Application of a Dragon's Blood Extract

Abstract

This invention discloses the application of a dragon's blood extract, specifically the application of the compound and its derivatives in the preparation of drugs for the prevention / treatment of diabetes, and the application of the dragon's blood extract in the preparation of drugs for the prevention / treatment of hearing loss. When used as a drug, the dragon's blood extract of this invention can be used directly or in the form of a pharmaceutical composition. The pharmaceutical composition contains 0.1% to 99%, preferably 0.5% to 90%, of the extract of this invention, with the remainder being pharmaceutically acceptable medicinal components, or pharmaceutically acceptable carriers and / or excipients that are non-toxic to humans and animals.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of pharmaceutical chemistry, and particularly relates to an application of a dragon's blood extract. BACKGROUND

[0002] Hearing loss as the fourth largest global disability cause has become a serious disease that harms human health. Hearing loss can be divided into conductive hearing loss and sensorineural hearing loss according to the damaged function, and most hearing loss can be attributed to sensorineural hearing loss. The main pathological changes are spiral organ damage, hair cell loss and supporting cell damage. Inner ear hair cells located in the organ of Corti are important receptors for transmitting sound into the auditory nervous system, and once damaged, they cannot regenerate, which will lead to permanent hearing loss. Therefore, protecting hair cells is of great significance to maintaining normal hearing.

[0003] Aging, continuous exposure in a noisy environment and drug ototoxicity are important pathogenic factors leading to hearing loss, and drug-induced hearing loss is particularly worth paying attention to. Aminoglycoside antibiotics are one of the commonly used antibiotics in clinical practice, and are widely used in the treatment of infections of the urogenital system, gastrointestinal tract, lower respiratory tract and other infectious diseases caused by Escherichia coli, Klebsiella pneumoniae, enterobacter and the like due to their low price and good antibacterial effect on many gram-negative bacilli. Neomycin, as a type of antibiotic, has been used in clinical practice for a long time due to its wide antibacterial spectrum, and its side effects of damaging the inner ear of patients, leading to hearing impairment and tinnitus, are also relatively clear. Therefore, neomycin is also used as a modeling agent for hearing loss research and a tool drug for hearing protection drug research.

[0004] Dragon's blood was discovered in Yunnan Province, China in 1972, and is the main source of "dragon's blood" in the minority medicine Dai medicine. It is mainly produced in Yunnan, Guangxi, Hainan and other places in China, and is known as the "sacred medicine" with the function of activating blood and resolving stasis. Modern pharmacological studies have shown that the effective components of dragon's blood have the pharmacological effects of activating blood and resolving stasis, treating colitis, resisting liver and lung fibrosis, effectively promoting wound healing, and protecting central nervous cells. Dragon's blood is mainly used in the treatment of cardiovascular diseases, dermatological diseases, gynecological diseases, digestive system diseases and anorectal diseases in clinical practice. The effect of the dragon's blood extract involved in the present patent in hearing protection has not been reported so far. SUMMARY

[0005] The purpose of the present application is to provide an application of a dragon's blood extract; and the second purpose is to provide a pharmaceutical composition.

[0006] The first purpose of the present application is achieved by the application of the dragon's blood extract in the preparation of a drug for preventing / treating hearing loss.

[0007] The second object of the present application is achieved in that the pharmaceutical composition is a pharmaceutical composition comprising a therapeutically effective amount of the extract of dragon's blood with a pharmaceutically acceptable salt or carrier.

[0008] The application of the extract of dragon's blood in the preparation of a medicament for preventing and treating hearing impairment is prepared by the following method: 10 g of dragon's blood is separated by silica gel column elution with petroleum ether: ethyl acetate solvent according to 4:1, 3:2, 2:3, 1:4, ethyl acetate, and methanol, and 6 component samples are obtained after drying, wherein the methanol sample is the protected component.

[0009] The extract of dragon's blood can be directly used or used in the form of a pharmaceutical composition when used as a drug. The pharmaceutical composition contains 0.1-99%, preferably 0.5-90%, of the extract of dragon's blood, and the rest is a pharmaceutically acceptable salt or a non-toxic pharmaceutically acceptable carrier and / or excipient for humans and animals.

[0010] Oral solid or liquid preparations such as powders, tablets, sugar-coated tablets, capsules, solutions, syrups, dripping pills, etc. can be used.

[0011] Injection solid or liquid preparations such as powder injection, solution injection, etc. can be used.

[0012] In order to better understand the essence of the present application, the biological activity of the drug composed of the extract of dragon's blood and a pharmaceutically acceptable carrier or excipient will be used to illustrate its application in the medical field.

[0013] HEI-OC1 cell viability test:

[0014] Mouse HEI-OC1 cells were inoculated into a 96-well plate, and after the cells adhered, the culture solution containing 20 mM neomycin was used to induce damage, and the culture solution without neomycin was set as the normal control group. After 24 h of induction, the culture solution was removed, the normal group and the model group (Neo) were replaced with high-sugar DMEM culture solution containing 10% fetal bovine serum, 100 ul / well; the extract of dragon's blood treatment group was prepared by mixing the extract of dragon's blood with 10% fetal bovine serum in high-sugar DMEM culture solution to a final concentration of 1.25, 2.5, 5, 10 and 20 μg / mL, 100 ul / well. After 24 h of drug treatment, the culture medium was discarded, 10% CCK-8 solution was prepared with serum-free culture medium and added to the cell plate, 100 ul / well, incubated at 37°C in the dark for half an hour, and then the absorbance value of each well was measured at 450 nm on a microplate reader, and the OD value of the culture medium containing 10% CCK-8 was measured as a blank well. The cell viability was determined according to the following formula:

[0015] Cell viability (%) = [OD (experimental group) - OD (blank control group)] / [OD (control group) - OD (blank control group)] × 100%.

[0016] The results showed that, compared with the normal group (100%), the cell viability of the neomycin-induced injury model group was significantly reduced (cell viability value decreased to 58%, compared with the normal group). ### P <0.001), the treatment groups treated with dragon's blood extract showed a significant increase in the cell viability of HEI-OC1 cells (approximately 200% compared to the model group) within the concentration range of 1.25–20 μg / mL. *** P <0.001), reaching its maximum value in the 20 μ / mL concentration group (213%, compared to the model group). *** P <0.001). Attached Figure Description

[0017] Figure 1 Figure showing the effect of neomycin-induced damage on cell viability of mouse HEI-OC1 hair cells;

[0018] Among them, NC was the normal control group; Neo (20 mM) was the 20 mM neomycin-induced injury model group; and Dragon's Blood Extract was used as a post-treatment group for neomycin-induced injury (five concentrations: 1.25, 2.5, 5, 10, and 20 μg / mL). Detailed Implementation

[0019] The present invention will be further described below with reference to embodiments, but this is not intended to limit the present invention in any way. Any modifications or substitutions made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0020] The application of the dragon's blood extract described in this invention in the preparation of drugs for the prevention / treatment of hearing loss.

[0021] The dragon's blood extract is the methanol extract of dragon's blood. The specific preparation method is to use dragon's blood as raw material, and perform gradient elution with petroleum ether-ethyl acetate solutions with volume ratios of 4:1, 3:2, 2:3, and 1:4. Then, it is eluted and separated sequentially with pure ethyl acetate and pure methanol, and dried under reduced pressure to obtain six fractions of dragon's blood extract, wherein the methanol extract is the dragon's blood extract described in this invention.

[0022] The pharmaceutical composition of the present invention comprises a therapeutically effective dose of the dragon's blood extract with the addition of a pharmaceutically acceptable salt or carrier.

[0023] The invention will be further illustrated below with specific implementation examples:

[0024] Example 1

[0025] The experiment of the effect of the dragon's blood extract on the viability of HEI-CO1 auditory hair cells induced by neomycin damage:

[0026] The dragon's blood extract is prepared as follows:

[0027] 10 g of dragon's blood purchased from the medicinal material market was weighed, and eluted and separated by silica gel column with petroleum ether: ethyl acetate solvent system according to 4:1, 3:2, 2:3, 1:4, pure ethyl acetate, and methanol. After drying, six component segment samples were finally obtained, and the methanol segment was the patented component. The dragon's blood methanol segment sample was accurately weighed, dissolved in DMSO with a final concentration of 50 mg / mL, and stored for standby.

[0028] Experimental instruments: ordinary inverted optical microscope (Leica, Germany), ultrapure water system, clean bench (Thermo, USA), low-temperature storage box (Haier, China), automatic high-pressure sterilization pot (Hirayama, Japan), cell carbon dioxide incubator (Thermo, USA), pipette gun (Eppendorf, Germany), enzyme label instrument (PerkinElmer, USA), electronic balance (Denver Instrument, USA), ultra-low temperature refrigerator (Haier, China).

[0029] Experimental reagents: DMEM medium (Biological Industries, Israel), fetal bovine serum (FBS, Biological Industries, Israel), streptomycin / penicillin (Biological Industries, Israel), neomycin (Sigma, USA), 0.25% trypsin (Biological Industries, Israel), 96-well plate cell culture plate (NEST), DMSO (Sigma, USA), PBS, cell culture dish (Thermo, USA), centrifuge tube (Corning, USA), and CCK-8 kit (Biosharp, Guangzhou).

[0030] Experimental method: HEI-CO1 cells were cultured in high-glucose DMEM medium containing 10% fetal bovine serum in a 37°C, 5% CO2 constant temperature incubator. When the cell density reached 70% to 80%, the cells were passaged. The best growing HEI-OC1 cells were inoculated in a 96-well plate, and after 24 hours, the culture medium was removed and replaced with 10% fetal bovine serum high-glucose DMEM medium containing 20 mM neomycin, 100 ul / well, to induce damage and establish a hearing damage cell model. A normal control group was set up in parallel, and the cells in this group were replaced with normal 10% fetal bovine serum high-glucose DMEM medium, 100 ul / well. After 24 hours of treatment, the culture medium was removed, and the normal group and the model group (Neo) were replaced with 10% fetal bovine serum high-glucose DMEM medium, 100 ul / well; the Longxuejie extract treatment group was prepared with 10% fetal bovine serum high-glucose DMEM medium to a final concentration of 1.25, 2.5, 5, 10 and 20 μg / mL of Longxuejie extract, 100 ul / well. After 24 hours of drug treatment, the culture medium was discarded, and 10% CCK-8 solution was added to the cell plate without serum, 100 ul / well, and incubated at 37°C for half an hour in the dark. Then the absorbance value of each well was measured at 450 nm on a microplate reader, and the OD value of the culture medium containing 10% CCK-8 was also measured as a blank well. The cell viability was determined according to the following formula:

[0031] Cell viability (%) = [OD (experimental group) - OD (blank control group)] / [OD (control group) - OD (blank control group)] x 100%.

[0032] The above normal group, model group and series concentration Longxuejie extract treatment group were set up with 3 repeated holes, and the experimental results were shown as mean ± standard error, and the data were analyzed by GraphPad Prism 9.5 software. Student's t- test and one-way ANOVA were used for statistical analysis. P <0.05 was considered to be a significant difference, which was statistically significant. The results are shown in the figure as follows: compared with the control group, the cell viability value of the 20 mM neomycin-induced model group was significantly reduced to 58% (### P <0.001); Longxuejie extract at each concentration effectively improved the cell viability, and the protective effect was the largest at a concentration of 20 μg / mL, with a cell survival rate of 213% (*** P<0.001). It is shown that the Longxuejie extract has obvious reversal effect on HEI-OC1 cell damage caused by neomycin, and shows a dose-dependent relationship.

[0033] Embodiment 2

[0034] Take Longxuejie extract, add excipient at a weight ratio of 1:1, granulate and press into tablets.

[0035] Embodiment 3

[0036] Take Longxuejie extract, and make into capsules according to conventional capsule preparation method.

[0037] Embodiment 4

[0038] Take Longxuejie extract, add excipient at a weight ratio of 1:2, granulate and press into tablets.

[0039] Embodiment 5

[0040] Take Longxuejie extract, add excipient at a weight ratio of 1:3, granulate and press into tablets.

[0041] Embodiment 6

[0042] Tablets: Longxuejie extract 100 mg

[0043] Starch 100 mg

[0044] Corn syrup 17% q.s.

[0045] Magnesium stearate q.s.

[0046] Embodiment 7:

[0047] Capsules: Longxuejie extract 100 mg

[0048] Starch 100 mg

[0049] Magnesium stearate q.s.

[0050] Preparation method: mix Longxuejie extract with auxiliary agent, sieve, mix uniformly in a suitable container, and fill the obtained mixture into hard gelatin capsules.

Claims

1. Use of a Dragon's Blood extract, characterized in that, The application of the dragon's blood extract in the preparation of a drug for preventing / treating hearing impairment; The dragon's blood extract is a methanol extraction section of dragon's blood, and the specific preparation method is as follows: taking dragon's blood as raw material, performing gradient elution with a petroleum ether-ethyl acetate solution with a volume ratio of 4:1, 3:2, 2:3 or 1:4, then sequentially performing elution and separation with pure ethyl acetate and pure methanol, and performing drying under reduced pressure to obtain six component sections of dragon's blood extract, wherein the methanol extraction section of dragon's blood extract is the dragon's blood extract.

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