Application of perilla lactone in the preparation of drugs to delay the progression of Parkinson's disease

Abstract

This invention relates to the field of biomedical technology. Specifically, it provides the application of perilla lactone in the preparation of drugs that delay the progression of Parkinson's disease. Perilla lactone can reduce the pathogenic protein α-synuclein, enhance the enzyme activities of superoxide dismutase, catalase, and glutathione peroxidase, and optimize mitochondrial membrane potential levels, thereby achieving neuroprotective effects and delaying the progression of Parkinson's disease.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of perillaldehyde in the preparation of drugs to delay the progression of Parkinson's disease. Background Technology

[0002] Parkinson's disease (PD) is a common chronic progressive degenerative disease of the central nervous system, primarily affecting middle-aged and elderly individuals. A 2014 meta-analysis showed that the prevalence of PD in the general population of my country was 190 per 100,000. The incidence of PD increases exponentially with age, reaching 1%-2% in people over 65 years of age and as high as 3%-5% in those over 85 years of age. Therefore, the number of PD patients is expected to continue to grow in the long term. Pathologically, the main characteristics of PD are the loss of dopaminergic neurons in the substantia nigra pars compacta and the accumulation of misfolded α-synuclein (α-syn) in the cytoplasmic inclusions of Lewy bodies. In PD, α-synuclein misfolds to form β-sheet-rich amyloid aggregates. These amyloid aggregates accumulate and form intracellular inclusion bodies. The intermediates formed during this aggregation process are toxic oligomers and protofibrils that impair the function of mitochondria, lysosomes, and proteasomes, disrupt biomembranes and the cytoskeleton, cause oxidative stress damage, and lead to neuronal apoptosis due to the presence of misfolded protein aggregates. Ultimately, this alters synaptic function and causes neuronal degeneration.

[0003] Currently, the main drugs used to treat Parkinson's disease (PD) include levodopa, piribedil, and selegiline. However, these drugs are prone to adverse reactions such as damage to the central and peripheral nervous systems, diarrhea, and tachycardia. As the disease progresses to the middle and late stages, many patients continuously increase the duration, dosage, or types of medication they take, leading to increased toxic side effects and ultimately severely impacting their quality of life. In contrast, active ingredients derived from traditional Chinese medicine and other natural drugs have become an important source for drug development due to their diverse pharmacological activities, fewer side effects, and multi-target and multi-pathway therapeutic characteristics.

[0004] Sclareolide (SCLA) is a sesquiterpene lactone derived from *Salvia sclare*, a fragrant plant belonging to the Lamiaceae family. Due to its unique structure, aroma, and pharmacological activities, it is an important raw material in the pharmaceutical, fragrance, food, and cosmetic industries. Previous studies have shown that sclareolide possesses biomedical activities such as antifungal, antibacterial, anticancer, and anti-inflammatory activities; however, its mechanism of action on neurodegenerative diseases remains unclear. Summary of the Invention

[0005] The purpose of this invention is to provide the application of perilla lactone in the preparation of drugs that delay the progression of Parkinson's disease. Perilla lactone can delay paralysis mediated by α-syn, reduce the aggregation of pathogenic protein α-syn, increase the activity of various enzymes, and improve mitochondrial dysfunction, thus having a good therapeutic effect on Parkinson's disease.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides the application of perilla lactone in the preparation of drugs that delay the progression of Parkinson's disease.

[0008] Preferably, the final concentration of the styracil lactone is 0.005-0.02%.

[0009] Preferably, the perilla lactone can reduce the pathogenic protein α-synuclein, enhance the enzyme activities of superoxide dismutase, catalase and glutathione peroxidase, and improve mitochondrial function.

[0010] This invention also provides the application of perilla lactone in the preparation of neuroprotective drugs.

[0011] By adopting the above technical solution, the present invention has the following beneficial effects:

[0012] The perilla lactone described in this invention can delay the progression of Parkinson's disease. In this embodiment, using a Caenorhabditis elegans (PD) model, perilla lactone administration enhanced the overall viability of the PD nematodes, specifically showing a significant increase in head-shaking frequency, average movement rate, and swallowing frequency. Simultaneously, quantitative real-time analysis of the pathogenic protein α-syn in PD nematodes showed that perilla lactone administration significantly reduced α-syn expression, suggesting that perilla lactone possesses good neuroprotective activity. Biochemical results showed that perilla lactone significantly increased glutathione (GSH) levels in PD nematodes and significantly enhanced the enzyme activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-PX). Furthermore, perilla lactone also increased adenosine triphosphate (ATP) levels and mitochondrial membrane potential (MMP) levels in PD nematodes. Therefore, perilla lactone may exert its neuroprotective effect by enhancing antioxidant capacity and improving mitochondrial dysfunction. Attached Figure Description

[0013] Figure 1 The changes in head swing velocity of the Caenorhabditis elegans PD model in different treatment groups;

[0014] Figure 2 The changes in the average movement rate of the Caenorhabditis elegans PD model in different treatment groups;

[0015] Figure 3 The changes in swallowing frequency in the Caenorhabditis elegans PD model under different treatment groups;

[0016] Figure 4 Representative fluorescence images of α-syn aggregation in the body wall cells of the Caenorhabditis elegans PD model from different treatment groups;

[0017] Figure 5 Figure showing the quantitative analysis of the average number of α-syn in the body wall cells of the Caenorhabditis elegans PD model in different treatment groups;

[0018] Figure 6 SOD activity in Caenorhabditis elegans PD model under different treatment groups;

[0019] Figure 7 CAT activity in the Caenorhabditis elegans PD model under different treatment groups;

[0020] Figure 8 GSH levels in the Caenorhabditis elegans PD model of different treatment groups;

[0021] Figure 9 GSH-PX activity in the Caenorhabditis elegans PD model under different treatment groups;

[0022] Figure 10 ATP levels in a Caenorhabditis elegans PD model under different treatment groups;

[0023] Figure 11 The MMP levels in the Caenorhabditis elegans PD model were observed in different treatment groups. Detailed Implementation

[0024] This invention provides the application of perilla lactone in the preparation of drugs that delay the progression of Parkinson's disease.

[0025] In this invention, the structural formula of the perilla lactone is shown in formula (Ⅰ).

[0026]

[0027] In this invention, the final concentration of the styracil lactone is preferably 0.005-0.02%, more preferably 0.008-0.015%, and even more preferably 0.01%.

[0028] In this invention, the biological model used for the application of the perillaldehyde is preferably the *C. elegans* Parkinson's disease model, specifically the *C. elegans* NL5901 mutant. The *C. elegans* NL5901 mutant described in this invention is a nematode with a defective phenotype that expresses α-syn in its muscle somatic cells.

[0029] In this invention, the perilla lactone achieves its effect of delaying the progression of Parkinson's disease by inhibiting the aggregation of pathogenic proteins, enhancing antioxidant activity, and improving mitochondrial dysfunction. The perilla lactone of this invention can improve the feeding ability and overall vitality of nematodes in a Parkinson's disease model, while reducing the aggregation of the pathogenic protein α-syn; it can significantly increase the activity of various enzymes, including SOD, CAT, and GSH-PX, and also enhance in vivo ATP levels and optimize mitochondrial membrane potential.

[0030] This invention also provides the application of perilla lactone in the preparation of neuroprotective drugs.

[0031] In this invention, the neuroprotective diseases include Parkinson's disease, and the neuroprotective drugs can exert neuroprotective effects by reducing the expression of the pathogenic protein α-syn, enhancing antioxidant capacity, and improving mitochondrial dysfunction.

[0032] In this invention, the drug is prepared by combining perillaldehyde alone with or in combination with other active ingredients or excipients. The dosage forms of the drug of this invention include oral liquids, tablets, pills, granules, capsules, injections, drop pills, syrups, and ointments.

[0033] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0034] Example 1 (one)

[0036] 1. Experimental Materials

[0037] NL5901 mutant eggs were randomly divided into 3 groups and placed on 3 different nematode growth mediums (NGM) for 3 days at 20℃.

[0038] The three culture media were the blank control group (OP50), the solvent control group (ETOH), and the succinyl lactone group (SCLA).

[0039] Preparation of SCLA culture medium: SCLA (purchased from Aladdin, purity ≥97%) was diluted to 0.2M (i.e., 0.05g / mL) with anhydrous ethanol, and then mixed with E. coli OP50 bacterial suspension (the OD of the E. coli OP50 bacterial suspension was...). 600 Mix thoroughly to form a working solution with a concentration of 400 μM (0.6%), then coat it onto NGM, air dry, seal, and store at 4°C for later use.

[0040] Preparation of solvent control (ETOH) medium: Anhydrous ethanol was mixed with E. coli OP50 bacterial suspension to obtain a working solution with a final concentration of 0.2% ETOH. The solution was spread on NGM, dried, sealed, and stored at 4°C for later use.

[0041] Preparation method of blank control group (OP50) culture medium: E. coli OP50 bacterial suspension was directly spread on NGM, dried and sealed, and stored at 4℃ for later use.

[0042] 2. Measurement Method

[0043] The three groups of NL5901 nematodes obtained from the above culture were subjected to head swing, swallowing frequency, movement speed, and in vivo α-syn aggregation measurements, as well as biochemical analysis.

[0044] Head swing measurement: The number of times the nematode's head swings within 30 seconds is observed and recorded under a biological microscope. The standard for measurement is that the nematode's head swings from the left to the right and then back to the left, which is recorded as one swing.

[0045] Swallowing frequency measurement: The number of swallowing movements of nematodes within 30 seconds was observed and recorded under a biological microscope.

[0046] Movement speed measurement: 20-30 NL5901 nematodes of the same size were selected from each group and placed in an NGM containing the sample and used for video recording. The video was recorded using the Wormlab nematode video acquisition system, and then the average movement speed of the nematodes was analyzed using Wormlab as the analysis software based on the acquired video.

[0047] In vivo α-syn aggregation assay: At least 15 NL5901 nematodes were anesthetized and fixed on a glass slide, and photographed using an upright fluorescence microscope at 20× magnification. The images were analyzed using ImageJ software.

[0048] Biochemical analysis: NL5901 nematodes from each group were homogenized using an automated tissue homogenizer, centrifuged at low temperature (4℃, 8000 r / min, 10 min), and the supernatant was collected and stored at 4℃ for analysis. Measurements were performed according to the kits provided (purchased from Nanjing Jiancheng Bioengineering Institute; ATP and MMP kits were purchased from Beyotime Biotechnology Co., Ltd.), including adenosine triphosphate (ATP) content, catalase activity, superoxide dismutase activity, glutathione content, glutathione peroxidase activity, and mitochondrial membrane potential. Results were standardized by protein concentration.

[0049] 3. Data Processing

[0050] All experiments were repeated at least three times. Results are expressed as mean ± standard deviation (mean ± SD). One-way ANOVA was used to analyze significance using GraphPad Prism version 9.0.0 (GraphPad Software, Inc., San Diego, CA). A p-value less than 0.05 was considered statistically significant. * indicates statistically significant differences (*p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001).

[0051] (II) Experimental Results

[0052] 1. Perilla lactone can delay α-synuclein-mediated paralysis in nematodes.

[0053] The locomotor behavior of *Caenorhabditis elegans* is related to the basic functions of its nervous system. To observe the inhibitory effect of perillaldehyde on the toxicity of α-syn protein, this invention evaluated changes in the overall vitality of nematodes during paralysis by detecting head swing speed and average locomotor speed in a nematode model. Experimental results showed that perillaldehyde intervention significantly increased the head swing speed of nematodes (e.g., ...). Figure 1 Further observation was conducted on the effect of perillaldehyde on the motility rate of PD nematodes. Compared with the control, the average motility rate of NL5901 nematodes increased by 39% after perillaldehyde treatment (e.g., Figure 2 ).

[0054] The pharyngeal pump is a neuromuscular pump located at the anterior end of the nematode's digestive tract. During feeding, nematodes obtain food from the external environment through the rhythmic contraction of the pharyngeal pump. The vibration of the pharyngeal pump is controlled by neurons and neurotransmitters. This invention uses the pharyngeal pump vibration frequency to measure the effect of perillaldehyde on the feeding behavior of nematodes, thereby indirectly evaluating whether perillaldehyde has a role in mitigating neurotoxicity in PD nematodes. The results showed that after perillaldehyde intervention, the pharyngeal pulsation frequency of PD nematodes was significantly increased (e.g., ...). Figure 3 ).

[0055] To investigate the regulatory effect of perilla lactone on α-syn aggregation in a PD nematode model, this invention used the PD transgenic nematode NL5901 to observe α-syn aggregation and quantified the number of α-syn in the nematodes using ImageJ software. The α-syn in NL5901 nematodes was labeled with GFP, thus its distribution in vivo could be observed under a microscope, as shown in (…). Figure 4 Quantitative results showed that treatment with perilla lactone significantly reduced the accumulation of α-syn in NL5901 nematodes (e.g., Figure 5 ).

[0056] 2. Perilla lactone can enhance the antioxidant capacity of PD nematodes.

[0057] In patients with Parkinson's disease (PD), protein aggregation toxicity leads to an increase in reactive oxygen species (ROS), causing the oxidative products generated by ROS-induced oxidation reactions to exhibit varying degrees of cytotoxicity. The antioxidant defense system plays a crucial role in responding to oxidative damage. Reduced glutathione (GSH) is the most important non-enzymatic antioxidant in the body, possessing multiple important physiological functions, including scavenging free radicals, detoxification, promoting iron absorption, maintaining the integrity of erythrocyte membranes, maintaining DNA biosynthesis, normal cell growth and development, and cellular immunity. Superoxide dismutase (SOD), catalase (CAT), and GSH-PX are the main enzymes protecting cells from ROS-induced oxidative stress damage in the human body. Experimental results show that treatment with perilla lactone significantly increased SOD, CAT, GSH, and GSH-PX levels in PD nematodes (e.g., ...). Figure 6-9 ).

[0058] 3. Perilla lactone can improve mitochondrial dysfunction in PD nematodes.

[0059] Mitochondria, as vital organelles involved in various cellular life activities, play a crucial role in the development of the nervous system. Numerous studies have shown that the pathogenic protein α-syn in PD interacts with mitochondria, inducing mitochondrial dysfunction and reducing ATP and MMP levels. This invention experimentally demonstrates that treatment with perilla lactone significantly increases ATP and MMP levels in PD-positive nematodes (e.g., ...). Figure 10-11 The results suggest that this substance can improve mitochondrial dysfunction in PD nematodes.

[0060] The above results indicate that, compared with the control group, treatment with perilla lactone significantly improved the overall viability of PD nematodes. Further experiments suggest that this substance may exert a neuroprotective effect by enhancing antioxidant capacity and improving mitochondrial dysfunction, indicating its great potential to alleviate Parkinson's disease.

[0061] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of perilla lactone in the preparation of drugs to delay the progression of Parkinson's disease.

2. The application according to claim 1, characterized in that, The final effective concentration of the perillaldehyde is 0.005-0.02%.

3. The application according to claim 1, characterized in that, The sage lactone can reduce the pathogenic protein α-synuclein, enhance the enzyme activities of superoxide dismutase, catalase and glutathione peroxidase, and improve mitochondrial function.

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