Application of the compound curcumene in the preparation of anti-Alzheimer's disease drugs

Abstract

This invention belongs to the field of new drug development technology, specifically to the use of the compound curcumene in the preparation of anti-Alzheimer's disease drugs. This invention first discovered that curcumene, within a certain dosage range, can inhibit Aβ-induced neural damage, increase neuronal cell survival, and improve memory and cognitive abilities in Alzheimer's disease model mice. Therefore, curcumene has a promising therapeutic effect in the treatment of Alzheimer's disease.

Description

Technical Field

[0001] The present invention belongs to the technical field of new drug research and development, and particularly relates to the use of the compound curcumene in the preparation of anti-Alzheimer's disease drugs. Background Art

[0002] Alzheimer's disease (AD) is one of the most common neurodegenerative diseases. Currently, the global prevalence of AD has reached 54 million, and with the aging population, this number is expected to exceed 152 million by 2050. The increasing prevalence of AD has led to a growing socioeconomic burden. Currently, FDA-approved AD treatments only slow disease progression and improve symptoms, not cure them completely. These treatments are also associated with serious side effects, such as hepatotoxicity with tacrine and gastrointestinal side effects such as nausea and vomiting with rivastigmine. Therefore, the development of anti-AD drugs with minimal side effects and significant therapeutic efficacy is urgent.

[0003] Curcuma oil is a volatile oil extracted from the rhizome of the traditional Chinese medicine Curcuma zedoaria. Its preparations have long been used clinically for anti-tumor and antiviral effects. Curcumene (CAS: 17910-09-7) is a key component of zecuma oil, and current research on curcumene focuses on its anti-tumor and antiviral activities. Summary of the Invention

[0004] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide an application of the compound curcumene in the preparation of an anti-Alzheimer's disease drug.

[0005] The present invention provides the use of the compound curcumene in the preparation of an anti-Alzheimer's disease drug. The chemical formula of the curcumene is as follows:

[0006] .

[0007] Furthermore, for SH-SY5Y cells, 5-20 μM curcumene could reverse AD damage without cytotoxicity.

[0008] The present invention first discovered that curcumene, within a certain dosage range, can inhibit Aβ-induced nerve damage, increase nerve cell survival rate, and improve memory and cognitive ability in AD model mice. Therefore, curcumene has a good effect in the treatment of AD. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, without paying any creative work, other drawings obtained based on these drawings still fall within the scope of the present invention.

[0010] Figure 1 Figure 3. The protective effect of curcumene on Aβ-induced SH-SY5Y cells. (A) Viability of SH-SY5Y cells. SH-SY5Y cells were plated in 96-well plates. After 24 hours, curcumene was applied to the cells at different concentrations (2.5-40 μM). Cell viability was determined by MTT assay. (B) Curcumene inhibited Aβ-induced damage in SH-SY5Y cells. Curcumene (5, 10, and 20 μM) was applied to the cells. Cell viability was determined by MTT assay. Data are mean ± SD, n ≥ 3. ### P < 0.001, *** P < 0.001, ** P < 0.01, * P < 0.05.

[0011] Figure 2 Results of the Y-maze test; (A) Number of arm entries in each group in the Y-maze test; (B) Spontaneous alternation rate of mice in each group in the Y-maze test; Data are mean ± SD, n = 8, ### P < 0.001, *** P < 0.001, ** P < 0.01, * P < 0.05;

[0012] Figure 3 Results of the water maze test; (A) Escape latency of mice in each group during the navigation phase of the water maze test; (B) Percentage of time spent in the target quadrant of mice in each group during the water maze test; Data are mean ± SD, n = 8, ### P < 0.001, ***P < 0.001, **P < 0.01, *P < 0.05. DETAILED DESCRIPTION

[0013] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be described in further detail below with reference to the accompanying drawings.

[0014] Example 1 Curcumene reverses Aβ-induced SH-SY5Y cell damage:

[0015] Human neuroblastoma cells (SH-SY5Y) were stimulated with amyloid beta (Aβ) as an AD injury model. When the cell density reached 80%, oligomerized Aβ working solution was added to a final concentration of 5 μM and the cells were cultured in a 37°C, 5% CO2 incubator.

[0016] 1. Experimental Grouping

[0017] SH-SY5Y cells were divided into five groups:

[0018] a) Blank control group: DMEM complete medium was replaced once during pretreatment of other groups;

[0019] b) Model group: prepared according to the above AD model method;

[0020] c) Curcumene (5 μM) intervention group: Curcumene working solution was diluted with complete culture medium to a final concentration of 5 μM, and cultured in a 37°C, 5% CO2 incubator for 4 hours. Subsequently, oligomerized Aβ working solution was added and cultured for another 24 hours.

[0021] d) Cell model grouping: Curcumene (10 μM) intervention group: Curcumene working solution was diluted with complete culture medium to a final concentration of 10 μM, and cultured in a 37°C, 5% CO2 incubator for 4 hours. Subsequently, oligomerized Aβ working solution was added and cultured for another 24 hours.

[0022] e) Curcumene (20 μM) intervention group: Curcumene working solution was diluted with complete culture medium to a final concentration of 20 μM, and cultured in a 37°C, 5% CO2 incubator for 4 h. Subsequently, oligomerized Aβ working solution was added and cultured for another 24 h.

[0023] 2. Cell Viability Assay

[0024] Cells were seeded in a 96-well plate at 10,000 cells per well and surrounded by PBS. When cells reached 80% confluence, they were pretreated according to the groupings described above. Oligomeric Aβ was added and the cells were incubated in an incubator for 24 hours. A mixture was prepared by diluting the MTT working solution 1:9 with complete culture medium. After removing the cell supernatant, 100 μL of the mixture was added to each well and incubated in an incubator for 3 hours. The remaining liquid was carefully removed from the wells. 150 μL of DMSO was added to each well and shaken for 5 minutes. The OD values ​​were measured using a microplate reader at a wavelength of 490 nm.

[0025] like Figure 1 As shown, curcumene showed no cytotoxicity within the 2.5-40 μM range. Subsequently, when SH-SY5Y cells were damaged by 1.25 μM Aβ, curcumene at 5 μM, 10 μM, and 20 μM significantly reversed this damage in a dose-dependent manner. In vitro results indicate that curcumene has significant anti-AD activity.

[0026] Example 2 Curcumene alleviates memory and cognitive impairment in APP / PS1 gene mice:

[0027] APP / PS1 transgenic mice are a commonly used AD model. These mice spontaneously produce excessive Aβ during growth, leading to typical pathological changes in Alzheimer's disease. In this example, C57BL / 6 mice were used as a blank control group, and APP / PS1 transgenic mice were used as a model group.

[0028] 1. Experimental Grouping

[0029] The experiment was divided into five groups, with 6 mice in each group.

[0030] a) Blank control group: C57BL / 6 mice were given normal saline by gavage for 30 days.

[0031] b) Model group: APP / PS1 mice were given normal saline by gavage for 30 days.

[0032] c) Curcumene (20 mg / kg) intervention group: APP / PS1 mice were continuously gavaged with curcumene (20 mg / kg) for 30 days.

[0033] d) Curcumene (40 mg / kg) intervention group: APP / PS1 mice were given curcumene (40 mg / kg) by oral gavage for 30 days.

[0034] e) Donepezil (DPZ, 40 mg / kg) intervention group: APP / PS1 mice were continuously gavaged with the positive drug donepezil for 30 days.

[0035] 2. Water Maze Experiment

[0036] The Morris water maze experiment mainly detects the learning and memory ability of the test animals for spatial position and direction by finding a hidden platform. The water maze device consists of a circular constant temperature water pool and a real-time recording camera (on the upper side of the pool). During the experiment, the water depth in the pool was 30 cm. Since the experimental animals were white, ink was added to make the water color black for easy observation. The circular pool is divided into four quadrants. A 6 cm radius underwater hidden platform is placed 1 cm underwater in the center of the first quadrant. Objects of different colors and shapes are placed around the pool to facilitate the memory of mice. The water maze experiment is divided into two parts:

[0037] Positioning and navigation experiment: This phase lasted for 5 days, with each mouse trained twice daily at a fixed time. At the beginning of training, the center of the diagonal quadrants 1 and 3 was used as the placement point. The mouse was placed in the water facing the pool wall and allowed to explore freely for 90 seconds. If the mouse took less than 90 seconds to find the platform, the latency was recorded as the time it took to find the platform; if it took more than 90 seconds, the latency was recorded as 90 seconds. The experimenter then guided the mouse to the platform, allowing it to rest on the platform for 15 seconds before the next trial. The mouse's latency was recorded twice daily for 5 days of training.

[0038] Spatial exploration experiment: On the second day after the completion of the previous phase of the experiment, the hiding platform was removed and the mice were placed facing the central wall of the third quadrant as the release point. They were allowed to explore freely for 90 s, and their swimming speed and the percentage of time they stayed in the target quadrant were measured.

[0039] In the water maze experiment, Figure 3As shown in (A), during the 5-day positioning navigation, the escape latency of mice to reach the platform gradually shortened over time, and the training time of the model group in the last 3 days was significantly longer than that of the blank group. The escape latency of all drug-treated groups began to shorten significantly on the 3rd day. In the spatial exploration experiment, Figure 3 As shown in (B), mice in the blank group spent a longer time in the target quadrant, while mice in the model group failed to quickly locate the previous platform (p < 0.001). Compared with the model group, mice in the drug-treated and positive drug-treated groups spent significantly longer time in the target quadrant. These results suggest that curcumene treatment can significantly reverse cognitive impairment.

[0040] 3. Y-maze experiment

[0041] The Y-maze test is used to examine short-term memory and locomotor activity. The Y-maze consists of three arms (A, B, and C) of equal length (30 cm), width (5 cm), and height (12 cm) at an angle of 120° to each other. The arms are placed in a noise-free experimental chamber. Each mouse enters from the end of one arm and can freely enter all three arms during the 8-minute test. Two trained observers manually record the order and total number of arm entries. Spontaneous alternation behavior is defined as consecutive entries into all three arms, including the following six conditions: ABC, ACB, CAB, BCA, CBA, and BAC. Between trials, the maze arms are thoroughly cleaned with 75% ethanol, and the bottom of the arms are evenly covered with clean bedding to remove residual odor and secretions from the previous mouse. The spontaneous alternation rate (%) of the mouse is calculated as the number of spontaneous alternations / (total number of arm entries - 2) × 100%.

[0042] In the Y maze, if Figure 2 As shown, there was no significant difference in the total number of arm entries between the groups, but there was a significant difference in the spontaneous alternation rate between the groups. All treatment groups with different ratios were able to significantly improve the cognitive impairment of the model mice.

[0043] The above disclosure is merely a preferred embodiment of the present invention and certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. Application of the compound curcumene as the sole active ingredient in the preparation of anti-Alzheimer's disease drugs.

2. Use of the compound curcumene according to claim 1 as the sole active ingredient in the preparation of an anti-Alzheimer's disease drug, characterized in that: For SH-SY5Y cells, 5-20 μM curcumene could reverse AD damage without cytotoxicity.

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