.15, 28.85, 25.37, 23.51, 18.75, 17.34, 13.71.

[0092] Acanthosessilioside K (ASS K), white powder, C 51 H 80 O 20 .MS: [M] + 1012.5521. 13 C NMR (100MHz, Pyridine-d5) δppm: 174.52, 174.29, 151.25, 148.75, 113.68, 110.76, 105.02, 102.62, 9 5.38, 78.81, 78.21, 78.15, 77.18, 76.54, 76.51, 75.32, 74.19, 73.91, 72.73, 72.53, 71.59, 70.34, 69. 51, 69.35, 64.52, 63.18, 61.26, 50.41, 47.55, 44.31, 43.21, 41.92, 41.23, 40.18, 39.51, 38.32, 34.81, 34.71, 31.06, 29.97, 26.45, 25.92, 25.71, 23.67, 20.41, 19.42, 19.32, 18.47, 16.31, 14.92, 13.73.

[0093] Unless otherwise specified, other materials and reagents can be purchased in the usual way.

[0094] Studies have found that long-term administration of high doses of D-galactose can induce aging symptoms in animals, and D-galactose is often used to establish aging animal models. D-galactose produces too much ROS during metabolism, leading to oxidative stress, which in turn leads to cell apoptosis. Oxidative stress can damage biological molecules in cells, such as DNA, proteins, and lipids, thereby accelerating cell aging. D-galactose can participate in the regulation of aging by affecting the expression of certain genes. D-galactose can activate MKK3 and then activate p38. Activated p38 can phosphorylate p53, enhance the stability and transcriptional activity of p53, thereby inducing the expression of cell cycle arrest-related gene p21, causing cells to stagnate in the G1 phase. The sensorineural presbycusis model can be replicated by D-galactose modeling. Therefore, this embodiment uses D-galactose to induce ear hair cells or mice to establish an aging model.

[0095] Chiisanoside (CSS) and its analogs used in the examples of the present invention are homemade in the laboratory, with a purity of more than 98%. The specific identification data are shown above.

[0096] Example 1 CSS and its analogs resist D-galactose-induced senescence of ear hair cells by inhibiting cell apoptosis

[0097] 1. Experimental Methods

[0098] 1. Experimental cell lines

[0099] Ear hair cells (HEI-OC1) were donated by the Third Hospital of Jilin University.

[0100] 2. Effects of Chiisanoside (CSS) and its analogs on HEI-OC1 cell viability

[0101] The cells were cultured using conventional methods, and the CCK-8 method was used to detect the effects of different concentrations of Chiisanoside (CSS) and its analogs (6.25, 12.5, 25, 50, and 100 μM) on the viability of HEI-OC1 cells.

[0102] 3. Effects of Chiisanoside (CSS) and its analogs on cell activity of D-galactose-induced senescent otic hair cells

[0103] The cells were cultured using conventional methods. After 24 hours, D-galactose (60 mg / mL, determined by experimental exploration) was used to induce cell senescence. After 12 hours, different concentrations of Chiisanoside (CSS) and its analogs (6.25, 12.5, 25, 50, and 100 μM) were administered. After 24 hours, the viability of HEI-OC1 cells was determined using the CCK-8 method.

[0104] 4. Crystal violet staining

[0105] HEI-OC1 cells were seeded into 24-well plates at 5 × 10 4 Cells were incubated at 37°C and 5% carbon dioxide for 24 hours. A senescence model was established using 60 mg / mL D-galactose (source leaf biological, purity ≥ 99.0%). After 12 hours, a representative compound Chiisanoside (CSS, 12.5, 25, 50 μM) was used to treat the culture medium. After 24 hours, it was rinsed twice with PBS and fixed with a fixative for 30 minutes, and air-dried appropriately. After staining with 0.1% crystal violet for 20 minutes, the crystal violet stain was removed, and the cells were rinsed twice with PBS. The number and morphology of cells were observed under a fluorescence microscope.

[0106] 5. Live / dead cell analysis

[0107] HEI-OC1 cells were cultured at 5×10 4The cells were inoculated at a density of 1.577 nm in the wells in a 24-well plate, and an aging model was established using D-galactose (60 mg / mL). After 12 h, the cells were treated with a culture medium containing the representative compound Chiisanoside (CSS, 12.5, 25, and 50 μM). After 24 h, the cells were stained with FDA (0.02 mg / mL) and PI (0.02 mg / mL) dyes, incubated in the dark for 10 min, and the cells were observed under a fluorescence microscope.

[0108] 6. Flow cytometric analysis of apoptosis

[0109] The cells were plated at 5 × 10 4 The cells were inoculated at a density of 100 μg / mL in a 6-well plate and cultured for about 24 h after attachment. D-galactose (60 mg / mL) was used to establish an aging model. After 12 h, the cells were treated with a culture medium containing the representative compound Chiisanoside (CSS, 12.5, 25, and 50 μM), while the cells in the model group were not given any drug. At the same time, a blank group and a group treated with Chiisanoside (CSS, 25 μM) and shTRAF2 (sh TRAF2 gene overexpression plasmid and adenovirus were designed and provided by Shanghai Heyuan Biotechnology Co., Ltd.) were established. After 24 h, 400 μL of Annexin V binding solution was added to each cell sample tube to suspend the cells, and 5 μL of Annexin V-FITC staining solution was added for incubation (protected from light, 15 min), followed by 10 μL of PI (propidium iodide) staining solution for continued incubation (protected from light, 5 min). The cell suspension was filtered through a 200-mesh filter into a flow tube and immediately detected by flow cytometry. The entire detection process should be completed within 1 h to avoid fluorescence quenching. The data were then analyzed using FlowJo10 software.

[0110] 7. Transcriptomics sequencing

[0111] HEI-OC1 cells were collected at 1×10 6 The cells / well were inoculated in a 6-well plate at a density of 24 h, and an aging model was established using D-galactose (60 mg / mL). After 12 h, the cells were treated with culture medium containing the representative compound Chiisanoside (CSS, 25 μM), while the cells in the model group were not given any medication. A blank group was also established, and the following operations were performed.

[0112] 7.1 RNA extraction and identification

[0113] After the aging model was established with D-galactose and treated with the test drugs for 24 h, total RNA was extracted from HEI-OC1 cells using TRIzol reagent. RNA concentration and purity were measured using NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific).

[0114] 7.2qRT-PCR

[0115] Total RNA extracted by Trizol method was taken from each group, and after the concentration was determined, cDNA was synthesized according to the instructions of the reverse transcription kit. The reaction system was prepared according to the instructions, and cDNA was amplified and detected using qRT-PCR instrument. The relative expression level of the target gene was calculated according to the cycle threshold (Ct) value.

[0116] 8. ROS content analysis

[0117] HEI-OC1 cells were cultured at 5×10 4 The cells were inoculated at a density of 1.57 nm in the wells into a 24-well plate for 24 h, and an aging model was established using D-galactose (60 mg / mL). 12 h later, the representative compound Chiisanoside (CSS, 12.5, 25, 50 μM) and the culture medium combined with Chiisanoside (CSS, 25 μM) and shTRAF2 were used to treat the cells. After 24 h, a reactive oxygen species detection kit was used to detect the content of reactive oxygen species in HEI-OC1 cells using fluorescent probes DCFH-DA and DAPI.

[0118] 9. Mitochondrial membrane potential detection

[0119] HEI-OC1 cells were cultured at 5×10 4 The cells were inoculated in a 24-well plate at a density of 1.5 μg / mL. After 24 hours, D-galactose (60 mg / mL) was used to establish an aging model. After 12 hours, representative compounds Chiisanoside (CSS, 12.5, 25, 50 μM) and Chiisanoside (CSS, 25 μM) combined with shTRAF2 were used to treat the culture medium. After 24 hours, JC-1 was used to detect the mitochondrial membrane potential (ΔΨm) in the cells. When the mitochondrial membrane potential is high, the JC-1 probe emits red fluorescence; when the mitochondrial membrane potential is too low, the JC-1 probe emits green fluorescence, which is observed and photographed with a fluorescence microscope. The excitation wavelengths of red and green fluorescence are 530 and 580 nm, respectively.

[0120] 10. Detection of relevant biochemical indicators

[0121] Measurement of LDH, HMBG-1, MDA, GSH, SOD, Ca 2+ HEI-OC1 cells (15×10 4 Cells were collected and the culture medium was used to detect LDH and HMBG-1, and cells were lysed and used for MDA, GSH, SOD, Ca 2+ Detection.

[0122] 11. Analysis of related protein expression

[0123] HEI-OC1 cells were inoculated in 6-well plates and pretreated with representative compounds Chiisanoside (CSS, 12.5, 25, 50 μM) and Chiisanoside (CSS, 25 μM) combined with shTRAF2 for 24 hours. The culture medium was discarded, and the cells were washed three times with pre-cooled PBS. 200 μL of cell lysis buffer was added. After the cells were completely lysed, the cells were scraped off with a pipette tip and aspirated into a sterile 1.5 mL centrifuge tube. Centrifuged at 12500 rpm for 25 minutes at 4°C, the precipitate was removed, the supernatant was collected, and stored at -80°C (the cell lysis process must be performed on an ice box).

[0124] The protein concentration of each sample was detected using the BCA kit.

[0125] The protein expression was detected using TRAF2, PERK, and Caspase-3 protein content detection kits. The protein was quantified using the BCA method, followed by SDS-PAGE gel electrophoresis, membrane transfer, and blocking. The corresponding primary and secondary antibodies were added in sequence, and the luminescent solution was added before development on a developer. β-actin was used as a control, and the protein blot was analyzed using Image J software.

[0126] 12. Statistical processing

[0127] All data were analyzed using Prism 9 (GraphPad) statistical software. Data were expressed as Mean ± SD. The groups were compared using t-test combined with one-way analysis of variance.

[0128] 2. Experimental Results

[0129] 1. Effects of Chiisanoside (CSS) and its analogs on the viability of otic hair cells

[0130] Through measurement, Chiisanoside (CSS) and its analogs (6.25, 12.5, 25, 50, 100 μM) of the present invention did not show obvious cytotoxic activity to HEI-OC1 cells, but showed certain proliferative activity. The results are shown in Figure 1 .

[0131] 2. Protective activity of chiisanoside (CSS) and its analogs against D-galactose-induced senescent ear hair cells

[0132] From the perspective of cell protection activity against HEI-OC1, Chiisanoside (CSS) and its analogs showed good protective effects on the D-galactose-induced ear hair cell aging model, among which Chiisanoside (CSS), Divaroside (DVS), Sessiloside A1 (SSA) and Chiisanogenin (CSG) had better activity. After administration, the cell activity was even twice as high as that of the model group. The cell activity results are shown in Figure 2 , calculate EC 50 See Table 2 for values.

[0133] Table 2 EC of CSS and its analogs on D-galactose-induced otic hair cell senescence model 50 Value (μM)

[0134] Compound <![CDATA[EC 50 (μM)]]> Compound <![CDATA[EC 50 (μM)]]> CSS 7.61 CSG 19.46 DVS 14.37 SSA 8.62 ESPF 31.26 ESPG 21.89 ASSF 28.32 ASSK 35.46 1-DCS 30.46 24-HCS 29.56 22α-HCS 36.41 22α-HCG 25.32

[0135] Subsequent experiments were conducted using Chiisanoside (CSS) as a representative, and Divaroside (DVS), SessilosideA1 (SSA) and Chiisanogenin (CSG) had similar effects to CSS.

[0136] 3. Effect of Chiisanoside (CSS) on HEI-OC1 cell morphology

[0137] 3.1 Crystal violet staining

[0138] The crystal violet staining results of each component on HEI-OC1 cells are shown in Figure 3 , it was observed that the number of HEI-OC1 living cells in the model group was significantly lower than that in the control group, and the cytoskeleton was significantly damaged and deformed. In contrast, the viability of cells in the Chiisanoside (CSS) pretreatment group, especially the Chiisanoside (CSS, 25μM) pretreatment group, was significantly improved, and compared with the significant damage of cell morphology in the model group, the cells were arranged more neatly and had a regular morphology.

[0139] 3.2 Live and dead cell analysis

[0140] The results of double staining of HEI-OC1 cells with fluorescein diacetate (FDA) and propidium iodide (PI) are shown in Figure 4. It was observed that very few dead cells stained with PI (red) were observed in the blank group, while a significant number of dead cells stained with PI (red) were observed in the model group. The Chiisanoside (CSS) pretreatment group, especially Chiisanoside (CSS, 25 μM) pretreatment, significantly increased the uptake of FDA by living cells (green) and reduced PI staining of dead cells (red).

[0141] 3.3 Effect of Chiisanoside (CSS) on apoptosis of HEI-OC1 cells

[0142] Flow cytometric analysis Figure 5 As shown in the results, the percentage of apoptosis and necrosis of HEI-OC1 cells significantly increased to 49.14% after D-galactose treatment (P<0.01 compared with control). Compared with the model group, the apoptosis and necrosis rates were reduced to 37.34% (P<0.05), 13.72% (P<0.01), and 28.45% (P<0.01) after treatment with 12.5, 25, and 50 μM Chiisanoside (CSS), respectively. However, shTRAF2 blocked the effect of Chiisanoside (CSS, 25 μM). In summary, it was demonstrated that Chiisanoside (CSS) can inhibit apoptosis and resist irreversible damage caused by D-galactose by reducing the generation of TRAF2 in cells, and this protective effect may be related to the activation of TRAF2-related pathways. These results indicate that Chiisanoside (CSS) can effectively improve D-galactose-induced HEI-OC1 cell apoptosis and necrosis, and play a pre-protective role against D-galactose-induced aging of ear hair cells.

[0143] 4. Transcriptome differential analysis

[0144] DEGs in the model group (M) and chiisanoside group (CSS) were enriched by KEGG pathways. Figure 6As shown, the top 20 pathways with the most reliable enrichment significance (i.e., the smallest Q value) are selected to present the results. Among them, the present invention selects the most relevant and most significantly different apoptosis (Apoptosis), cellular senescence (Cell senescence), cell cycle (Cell cycle) and MAPK (MAPK signaling pathway) signaling pathways. Through gene expression differences, it was found that Chiisanoside (CSS) can significantly counteract the changes in the expression levels of the above-mentioned pathway-related genes TRAF2, PERK, Caspase-3, DAXX, MKK3, p38, and CyclinE induced by D-galactose, especially Chiisanoside (CSS, 25μM). The results are shown in Figure 7 shown.

[0145] 5. ROS content analysis

[0146] The results are as follows Figure 8 As shown in the figure, compared with the blank group, the fluorescence intensity of the D-galactose treatment group (model group) was significantly enhanced (P<0.01). Compared with the model group, the ROS fluorescence of the Chiisanoside (CSS) group was significantly weakened, especially Chiisanoside (CSS, 25μM). shTRAF2 significantly blocked the effect of Chiisanoside (CSS, 25μM), further indicating that the reversal of D-galactose-induced ROS production in ear hair cells by Chiisanoside (CSS, 25μM) is related to the effect on TRAF2.

[0147] 6. Mitochondrial membrane potential staining

[0148] The oxidation-sensitive dye DCFH-DA and JC-1 probe were used to detect the intracellular mitochondrial membrane potential δψm. Fig. 9 As shown in the figure, compared with the control group, the red-to-green ratio of fluorescence in the D-galactose treatment group (model group) was significantly weakened (P<0.01), indicating that the excessive production of ROS in HEI-OC1 cells led to an increase in mitochondrial membrane potential. In contrast, Chiisanoside (CSS) pretreatment significantly enhanced the red-to-green ratio of fluorescence, especially Chiisanoside (CSS, 25μM). shTRAF2 significantly blocked the effect of Chiisanoside (CSS, 25μM), further indicating that the change of mitochondrial membrane potential by Chiisanoside (CSS, 25μM) is related to the effect on TRAF2.

[0149] 7. Biochemical index detection

[0150] like Fig.10As shown, compared with the control group, the Ca 2+ , MDA, LDH, and HMBG-1 levels were significantly increased, while SOD and GSH levels were significantly decreased (P<0.01). 2+ , MDA, LDH, HMBG-1 exposure gradually decreased, while SOD and GSH exposure increased significantly. In particular, the effect of pretreatment with 25μM Chiisanoside was the most significant. And shTRAF2 can reverse the above effects of Chiisanoside (CSS), further proving that the changes of Chiisanoside (CSS, 25μM) on the above cell aging-related biochemical indicators are related to the effect on TRAF2.

[0151] 8. Determination of related protein expression

[0152] The results of western blotting for the activity verification of related proteins are as follows Fig.11 As shown. Compared with the blank group, the expression of TRAF2, PERK, and Caspase-3 proteins in HEI-OC1 in the D-galactose-induced aging model was significantly increased (P<0.01). Compared with the model group, the Chiisanoside (CSS) groups significantly reduced the expression of the above proteins, especially the effect of Chiisanoside (CSS, 25μM). Further use of shTRAF2 reversed the above effects of Chiisanoside (CSS, 25μM).

[0153] The above experimental results of this example show that Chiisanoside (CSS) and its analogs have significant effects on resisting D-galactose-induced aging of hair cells and reducing cell apoptosis, among which Chiisanoside (CSS, 25 μM) is the most significant. Its anti-aging and anti-apoptotic activity of hair cells is achieved by inhibiting the expression of TRAF2, PERK, and Caspase-3 proteins.

[0154] Example 2 CSS and its analogs resist D-galactose-induced senescence of otic hair cells by inhibiting cell cycle arrest

[0155] 1. Experimental Methods

[0156] 1. Flow cytometer analysis cycle

[0157] In this example, PI staining was used to detect the cell cycle distribution. HEI-OC1 cells were 1×10 6Cells / well were inoculated in a 6-well plate at a density of 1.5 μg / mL for 24 hours, and an aging model was established using D-galactose (60 mg / mL). After 12 hours, the representative compound Chiisanoside (CSS, 12.5, 25, 50 μM) was used for culture medium treatment. At the same time, a Chiisanoside (CSS, 25 μM) and shDAXX co-treatment group was established. After 24 hours, PI dye (50 μg / mL) was used for staining, and the cells were incubated in the dark for 30 minutes. The cell suspension was filtered through a 200-mesh filter into a flow tube, and then the cell cycle distribution was immediately detected by flow cytometry. The entire detection process should be completed within 1 hour to avoid fluorescence quenching.

[0158] 2. β-Galactosidase staining

[0159] β-Galactosidase staining was performed using a β-galactosidase staining kit (Beijing Solebow Technology Co., Ltd.).

[0160] HEI-OC1 cells were collected at 1×10 6 The cells / well were inoculated in a 6-well plate at a density of 1.547 cells / well for 24 hours. D-galactose (60 mg / mL) was used to establish an aging model. After 12 hours, the culture medium containing the representative compound Chiisanoside (CSS, 12.5, 25, and 50 μM) was used for treatment. At the same time, a combined treatment group of Chiisanoside (CSS, 25 μM) and shDAXX (shDAXX gene overexpression plasmid and adenovirus were designed and provided by Shanghai Heyuan Biotechnology Co., Ltd.) was established. After 24 hours, the fixative was added and fixed at room temperature for 15 minutes. The plates were washed three times with PBS, and 500 μL of working solution (consisting of solution A, solution B, solution C and X-Gal) was added to each well. The plates were placed in an oven at 37°C (without CO2) for overnight incubation. After 12 hours, the plates were observed and photographed under an inverted fluorescence microscope.

[0161] 3. Protein expression analysis

[0162] The extraction of total cell protein was the same as in Example 1. The protein concentration of each sample was detected using a BCA kit (Shanghai Biyuntian Biotechnology Co., Ltd.), and protein expression was detected using DAXX, MKK3, p38 and CyclinE protein content detection kits (Shanghai Biyuntian Biotechnology Co., Ltd.). After protein quantification using the BCA method, SDS-PAGE gel electrophoresis, membrane transfer, and blocking were performed, and the corresponding protein primary and secondary antibodies were added in sequence. After adding luminescent liquid, the protein was developed on a developer, and β-actin was used as a control, and the protein blot was analyzed using Image J software.

[0163] 4. Statistical processing

[0164] All data were analyzed using Prism 9 (GraphPad) statistical software. Data were expressed as Mean ± SD. The groups were compared using t-test combined with one-way analysis of variance.

[0165] 2. Experimental Results

[0166] 1. Flow cytometry for cell cycle detection

[0167] The cell cycle was arrested in the G1 phase and the cells underwent senescence. Flow cytometry was used to detect the cell cycle arrest of HEI-OC1 cells in each group. The results are as follows Fig.12 As shown. Compared with the control group, the proportion of cells in the G1 phase in the D-galactose group (model group) increased, the proportion of cells in the G2 phase decreased, and the cell cycle was arrested in the G1 phase. This phenomenon was significantly reversed when the cells were treated with Chiisanoside (CSS), and Chiisanoside (CSS, 25μM) was particularly significant. shDAXX blocked the above therapeutic effects of Chiisanoside (CSS, 25μM). In summary, the experiment proved that Chiisanoside (CSS) can inhibit the cell cycle arrest of ear hair cells caused by D-galactose by acting on DAXX.

[0168] 2. Senescence-related β-galactosidase staining

[0169] Senescence-associated β-galactosidase (SA-β-gal) is an enzyme closely related to cell aging. SA-β-gal is widely considered to be one of the reliable biomarkers of cell aging. In senescent cells, its activity is significantly enhanced, while its activity is lower in normal cells or proliferating cells. By detecting the activity of SA-β-gal in cells, it is possible to intuitively determine whether the cells have entered a senescent state. The experimental results are as follows Fig.13 As shown in the figure, after HEI-OC1 cell senescence was induced by D-galactose (model group), the number of SA-β-gal positive cells was significantly higher than that in the control group (P<0.01). Chiisanoside (CSS) significantly reversed this phenomenon after treating ear hair cells, especially Chiisanoside (CSS, 25μM). shDAXX blocked the above therapeutic effect of Chiisanoside (CSS, 25μM). In summary, the experiment proved that Chiisanoside (CSS) can inhibit the senescence of ear hair cells caused by D-galactose by acting on DAXX.

[0170] 3. Determination of related protein expression

[0171] The results are as follows Fig.14As shown in the figure, compared with the blank group, after D-galactose induced HEI-OC1 cell senescence (model group), the expression levels of DAXX, p-MKK3, and p-p38 MAPK in ear hair cells were significantly increased (P<0.01), and the expression level of CyclinE was significantly decreased (P<0.01), which became the main reason for D-galactose-induced cycle arrest and cell senescence of ear hair cells HEI-OC1. Compared with the model group, the expression levels of DAXX, p-MKK3, and p-p38 MAPK in the Chiisanoside (CSS) pretreatment group were significantly decreased with the increase of dose, and the expression level of CyclinE was significantly increased, reaching a peak at Chiisanoside (CSS, 25μM). DAXX gene silencing (shDAXX) inhibited the effect of Chiisanoside (CSS, 25μM) to a certain extent.

[0172] In summary, Chiisanoside (CSS) inhibits the expression of DAXX, p-MKK3, and p-p38 MAPK by regulating the DAXX / MKK3 / p38 / CyclinE signaling pathway, thereby losing the blocking effect on CyclinE-CDK2, promoting the cell cycle from G1 to S phase, and initiating DNA replication, thereby inhibiting D-galactose-induced ear hair cell cycle arrest and cell senescence.

[0173] Example 3 CSS and its analogs protect against hearing loss induced by D-galactose in presbycusis-induced hearing loss mice by inhibiting inner ear cell apoptosis and cell cycle arrest

[0174] 1. Experimental Methods

[0175] 1. Experimental Animals

[0176] Adult male C57BL / 6 mice, weighing 20-22 g, were purchased from Changchun Yisi Experimental Animal Technology Co., Ltd. and raised in a controlled laboratory environment. They were randomly divided into groups, with 8 mice in each group.

[0177] 2. Establishment of a therapeutic model of senile senile mice

[0178] Adult male C57BL / 6 mice were selected and divided into five groups of equal number, namely control group, D-galactose group (D-Gal), high-dose treatment group (D-Gal+CSS-H), medium-dose treatment group (D-Gal+CSS-M), and low-dose treatment group (D-Gal+CSS-L). The control group was intraperitoneally injected with normal saline for eight consecutive weeks; the experimental group was intraperitoneally injected with 600 mg / kg D-galactose for eight consecutive weeks; the high-dose group, medium-dose group, and low-dose group were intraperitoneally administered with 50 mg / kg, 25 mg / kg, and 12.5 mg / kg of Chiisanoside (CSS) monomer compound after four weeks of intraperitoneal injection of 600 mg / kg D-galactose, and then intraperitoneally injected with 600 mg / kg D-galactose 2 hours later for four consecutive weeks.

[0179] 3. Auditory brainstem evoked potential detection of mouse hearing threshold

[0180] Auditory Brainstem Response (ABR) is an important electrophysiological examination method used to evaluate the function of the auditory system. By giving specific sound stimulation to the subject's ear, such as a click or a tone burst, the sound signal is transmitted to the inner ear along the auditory conduction pathway and to the brainstem via the auditory nerve, which will produce a series of potential changes related to the sound stimulation in different parts of the brainstem. These potential changes can be recorded by electrodes placed at specific locations on the scalp to form an auditory brainstem evoked potential waveform.

[0181] 3.1ABR audiometry

[0182] After drug administration, the mice were subjected to ABR testing to observe changes in hearing. A reference electrode, a recording electrode, and a ground electrode were inserted subcutaneously behind the mouse ear, and a speaker was placed on the side of the mouse's ear to be tested to record the waveform and observe changes in the mouse's hearing.

[0183] 3.2 Basement membrane staining

[0184] The cochlear basal membranes of the model mice were spread and immunofluorescence stained. Myosin VIIA (Myosin7A), phalloidin and 4',6-diamidino-2-phenylindole (DAPI) were used to compare the number of hair cells and cilia of the mice.

[0185] 4. Determination of related protein expression

[0186] The expression levels of TRAF2, PERK, Caspase-3, DAXX, MKK3, p38, CyclinE and other proteins in the homogenate of mouse cochlear tissue pretreated with the representative drug Chiisanoside (CSS) were detected. The mouse cochlear tissue was taken and added with lysis buffer to extract protein, and shTRAF2 and shDAXX were added respectively. The protein was quantified by BCA method and then SDS-PAGE gel electrophoresis, transfer and blocking were performed. The corresponding protein primary and secondary antibodies were added in sequence, and the luminescent solution was added and the protein was developed on a developer. β-actin was used as a control, and the protein blot was analyzed using Image J software.

[0187] 5. Statistical processing

[0188] SPSS statistical software package was used for t-test and analysis of variance.

[0189] 2. Experimental Results

[0190] 1. Hearing test of D-galactose-induced sensorineural presbycusis mice

[0191] The hearing of the D-galactose group was monitored after the model was successfully established. The results were as follows Fig.15 As shown. Compared with the control group, the hearing threshold of mice in the D-galactose model group was significantly increased (P<0.01), with an average of 90db, confirming that D-galactose-induced presbycusis in mice can lead to severe hearing loss. The hearing threshold of the group pretreated with Chiisanoside (CSS) decreased with the increase of the dosage, and reached a peak at a dose of 50mg / kg (P<0.01).

[0192] 2. Basement membrane staining

[0193] By labeling hair cells with Myosin7a and DAPI, it was found that the outer hair cells in the D-galactose injury group (model group) were significantly lost, among which the number of hair cells in the cochlear basal circle was the most seriously lost. The loss of hair cells in the chiisanoside (CSS) group was milder than that in the D-galactose injury group, and the survival rate of hair cells was Fig.16 .

[0194] 3. Expression levels of apoptosis-related proteins

[0195] The results are as follows Fig.17As shown in the figure, compared with the blank group, the expression of TRAF2, PERK, and Caspase-3 proteins in the cochlea of ​​D-galactose-induced mice was significantly increased. Compared with the model group, the expression of TRAF2, PERK, and Caspase-3 in the Chiisanoside (CSS) pretreatment group decreased significantly with the increase of dose, and reached a peak at a dose of 50 mg / kg. TRAF2 gene silencing reversed the above effects of Chiisanoside (CSS). This is consistent with the results of cell experiments, which further strongly indicates that Chiisanoside (CSS) inhibits the expression of TRAF2, PERK, and Caspase-3 proteins by regulating the TRAF2 / PERK / Caspase-3 signaling pathway, and inhibits the apoptosis of ear hair cells caused by D-galactose-induced endoplasmic reticulum stress, which causes sensorineural presbycusis in mice.

[0196] 4. Expression levels of cell cycle-related proteins in mouse cochlea

[0197] The results are as follows Fig.18 As shown in the figure, in order to further determine that Chiisanoside (CSS) plays an important regulatory role in D-galactose-induced ear hair cell cycle arrest by regulating the DAXX / MKK3 / p38 / CyclinE signaling pathway. The expression levels of DAXX, p-MKK3, p-p38 MAPK and CyclinE were evaluated by Western blotting. The results showed that compared with the blank group, in D-galactose-induced mice, the expression levels of DAXX, p-MKK3, and p-p38 MAPK in the cochlea were significantly increased, and the expression level of CyclinE was significantly decreased, which became the main cause of D-galactose-induced G1 phase arrest of cochlear inner hair cells and sensorineural presbycusis in mice. Compared with the model group, the expression levels of DAXX, p-MKK3, and p-p38 MAPK in the Chiisanoside (CSS) pretreatment group were significantly decreased, and the expression level of CyclinE was significantly increased, also showing a dose-dependent trend. DAXX gene silencing reversed the above effects of Chiisanoside (CSS). These results further indicate that Chiisanoside (CSS) regulates the DAXX / MKK3 / p38 / CyclinE signaling pathway, inhibits the expression of DAXX, p-MKK3, and p-p38 MAPK proteins, thereby losing the blocking effect on CyclinE-CDK2, promoting the cell cycle from G1 to S phase, and initiating DNA replication, thereby inhibiting D-galactose-induced ear hair cell cycle arrest and cell senescence, and thus inhibiting the occurrence of D-galactose-induced sensorineural presbycusis in mice.

[0198] In summary, Chiisanoside (CSS) inhibits the expression of TRAF2, PERK, and Caspase-3 proteins by regulating the TRAF2 / PERK / Caspase-3 signaling pathway, and inhibits the apoptosis of hair cells in mice caused by endoplasmic reticulum stress induced by D-galactose, which causes sensorineural presbycusis in mice. At the same time, Chiisanoside (CSS) also inhibits the expression of DAXX, p-MKK3, and p-p38 MAPK proteins by regulating the DAXX / MKK3 / p38 / CyclinE signaling pathway, thereby losing the blocking effect on CyclinE-CDK2, and promoting the cell cycle from G1 to S phase, initiating DNA replication, thereby inhibiting D-galactose-induced hair cell cycle arrest and cell senescence, and then inhibiting the occurrence of D-galactose-induced sensorineural presbycusis in mice.

[0199] Example 4 Tablet Preparation

[0200] Reagents: starch (pharmaceutical grade, Tianjin Jindong Tianzheng Fine Chemical Reagent Factory); citric acid (Shanghai McLean Biochemical Technology Co., Ltd.); magnesium stearate (Shanghai McLean Biochemical Technology Co., Ltd.).

[0201] Preparation method:

[0202] ① Preparation of 10% starch slurry: Dissolve 0.25g of citric acid in 25mL of pure water, add 2.5g of starch and disperse evenly, heat to gelatinize, and obtain 10% starch slurry.

[0203] ② Granulation: Mix 20g of Chiisanoside powder and 20g of starch evenly, then add 10% starch slurry and mix, grind evenly, make a soft material, sieve through a 16-mesh sieve for granulation, and dry at 60°C for 1h. After granulation through a 16-mesh sieve, add lubricant magnesium stearate and press into tablets with a shallow punch with a diameter of 10mm.

[0204] The obtained tablets are off-white in color, uniform in color, uniform in thickness, and moderate in hardness. The tablet weight and disintegration time meet the requirements. The obtained Chiisanoside tablets meet the requirements and can be used as tablets.

[0205] Example 5 Preparation of Suspension Injection

[0206] Reagents: polylactic acid (PLA, Shanghai Zhenzhun Biotechnology Co., Ltd.); polylactic acid-glycolic acid copolymer (PLGA, Shanghai Yuanye Biotechnology Co., Ltd.); poloxamer 188 (Xi'an Tianzheng Pharmaceutical Excipients Co., Ltd.); dichloromethane, methanol, acetonitrile, etc. (Tianjin Tiantai Chemicals Co., Ltd.).

[0207] Preparation method:

[0208] ① Preparation of polymer microparticles: Weigh 2.5 g of Chiisanoside and 2.5 g of carrier (PLA / PLGA) into a 50 mL round-bottom flask, add 5 mL of dichloromethane to dissolve, remove most of the organic solvent by reduced pressure distillation at 28 ° C, and then vacuum dry at 40 ° C for 24 h until the solvent is completely removed, grind, and pass through a sieve with a pore size of 150 μm to obtain Chiisanoside polymer microparticles.

[0209] ② Preparation of Chiisanoside suspension injection: Disperse 2.5 g Chiisanoside polymer microparticles in 250 mL of aqueous solution containing 10 g / L poloxamer 188 stabilizer under continuous stirring until the dispersion is complete. Grind the drug dispersion to the desired particle size, take out the Chiisanoside polymer microparticle suspension, and stir at 3000 r·min -1 The mixture was centrifuged for 1 min and dispersed with 10 mL of aqueous stabilizer solution to concentrate the preparation to about 25 g / L.

[0210] The obtained suspension injection has uniform particle size, and the water content and surface particle size of the preparation meet the requirements. The in vitro sustained release effect is good and the stability is good. The obtained C suspension injection meets the requirements and can be used as a suspension injection.

[0211] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of a triterpenoid saponin and its analogs in the preparation of a medicament for treating presbycusis, characterized in that: The triterpenoid saponins and their analogs have the general structure shown in Formula I: wherein R1 is selected from -H, -β-D-Glc, -β-D-Glc-(6←1)-β-D-Glc or -β-D-Glc-(6←1)-β-D-Glc-(6←1)-α-L-Rha; R2 is selected from -H, -OH or -O(CH2)2CH3; R3 is selected from -CH3 or -CH2OH; R4 is selected from -OH or -H; R5 is selected from -CH(CH2)CH3, -C(O)CH3 or -CH(CH3)COOH.

2. The use according to claim 1, characterized in that: The triterpenoid saponins and their analogs include Chiisanoside, Divaroside, SessilosideA1 or Chiisanogenin.

3. The use according to claim 1, characterized in that: The dosage forms of the drug include oral preparation and injection.

4. The use according to claim 3, characterized in that: The oral dosage form includes tablets.

5. The use according to claim 3, characterized in that: The injection includes a suspension injection.

6. The use according to claim 1, characterized in that: The presbycusis is sensorineural presbycusis.

7. The use according to claim 1, characterized in that: The triterpenoid saponin and its analogs achieve the treatment of presbycusis by inhibiting the aging of ear hair cells.

8. The use according to claim 7, characterized in that: The triterpenoid saponins and analogs thereof inhibit the aging of ear hair cells by inhibiting apoptosis and cell cycle arrest of ear hair cells.

9. Use of the triterpenoid saponin and its analogs as claimed in claim 1 or 2 in the preparation of inhibitors of TRAF2, PERK, Caspase-3, DAXX, p-MKK3 or p-p38 MAPK.

10. Use of the triterpenoid saponin and its analogs as claimed in claim 1 or 2 in the preparation of an activator of CyclinE or CyclinE-CDK2.

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