New indication application of guava leaf extract

Abstract

The invention aims to provide a new application of a guava leaf extract for indications, and particularly relates to a new application of the guava leaf extract for treating nervous system degenerative diseases, Parkinson's disease, anti-aging (used for medicines with beauty and health care effects), amyotrophic lateral sclerosis and metabolic diseases, and an effect of the guava leaf extract in brain nerve protection.

Description

Technical Field

[0001] A new indication for the use of guava leaf extract, belonging to the field of new indications for use in traditional Chinese medicine. Background Technology

[0002] Parkinson's disease (Parkinson's disease) is a common neurodegenerative disorder characterized by the gradual loss of dopaminergic neurons in the substantia nigra and the formation of Lewy bodies, ultimately leading to symptoms such as bradykinesia, tremor, rigidity, and balance disorders. Currently, clinical treatment primarily involves medication and deep brain stimulation to alleviate symptoms. However, because these methods struggle to prevent or reverse neuronal degenerative changes, achieving neuroprotection from a pathological mechanism perspective has become a hot topic and a challenge in Parkinson's disease research. 6-OHDA, a classic neurotoxin, is frequently used to construct in vitro and in vivo Parkinson's disease models. 6-OHDA selectively destroys dopaminergic neurons, causing neuronal damage and death by inducing oxidative stress, impairing mitochondrial function, and activating apoptosis pathways. PC12 cells, due to their neuronal-like differentiation characteristics and sensitivity to neurotoxins, are widely used as in vitro models for studying neuroprotective mechanisms and evaluating the anti-neural injury effects of candidate drugs. Therefore, inducing PC12 cell damage with 6-OHDA and studying its protective mechanism is of great significance for exploring the mechanisms of action of novel anti-Parkinson's disease drugs.

[0003] In recent years, natural medicines have attracted much attention due to their advantages such as multiple targets and low toxicity. Guava leaves, as a traditional Chinese medicine, have the effects of clearing heat and detoxifying, promoting blood circulation and removing blood stasis. Their methanol extract is rich in polyphenols and flavonoids, which have been proven to have significant pharmacological activity. Currently, the role of PGLM in the 6-OHDA-induced PC12 cell model and its specific molecular mechanism have not been systematically elucidated. Researchers are gradually recognizing the crucial role of mitochondrial dysfunction in neuronal cell damage. 6-OHDA-induced oxidative stress often first damages mitochondria, leading to a decrease in mitochondrial membrane potential, reduced ATP production, and excessive ROS production. This not only impairs cellular energy supply but also activates the apoptosis pathway. Mitophagy, as a cellular protective mechanism specifically designed to clear damaged mitochondria, is receiving increasing attention in response to this pathological process. Activating mitophagy can effectively clear damaged mitochondria, reduce ROS production, thereby lowering intracellular oxidative stress levels and protecting neurons from further damage.

[0004] The applicant has systematically searched and reviewed existing technical literature related to guava leaf extract. CN120771194A discloses a composition of guava leaf extract and its application in inhibiting mycoplasma. This patent describes how combining konjac extract and guava extract can significantly enhance the inhibitory activity against mycoplasma. CN114796319A discloses a method for preparing guava leaf extract and its application, using 60-80%... ethanol Reflux, concentrate under reduced pressure to obtain a concentrated solution, extract with petroleum ether and ethyl acetate, concentrate the ethyl acetate fraction under reduced pressure, and then separate by neutral alumina column chromatography. dichloromethane The extract obtained by concentrating and drying the fraction of guava leaf extract (methanol = 10:2) exhibits anti-inflammatory activity. Patent CN107468760A discloses that guava leaf extract contains 2α-hydroxyoleanolic acid, 2α-hydroxyursolic acid, and... asiatic acid It has anti-rotavirus effects. CN108852929A discloses a guava leaf extract with anti-allergic effects, obtained by vacuum drying, enzymatic hydrolysis, and subcritical water extraction. CN107468760A discloses a guava leaf extract containing 7-15wt% 2α-hydroxyl groups. Oleanolic acid 20-40 wt% 2α-hydroxyursolic acid and 6-14 wt% snow oxalic acid Application of extracts in the preparation of anti-rotavirus drugs. Chu Shuzhou, Study on the mechanism of guava leaf water extract in reducing blood glucose in db / db mice, Central South University, Study on the mechanism of guava leaf water extract in reducing blood glucose in db / db mice, Publication date: 2022-11-01. This paper discloses that guava leaf water extract can reduce blood glucose in db / db mice by increasing the sensitivity of hepatocytes to insulin and reducing net hemoglobin (HGP).

[0005] Therefore, the clinical applications of this drug are concentrated in areas such as mycoplasma inhibition, antiviral activity, and hypoglycemia. Preliminary work in this study has shown that PGLM (guava leaf extract) not only improves cell survival and reduces oxidative stress in a 6-OHDA-induced cell model, but may also clear damaged mitochondria and inhibit cell death by activating mitophagy. The specific mechanism may involve the regulation of autophagy-related proteins (such as PINK1, Parkin, and LC3), thereby promoting the activation of mitophagy. This finding provides a new perspective for exploring the role of guava leaf extract in neuroprotection and suggests its potential advantages in regulating intracellular mitochondrial homeostasis. Summary of the Invention

[0006] The purpose of this invention is to provide a new indication for the use of guava leaf extract, specifically involving a new use in the treatment of neurodegenerative diseases, Parkinson's disease, anti-aging (for cosmetic and health care products), amyotrophic lateral sclerosis, metabolic diseases, and its role in neuroprotection of the brain.

[0007] The technical solution of this invention patent application is as follows:

[0008] The application of a guava leaf extract in the preparation of a drug for treating neurodegenerative diseases.

[0009] Preferably, the guava leaf extract is used in the preparation of a medicament for treating neuroprotective diseases of the brain.

[0010] Preferably, the guava leaf extract can be used in drugs for treating Parkinson's disease.

[0011] Further optimization is the application of guava leaf extract in the preparation of drugs for treating anti-aging diseases.

[0012] Preferably, the preparation method includes the following steps:

[0013] Weigh 25g of dried guava leaves, crush them, soak them in 200mL of methanol for 24 hours, heat and reflux to extract, each extraction time is 1.5 hours, extract 3 times, combine the filtrates, and recover the solvent under reduced pressure to obtain guava leaf extract.

[0014] Preferably, the effective concentration of the guava leaf extract is 25–100 μg / mL.

[0015] Preferably, the guava leaf extract can be formulated into granules, capsules, tablets, or oral liquids.

[0016] Preferably, the guava leaf extract is used in the preparation of a medicament for treating amyotrophic lateral sclerosis (ALS).

[0017] Preferably, the guava leaf extract is used in the preparation of drugs for treating metabolic diseases.

[0018] The beneficial effects of the guava leaf extract of this invention:

[0019] (1) Cellular experiments showed that PGLM significantly improved the survival rate of 6-OHDA-induced PC12 cells, reversed the decrease in mitochondrial membrane potential, reduced intracellular ROS levels, and improved mitochondrial morphology. PGLM significantly increased the formation of autophagic spots, and the RFP-GFP-LC3 dual-fluorescence assay confirmed that it promoted autophagic flux. Western blotting results showed a significant increase in the LC3-II / I ratio. Colocalization experiments and a model based on Keima-Red mitochondrial-targeting fluorescent protein further confirmed that PGLM can activate mitophagy. In addition, Western blotting showed that PGLM upregulated the expression of PINK1 and Parkin proteins. In the intervention experiment, the protective effect of PGLM on 6-OHDA-induced PC12 cells was significantly weakened after the application of the autophagy inhibitor 3-MA or the mitophagy inhibitor AC220, suggesting that its mechanism of action mainly depends on the PINK1 / Parkin pathway-mediated mitophagy process.

[0020] (2) Transgenic nematode experiments showed that PGLM significantly enhanced autophagy activity. In BC12921 nematodes, this enhancement was reflected by a decrease in P62 protein accumulation; in DA2123 nematodes, PGLM promoted the formation of GFP-LGG-1 fluorescent spots; and in MAH215 nematodes, autophagy flux also increased significantly. In the SJZ42 nematode model of mitophagy, PGLM further promoted mitophagy. In NL5901 nematodes overexpressing α-Syn, PGLM effectively reduced α-Syn aggregation, lowered ROS levels, and improved motility. However, these protective effects were significantly lost when the autophagy-related gene vps-34 or the mitophagy gene pdr-1 was knocked out. In the 6-OHDA-induced BZ555 nematode model, PGLM can protect dopaminergic neurons, reduce ROS accumulation, and improve motor ability and food perception behavior. However, its neuroprotective effect is no longer observed after the corresponding gene is knocked out, further verifying that its effect depends on autophagy and mitochondrial autophagy pathways.

[0021] (3) In this study, a mouse model of Parkinson's disease was successfully established by micro-injecting 6-OHDA into the substantia nigra region of mice using stereotactic brain imaging. PGLM intervention significantly reduced apomorphine-induced rotational behavior, improved immobility during forced swimming and forelimb suspension test performance, suggesting its role in improving motor function. Further analysis revealed that PGLM could restore the expression of tyrosine hydroxylase (TH) in the substantia nigra, reduce abnormal α-Syn aggregation, and inhibit neuroinflammatory responses (decreased expression of GFAP and IBA1). More importantly, PGLM also promoted the expression of mitophagy-related proteins PINK1 and Parkin. These results further demonstrate that PGLM exerts a neuroprotective effect by regulating the PINK1 / Parkin signaling pathway to induce mitophagy.

[0022] The experimental results of this invention show that PGLM (guava leaf extract) exhibits significant neuroprotective effects in cells, *C. elegans*, and mouse PD models. Its mechanism may be closely related to the activation of the PINK1 / Parkin pathway-mediated mitophagy. This study not only provides new experimental evidence for the theory of mitochondrial homeostasis imbalance in PD but also offers new research ideas for developing neuroprotective agents based on traditional Chinese medicine resources, and lays the foundation for the application of PGLM in PD treatment.

[0023] (4) The experimental results show that with the increase of PGLM treatment concentration, the autofluorescence intensity of the intestinal region of nematodes (the main deposition site of lipofuscin) significantly decreased, indicating that PGLM can effectively clear or inhibit the accumulation of lipofuscin in aging nematodes. PGLM treatment significantly enhanced the motility of N2 nematodes in a dose-dependent manner, with the 300 μg / mL treatment group showing particularly significant effects. This result strongly suggests that PGLM can effectively delay age-related decline in motor function and maintain the health of the neuromuscular system. PGLM (guava leaf extract) can effectively improve the pharyngeal pump function of N2 nematodes and significantly enhance their feeding ability. This effect suggests that guava leaf extract delays age-related physiological decline by protecting neuromuscular junction function, maintaining neuronal health, or enhancing energy metabolism, thereby promoting healthy aging. Attached Figure Description

[0024] The accompanying drawings, which are provided to further illustrate the invention and constitute a part of this invention, are illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention.

[0025] Figure 1 : The safe concentration of PGLM for PC12.

[0026] Figure 2The concentration of 6-OHDA used to establish PC12 cell modeling was determined, and compared with the control group, ***p<0.001.

[0027] Figure 3 PGLM improves the survival rate of 6-OHDA-induced PC12 cells.

[0028] Figure 4 A, B: PGLM inhibited the damage of 6-OHDA to PC12 cells. Compared with the control group, **p<0.01, ***p<0.001. Magnitude: ×10, Scale bar: 200 μm.

[0029] Figure 5 :AF :PGLM inhibits 6-OHDA-induced damage to PC12 cells by mitophagy, ***p<0.001 compared with the control group. Magnitude: ×10 Scale bar: 200 μm.

[0030] Figure 6 :AD: Effect of PGLM on mitochondrial autophagy in SJZ42 nematodes, compared with the control group, **p<0.01 fold: ×20, scale bar: 100 μm.

[0031] Figure 7 A, B: PGLM inhibited 6-OHDA-induced dopaminergic neuronal damage in BZ555 nematodes. Compared with the control group, **p<0.01, ***p<0.001. Magnitude: ×10, Scale bar: 200 μm.

[0032] Figure 8 A, B: PGLM improved the food perception and motility of 6-OHDA-induced BZ555 nematodes, with **p<0.01 and ***p<0.001 compared with the control group.

[0033] Figure 9 A, B: PGLM alleviates 6-OHDA-induced dopaminergic neuronal damage in BZ555 nematodes by mitophagy. Compared with the control group, **p<0.01, ***p<0.001. Magnitude: ×10, Scale bar: 200 μm.

[0034] Figure 10 PGLM reduced the expression of α-Syn in NL5901 nematodes and improved their motility, with ***p<0.001 compared to the control group. Variable: ×10, Scale bar: 200 μm.

[0035] Figure 11 A, B: PGLM reduced ROS levels in NL5901 nematodes, p < 0.001 compared to the control group. Magnitude: ×10, Scale bar: 200 μm.

[0036] Figure 12 AC:PGLM reduced the level of α-Syn in nematodes by activating mitophagy, with ***p<0.001 compared with the control group. Magnitude: ×10, Scale bar: 200 μm.

[0037] Figure 13 :AE:PGLM improved 6-OHDA-induced behavioral disorders in mice, **p<0.01, ***p<0.001 compared with the control group.

[0038] Figure 14 FG: PGLM restored TH expression in the substantia nigra of mice induced by 6-OHDA. Compared with the control group, *p<0.05, **p<0.01, ***p<0.001. Magnitude: ×10, Scale bar: 200 μm.

[0039] Figure 15 A, B: PGLM induces mitochondrial autophagy in mice by activating 6-OHDA via PINK1 / Parkin, reducing α-Synuclein expression. Compared with the control group, the fold change is ×10, and the scale bar is 200 μm.

[0040] Figure 16 The effects of PGLM on indicators related to delaying aging in N2 nematodes: A, B: lipofuscin; CF: nematode body length; G: pumping frequency; H: motility; I: reproductive capacity. Detailed Implementation

[0041] To better understand the implementation of this invention, the following typical embodiments are provided for further explanation. The therapeutic effects of guava leaf extract (PGLM) are demonstrated below through pharmacodynamic studies.

[0042] Example 1

[0043] 1.1 Cytotoxicity assessment of PGLM on PC12 cells

[0044] To determine the appropriate PGLM concentration for subsequent experiments, the dose-response relationship of PGLM on PC12 cells was assessed using the MTT assay. Results are as follows: Figure 1 As shown, compared with the control group, when the PGLM concentration reached 400 μg / mL, PC12 cell viability decreased and cell growth was inhibited (p < 0.001). Although no significant decrease in cell viability was observed at a concentration of 200 μg / mL, cell growth was still inhibited to some extent. Therefore, to ensure that PGLM does not affect normal cell growth in subsequent experiments, the PGLM concentration should be controlled at 100 μg / mL or below.

[0045] 1.2 Optimal Concentration of 6-OHDA to Induce PC12 Cell Damage

[0046] 6-OHDA is a commonly used inducer for constructing PC12 cell injury models. To determine the appropriate modeling concentration, we used the MTT assay to detect the effect of different concentrations of 6-OHDA on PC12 cell viability, thus selecting the optimal modeling concentration for subsequent experiments. The experimental results are as follows: Figure 2 As shown, compared with the blank control group, 400 μM, 200 μM, 100 μM, and 50 μM 6-OHDA all affected PC12 cell viability (p < 0.001). When selecting the modeling concentration, we generally consider a modeling time of 24 hours and select the concentration with a cell viability of 50%-60% as the optimal modeling concentration. Based on the above criteria, the PC12 cell viability at 200 μM 6-OHDA was around 60%, and this was determined as the 6-OHDA modeling concentration for subsequent experiments.

[0047] 1.3 PGLM inhibits 6-OHDA-induced damage to PC12 cells

[0048] To verify that PGLM inhibits the damage of 6-OHDA to PC12 cells, we measured cell viability after 6-OHDA-induced PC12 cell damage in the presence or absence of PGLM. The results are as follows: Figure 3 As shown, compared with the control group, the survival rate of PC12 cells in the 6-OHDA group was significantly decreased (p<0.001), while the viability of PC12 cells was significantly increased after administration of PGLM (25, 50, 100 μg / mL) compared with the 6-OHDA group (p<0.01).

[0049] Subsequently, we used Hoechst / PI dye to detect cell mortality, and the results were as follows: Figure 4 As shown in Figures A and B: Compared with the control group, the number of PI-positive cells increased under 6-OHDA stimulation (p < 0.001), while the number of PI-positive cells decreased after administration of different concentrations of PGLM, showing a concentration-dependent relationship (p < 0.01, p < 0.001). Furthermore, we photographed the morphology of PC12 cells under white light. The results showed that compared with the blank control group, the number of PC12 cells in the 6-OHDA group was significantly reduced, and the cells were shrunken. After PGLM intervention, the reduction and shrunkenness of cells were improved, further demonstrating that PGLM inhibits 6-OHDA-induced damage to PC12 cells.

[0050] 1.4 PGLM inhibits 6-OHDA-induced damage in PC12 cells through mitophagy.

[0051] In our experiments, we used the autophagy and mitophagy inhibitors 3-MA and AC220 to explore the effects of PGLM-mediated mitophagy on 6-OHDA-induced cell damage. PC12 cells treated with 6-OHDA showed significant morphological changes. See the attached table for experimental results. Figure 5 Impaired cell activity, such as shrinkage and impaired cell integrity, indicates cell death. PGLM treatment significantly improved cell viability compared to the 6-OHDA group (p < 0.001). However, the protective effect of PGLM was counteracted by the use of AC220 and 3-MA. Hoechst / PI staining showed a significant increase in PI-positive cells in the 6-OHDA group, while PGLM treatment reduced the number of PI-positive cells, but this effect was reversed by AC220 and 3-MA (p < 0.001). Cellular ROS levels were detected using Hoechst / DHE staining. 6-OHDA treatment significantly increased the number of DHE-positive cells (p < 0.001), indicating increased ROS production. PGLM treatment significantly reduced the number of DHE-positive cells, indicating decreased ROS levels, but the addition of the AC220 and 3-MA inhibitors reversed this effect. These results suggest that mitophagy plays a crucial role in the protective mechanism of PGLM against 6-OHDA-induced PC12 cell damage. In summary, PGLM effectively combats 6-OHDA-induced cell death and ROS generation in PC12 cells. These neuroprotective effects are mediated by the induction of mitophagy.

[0052] Example 2

[0053] 2.1 Effects of PGLM on mitochondrial autophagy in SJZ42 nematodes

[0054] In this study, we used *Caenorhabditis elegans* SJZ42 expressing the rgef-1p:tomm-20:Rosella construct, which reduced the visualization and quantification of mitophagy through a pH-sensitive dual-fluorescent reporter gene targeting mitochondria. Mitophagy activity was assessed by measuring the ratio of GFP to DsRed fluorescence intensity. See attached notes. Figure 6 Treatments with A, B, PGLM, and CCCP significantly reduced the GFP / DsRed ratio (p < 0.01), indicating active mitophagy. Secondly, see the attached instructions. Figure 6 In C and D, after feeding the SJZ42 nematode with double-stranded RNA bacteria Pink1 and pdr-1 that target the corresponding genes, PGLM could not reverse the GFP / DsRed ratio (p < 0.01), indicating that PGLM can inhibit mitochondrial autophagy in SJZ42 nematode.

[0055] 2.2 Protective effect of PGLM6-OHDA on dopaminergic neurons of BZ555 nematode

[0056] 2.2.1 PGLM inhibits 6-OHDA-induced dopaminergic neuron damage in BZ555 nematodes

[0057] 6-OHDA has a high affinity for dopamine transporters, enabling it to enter dopaminergic neurons via these transporters and accumulate intracellularly, thereby selectively damaging dopaminergic neurons. BZ555 is a transgenic nematode expressing dopaminergic neurons fused with GFP; the intensity of GFP fluorescence can be used to indirectly measure indicators of dopaminergic neurons. In our experiments, we found... (The text abruptly ends here.) Figure 7 Compared with the control group, the 6-OHDA group showed weaker head fluorescence and smaller cell bodies in the BZ555 nematodes (p < 0.001), indicating that the DA neurons of the BZ555 nematodes were significantly and severely damaged at this time. After administration of PGLM and L-dopa (positive control), the head fluorescence of the BZ555 nematodes recovered and the cell bodies became larger (p < 0.001), suggesting that PGLM has an inhibitory effect on 6-OHDA-induced damage to DA neurons.

[0058] 2.2.2 PGLM improves food perception and motility in 6-OHDA-induced BZ555 nematodes

[0059] Food perception and motor function are important behavioral indicators for assessing the functional status of dopaminergic neurons in nematodes. Under normal conditions, when dopaminergic neurons are functioning well, nematodes exhibit strong food perception and active motor behavior (such as body swaying frequency). However, when dopaminergic neurons are damaged, both food perception and motor function significantly decline. Therefore, by detecting these two indicators, the degree of damage to dopaminergic neurons and their functional recovery can be indirectly reflected. Experimental results are shown in the attached instruction manual. Figure 8 As shown in Figures A and B, compared with the blank control group, the nematode motility of the 6-OHDA treatment group was significantly reduced, manifested by a significant decrease in the number of body swings and a decline in food perception (p < 0.01, p < 0.001). However, after intervention with PGLM and L-dopa (positive control drug), the nematode motility was significantly improved, the number of body swings was significantly increased, and the food perception ability was enhanced (p < 0.01, p < 0.001). This result indicates that PGLM restores their food perception behavior and motility.

[0060] 2.2.3 PGLM alleviates 6-OHDA-induced damage to dopaminergic neurons in BZ555 nematodes through mitophagy.

[0061] The expression of DA neurons was observed by feeding bacteria containing double-stranded RNA (dsRNA) targeting the corresponding gene to nematodes BZ555. The experimental results are shown in the attached instructions. Figure 9As shown in Figures A and B, for the Ctrl group, the DA neuron damage was significant in the 6-OHDA group (p < 0.001). Compared with the 6-OHDA group, PGLM improved the DA neuron damage, but after feeding with double-stranded RNA bacteria vps34 and pdr-1 targeting the corresponding genes, PGLM did not show the above effect (p < 0.01, p < 0.001). This suggests that PGLM can improve the expression of DA neurons in BZ555 nematodes through mitophagy.

[0062] 2.3 PGLM inhibits the production of α-Syn in NL5901 nematodes.

[0063] 2.3.1 PGLM reduces α-Syn expression and enhances motility in NL5901 nematodes.

[0064] NL5901 contains overexpressed α-Syn, and its fluorescence intensity reflects the α-Syn content. We collected samples from NL5901 adults on the fifth day of their adult stage for verification. The experimental results are shown in the attached figure. Figure 10 AC analysis showed that, compared with the blank control group (Ctrl), the L-dopa positive drug group exhibited decreased fluorescence and enhanced body movement ability; different concentrations of PGLM all reduced α-Syn expression, with 300 μg / mL PGLM showing the best effect (p < 0.001). This suggests that PGLM can reduce α-Syn expression and improve the motility of NL5901 nematodes.

[0065] 2.3.2 PGLM reduces ROS levels in NL5901 nematodes

[0066] The aggregation of α-Syn leads to an increase in ROS. We used a DHE reactive oxygen species probe to measure the ROS of NL590 nematodes. See the attached notes for results. Figure 11 As shown in A and B, the red fluorescence intensity of the PGLM group was significantly reduced in the Ctrl group (p < 0.001). When the drug concentration was 300 μg / mL, the red fluorescence intensity of NL5901 nematodes was comparable to that of the positive drug L-dopa group in reducing ROS levels, indicating that PGLM can reduce ROS levels in NL5901 nematodes.

[0067] 2.3.3 PGLM may effectively reduce the expression level of α-Syn by activating the mitophagy pathway.

[0068] In the NL5901 nematode model, changes in the expression of α-synuclein (α-Syn) were observed by feeding bacteria containing double-stranded RNA (dsRNA) targeting specific genes. Experimental results are attached as described in the appendix. Figure 12The results showed that PGLM significantly reduced α-Syn protein levels compared to the control group (p < 0.001), with effects comparable to L-dopa, while also significantly improving nematode motility. However, PGLM did not exhibit the aforementioned effects of reducing α-Syn and improving motility after feeding bacteria with dsRNA targeting the vps34 and pdr-1 genes. These results suggest that PGLM may effectively reduce α-Syn expression levels by activating the mitophagy pathway.

[0069] Example 3

[0070] 3.1 PGLM improves 6-OHDA-induced behavioral disorders in mice

[0071] Behavioral disorders in animals can indirectly reflect their pathological condition; see the instruction manual appendix. Figure 13 -A is a cross-section of a mouse brain, for illustrative purposes only. Figure 13 -B represents the timeline of adaptive feeding, surgery, drug intervention, and behavioral data collection in mice. We induced rotation in mice through subcutaneous injection of APO; APO induced mice to rotate laterally towards the healthy side, and behavioral scores were then calculated. (See attached image.) Figure 13 As shown in -C: Compared to the sham-operated group, the number of rotations per minute in the 6-OHDA group was significantly increased (p < 0.001). Compared to the model group, the number of rotations in mice was significantly decreased after administration of L-dopa. Administration of PGLM showed no significant difference compared to the 6-OHDA model group at 100 mg / kg, but increasing the drug concentration resulted in a significant decrease in mouse rotation (p < 0.001). Forced swimming and forelimb suspension tests were subsequently performed, as per the instructions. Figure 13 As shown in Figures D and E, compared with the sham-operated group, the 6-OHDA group showed significantly lower scores in forced swimming and forelimb suspension tests (p < 0.001), indicating severe neuronal damage that affected the mice's normal activity. Compared with the 6-OHDA model group, L-dopa administration increased scores, indicating improvement in the mice's behavioral impairment. In the forelimb suspension and forced swimming tests, no significant difference was observed at 100 mg / kg, but a significant difference was observed at 200 mg / kg, suggesting this concentration could be considered an effective dose for improving forelimb suspension and forced swimming tests in mice (p < 0.01). This suggests that PGLM can improve the 6-OHDA-induced behavioral impairment in mice.

[0072] 3.2 PGLM restores 6-OHDA-induced TH expression in the substantia nigra of mice

[0073] Tyrosine hydroxylase plays a crucial role in the dopamine production pathway and is involved in the development of various neurodegenerative diseases. As a limiting enzyme in dopamine synthesis in the brain, tyrosine hydroxylase levels are significantly reduced in the substantia nigra region of Parkinson's disease. Immunohistochemical quantitative analysis of tyrosine hydroxylase was used to assess the degree of damage to dopaminergic neurons. Experimental results are attached. Figure 14 As shown in F and G, neurochemical detection revealed that the density of TH-positive neurons in the substantia nigra pars compacta of 6-OHDA-induced Parkinson's disease model mice was significantly reduced compared to the blank control group, with lighter cytoplasmic staining and morphological degeneration or absence (p < 0.001). However, after PGLM treatment, the number of TH-positive neurons in the substantia nigra of the PGLM group significantly increased, and administration of 200 mg / kg significantly restored the effect to that of the positive drug L-dopa group (p < 0.05, p < 0.01), indicating that PGLM can inhibit the damage of 6-OHDA to dopaminergic neurons.

[0074] 3.3 PGLM induces mitophagy in mice by activating 6-OHDA via PINK1 / Parkin and reduces α-Synuclein expression.

[0075] To verify that PGLM inhibits 6-OHDA-induced damage in mice via the mitophagy pathway, the expression of PINK1 and Parkin in mouse brain tissue was detected using immunofluorescence. The results showed that PINK1 and Parkin expression was upregulated after PGLM intervention, indicating activation of mitophagy (see instruction manual appendix). Figure 15 -A). Meanwhile, in 6-OHDA-induced C57 mouse brain tissue, the fluorescence intensity of α-synuclein (α-Syn) was significantly enhanced, while PGLM intervention significantly reduced the fluorescence intensity of α-Syn, indicating that PGLM can effectively downregulate α-Syn expression (see instruction manual appendix). Figure 15 -B).

[0076] Example 4

[0077] 4.1 Effects of PGLM on lipofuscin content in nematodes

[0078] To investigate the effects of PGLM on aging-related indicators, we examined its effect on lipofuscin accumulation in wild-type N2 nematodes. Lipofuscin is a fluorescent aging pigment that accumulates in lysosomes, and its content increases with age; it is a classic biomarker for measuring the level of aging. Experimental results are shown in the attached instructions. Figure 16 As shown in Figures A and B, compared with the untreated control group, the autofluorescence intensity of the nematode intestinal region (the main deposition site of lipofuscin) was significantly reduced with increasing PGLM concentration, indicating that PGLM can effectively clear or inhibit the accumulation of lipofuscin in senescent nematodes.

[0079] 4.2 Effects of PGLM on nematode body length

[0080] Body length is a fundamental indicator for measuring the growth, development, and health status of nematodes. We measured the changes in body length of N2 nematodes on days 1, 3, and 5 under different concentrations of PGLM treatment. Experimental results are shown in the attached instruction manual. Figure 16 CF studies showed that PGLM treatment significantly promoted the body length of nematodes, and this effect was observed across different age groups. On day 1, compared with the control group, the nematodes in the 300 μg / mL PGLM treatment group showed a significant increase in body length, indicating that PGLM may promote early development. On day 3, the body length of nematodes in all concentration treatment groups (100, 200, 300 μg / mL) was significantly greater than that in the control group, showing that PGLM could maintain its growth-promoting effect in the adult stage. On day 5 of senescence, while the nematodes in the control group may have shown slight body wall laxity or atrophy due to aging, all PGLM treatment groups, especially the 300 μg / mL group, maintained significantly better body length. This suggests that PGLM not only promotes growth but may also help delay or counteract age-related body morphological changes.

[0081] 4.3 Effect of PGLM on the pumping frequency of nematodes

[0082] The pharyngeal pump is a core organ for feeding and digestion in nematodes, and its pumping frequency (i.e., the number of pharyngeal pumps) is an important indicator for assessing the physiological health status, neuronal function, and aging process of nematodes. Typically, the pharyngeal pump frequency decreases significantly with aging. We investigated the effect of PGLM on the pharyngeal pump movement of N2 nematodes. (See attached instruction manual). Figure 16 G recorded the pharyngeal pump frequency of nematodes on day 5 of adulthood in the control group and the 100, 200, and 300 μg / mL PGLM-treated groups. Statistical results showed that PGLM treatment effectively and dose-dependently increased the pharyngeal pump frequency of nematodes. This result indicates that PGLM can effectively improve the pharyngeal pump function of N2 nematodes and significantly enhance their feeding capacity. This effect suggests that PGLM may delay age-related physiological decline by protecting neuromuscular junction function, maintaining neuronal health, or enhancing energy metabolism, thereby promoting healthy aging.

[0083] 4.4 Effects of PGLM on the motility and reproductive capacity of nematodes

[0084] Decreased locomotor function and reduced fertility are typical manifestations of aging. To comprehensively assess the effects of PGLM on the physiological functions of nematodes, this study examined its effects on locomotor behavior and oviposition. (See attached instructions.) Figure 16As shown in Figure H, PGLM treatment dose-dependently increased the motility frequency (e.g., head swings or body flexions) of *N. nervosa*, with the most significant effect observed at a concentration of 300 μg / mL (p < 0.001), indicating that PGLM can effectively delay age-related motor function decline and help maintain neuromuscular health. Regarding reproductive capacity, see attached... Figure 16 As shown in Figure I, PGLM significantly increased oviposition in N2 nematodes during the peak reproductive period (days 1–3), exhibiting a dose-dependent increase. However, during days 4–5, oviposition in all PGLM treatment groups showed no significant difference from the control group, declining to a low level, consistent with the natural decline of the nematode reproductive system with age. These results indicate that PGLM can significantly optimize the reproductive performance of nematodes during their peak reproductive period, but does not alter the natural decline process of the reproductive system in the later stages of their life cycle, suggesting that its effect is mainly manifested in enhancing physiological functions during the peak reproductive period, rather than indefinitely prolonging the reproductive cycle.

Claims

1. Use of Psidium guajava leaf extract in the preparation of a medicament for treating neurodegenerative diseases.

2. Use according to claim 1, characterized in that, Use of the Psidium guajava leaf extract in the preparation of a medicament for treating brain neuroprotective diseases.

3. Use as claimed in claim 1, characterised in that, Use of the Psidium guajava leaf extract in the preparation of a medicament for treating Parkinson's disease.

4. Use of Psidium guajava leaf extract in the preparation of a medicament for treating anti-aging diseases.

5. The method of preparing Psidium guajava leaf extract as claimed in claim 1, wherein, The preparation method comprises the following steps: Weigh 25g of dried Psidium guajava leaves, crush them, and soak them in 200mL of methanol for 24 hours. Heat and reflux to extract, with each extraction time being 1.5 hours. Extract three times, combine the filtrates, and recover the solvent under reduced pressure to obtain Psidium guajava leaf extract.

6. Use according to any one of claims 1 to 4, characterized in that, The effective concentration of the Psidium guajava leaf extract is 25-100μg / mL.

7. Use according to any one of claims 1 to 4, wherein the compound is ###0002### The Psidium guajava leaf extract can be made into granules, capsules, tablets, and oral liquids.

8. Use as claimed in claim 1, characterised in that, Use of the Psidium guajava leaf extract in the preparation of a medicament for treating amyotrophic lateral sclerosis.

9. Use as claimed in claim 1, characterised in that, Use of the Psidium guajava leaf extract in the preparation of a medicament for treating metabolic diseases.

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