Application of EMA (Ethyl Methacrylate) in preparing medicine for protecting nerves or delaying senescence

Abstract

The invention belongs to the technical field of medicines, and particularly discloses application of EMA (Ethyl Methacrylate) in preparation of a medicine for protecting nerves or delaying senescence. Experiments prove that the EMA can reduce RAW264.7 cell inflammation, obviously prolong the life and aging related indexes (lipofuscin, autophagy, nuclear translocation and the like) of a caenorhabditis elegans model, improve neuron damage (tropism) of an Alzheimer's disease nematode model and improve the oxidative stress resistance of nematodes. Based on this, it is prompted that EMA may play an anti-aging role through a DAF-16 pathway and has a potential anti-AD role, and EMA can be used for preparing drugs for prolonging life or delaying aging.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical technology, and specifically discloses an application of EMA in preparing a neuroprotective or anti-aging drug. Background Art

[0002] Aging is a complex and irreversible degenerative process that occurs in various body tissues and organs. It can be reflected in various aspects such as lifespan, motor ability and reproductive ability. Studies have shown that aging is always accompanied by various chronic diseases, such as neurodegenerative diseases: AD cognitive decline, cardiovascular disease, etc. Fortunately, drugs, genetic or dietary interventions that can extend lifespan will help delay the onset of the disease. In recent decades, the use of the model organism Caenorhabditis elegans as a model system for the study of aging, longevity mechanisms and drug screening has been widely recognized.

[0003] AD is a progressive neurodegenerative disease that affects cognition and behavior, with memory impairment being the most common initial symptom. So far, the pathogenesis of AD has not been elucidated, but increasing evidence suggests that extracellular senile plaques caused by the accumulation of β-amyloid protein (Aβ) are the key histopathological features of AD, and the neurotoxicity of Aβ may play a core role in the pathogenesis of AD.

[0004] C. elegans is an ideal biological model for aging research due to its short life cycle, easy manipulation, ability to simulate natural aging, small size, rapid reproduction, and simple and developed reproductive and nervous systems. In addition, C. elegans will show slow behavior and decreased physiological function as it ages, which is very similar to higher mammals. As C. elegans ages, muscle atrophy and muscle tissue gradually become damaged, and oxidized proteins and lipofuscin gradually accumulate in the body. The characteristics of aging are generally similar to those of other species. C. elegans is highly homologous to humans at the aging-related and protein levels, and is therefore also called an ideal model organism for pharmacology and drug-induced gene expression.

[0005] EMA (12-Ethoxy-Marchantin A) is derived from the common spore plant of the genus Marchantia in the family Marchantaceae. EMA belongs to the phenolic compound family of stilbene compounds. Studies have reported that EMA has multiple pharmacological activities such as antibacterial, antitumor and antioxidant activities, but there is no literature report on its use in neuroprotective or anti-aging drugs. Summary of the invention

[0006] In view of the above problems, the purpose of the present invention is to provide a phenolic compound EMA for use in the treatment of neuroprotection or anti-aging drugs. The phenolic compound EMA has obvious pharmacological effects, and has the effects of significantly reducing RAW264.7 cell inflammation, restoring Caenorhabditis elegans neuronal damage, and prolonging the lifespan of Caenorhabditis elegans, and is safe and has no toxic side effects on Caenorhabditis elegans.

[0007] Specifically, the present invention conducts the following research:

[0008] First, the present invention provides the anti-inflammatory ability of EMA on RAW264.7 cells, and provides the ability of EMA to restore neuronal damage in Caenorhabditis elegans, that is, the role of EMA in restoring neuronal function is determined by the Caenorhabditis elegans chemotaxis experiment. The effect of EMA on extending the healthy lifespan of nematodes is provided, that is, the role of EMA in improving the body function of nematodes is determined by lifespan and egg laying.

[0009] In addition, in the oxidative stress experiment, EMA significantly improved the stress resistance of nematodes, indicating that EMA can protect nematodes from oxidative damage; in the autophagy fluorescence experiment, EMA significantly increased the fluorescent spots of LGG::GFP in nematodes, indicating that EMA can promote the occurrence of autophagy in nematodes; in the nuclear translocation experiment, EMA significantly increased the nuclear entry of DAF-16 in nematodes, and in the qRT-PCR experiment, EMA upregulated the expression of SKN-1 pathway-related genes and autophagy-related genes, and upregulated the expression of hsp-16.2 genes. The present invention first discovered that the phenolic compound EMA has new uses in neuroprotection and / or anti-aging, as follows:

[0010] In terms of neuroprotection:

[0011] (a) Reduced the inflammatory response of RAW264.7 cells;

[0012] (b) The head swing frequency of BR5270 / BR5271 nematodes was increased, alleviating movement disorders;

[0013] (c) increased the chemotaxis index of CL2355 / CL2122 nematodes and restored the function of damaged neurons;

[0014] In terms of anti-aging

[0015] (a) Extended the lifespan of healthy nematode N2;

[0016] (b) Increased egg production of healthy nematode N2;

[0017] (c) Improved the anti-oxidative stress capacity of healthy nematode N2;

[0018] (d) Increased LGG::GFP autophagy fluorescence spots in the autophagy-induced nematode DA2123;

[0019] (e) Increased nuclear translocation of DAF-16 transgenic nematode TJ356;

[0020] (f) The expression of SKN-1 and related downstream genes was upregulated, and the expression of autophagy genes was upregulated.

[0021] The EMA of the present invention is obtained by extracting Marchantia polymorpha L. The extraction method is as follows: 1.1 kg of fresh Marchantia polymorpha L is extracted with methanol (10 L x 3 times) at room temperature to obtain a crude extract, the crude extract is dissolved in an aqueous solution, further extracted with an equal volume of ethyl acetate, the extraction layer is placed on an MCI gel column, and gradient eluted with a methanol-water system (50%, 70%, 80%, 100% methanol) in sequence to obtain 4 fractions. The fraction obtained by eluting with 80% methanol is further purified on RP-HPLC using 45% MeCN (2.0 mL / min) as an eluent to obtain compound EMA (24.7 mg).

[0022] The concentration of EMA in the preparation of neuroprotective or anti-aging drugs is 5-80 μM.

[0023] The present invention has the following beneficial effects:

[0024] (1) The present invention uses the model organism Caenorhabditis elegans as a model to simulate natural aging and study the effects of the phenolic compound EMA on related indicators accompanying the aging process, thereby revealing the diseases and molecular mechanisms that EMA may improve and regulate.

[0025] (2) The present invention screens out the optimal concentration of EMA for the study of neuroprotection or anti-aging effects based on nematode egg-laying and lifespan experiments, verifies the neuroprotective effect of EMA on nematodes through head swing experiments and chemotaxis experiments; verifies the anti-stress effect of EMA on nematodes through oxidative stress experiments; verifies the potential pathway of EMA through nuclear translocation experiments; and analyzes the mechanism of action of EMA on nematodes through qRT-PCR, providing a new application for EMA and a strong theoretical basis for the research and development of EMA in neuroprotection and / or anti-aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Effect of EMA on the inflammatory response of RAW264.7 cells.

[0027] Figure 2 Effects of EMA on head shaking of nematodes BR5270 and BR5271.

[0028] Figure 3 Effects of EMA on the chemotaxis index of CL2355 and CL2122 nematodes.

[0029] Figure 4 Effects of EMA on the lifespan of N2 nematodes.

[0030] Figure 5 Effect of EMA on egg laying in N2 nematodes.

[0031] Figure 6 Effects of EMA on lipofuscin deposition in N2 nematodes

[0032] Figure 7 Effects of EMA on oxidative stress in N2 nematodes.

[0033] Figure 8 Effects of EMA on autophagic fluorescent spots in DA2123 nematodes.

[0034] Fig. 9 Effect of EMA on nuclear translocation in TJ356 nematodes.

[0035] Fig.10 Effects of EMA on genes related to the SKN-1 pathway.

[0036] Fig.11 Effects of EMA on autophagy and hsp-16.2-related genes. DETAILED DESCRIPTION

[0037] The present invention is further described in detail by the following examples, but it should be noted that the scope of the present invention is not limited by these examples.

[0038] Example 1 Preparation of EMA

[0039] 1.1 kg of fresh Marchantia polyphylla was extracted with methanol (10 L x 3 times) at room temperature to obtain 21 g of crude extract, which was dissolved in aqueous solution and further extracted with an equal volume of ethyl acetate. The extract was placed on an MCI gel column and gradient eluted with a methanol-water system (50%, 70%, 80%, 100% methanol) to obtain 4 fractions. The fraction obtained by eluting with 80% methanol from the gel column was further purified on RP-HPLC using 45% MeCN (2.0 mL / min) as the eluent to obtain compound EMA (24.7 mg).

[0040] Example 2 Cultivation and treatment of RAW264.7 cells

[0041] Experimental cells: RAW264.7 is a mouse mononuclear macrophage leukemia cell line, which is one of the most commonly used in vitro models for inflammation research.

[0042] Experimental reagents: Prepare 100mL MTT solution

[0043] Use a balance to accurately weigh 0.5 g of MTT powder into a 250 mL beaker, add 100 mL of DDW, stir continuously, and dispense 1 mL into 1.5 mL brown centrifuge tubes after complete dissolution, and freeze at -20°C for later use.

[0044] Cell culture and treatment: RAW264.7 cells in good culture state after recovery were inoculated on 96-well plates for 12 hours, pretreated with different concentrations of EMA (5μM, 10μM, 20μM), and co-treated with LPS (200ng / mL) for 24 hours. 10μL MTT solution was added to each well and cultured for 4 hours. After 4 hours, the supernatant was aspirated and discarded by a vacuum pump against the well wall, and 100μL DMSO was added and shaken evenly. The absorbance between 570nm and 630nm was detected using an ELISA reader. The survival rate was calculated based on the average absorbance of 3 duplicate wells in each group, and the experiment was repeated 3 times for each group of samples.

[0045] The experimental results show that Figure 1 As shown in B, the effects of different concentrations of EMA (2.5-100 μM) on cell toxicity were detected. The results showed that concentrations greater than 50 μM had a certain degree of toxicity to cells. Figure 1 As shown in Figure C, the toxicity of LPS at different concentrations to cells was detected. As the concentration of LPS gradually increased, the survival rate of RAW264.7 cells did not decrease significantly, and the damage to the cells was not significant. Figure 1 As shown in D, co-treatment of different concentrations of EMA and LPS had no effect on the survival rate of RAW264.7 cells. Figure 1 As shown in E, different concentrations of EMA treatment can significantly reduce the content of NO produced by LPS-induced cells. Figure 1 As shown in Figure F, different concentrations of EMA can reduce the expression of the inflammatory factor PGE2. The above results show that EMA has a good anti-inflammatory effect and can reduce cellular inflammatory response.

[0046] Example 3 Experimental strains of Caenorhabditis elegans and culture medium preparation process

[0047] The experimental nematode strains are: N2 nematodes: wild-type nematode strain; BR5270 nematodes: genotype byIs161 [rab-3p:: F3(delta)K280+myo-2p:: mCherry] transgenic nematodes with human Tau protein, BR5271 nematodes: genotype: byIs162 [rab-3p:: F3(delta)K280 I277P I380P+myo-2p:: mCherry], often used as a positive control for BR5270 nematodes; DA2123 nematodes: genotype adIs2122

[0048] [lgg-1p::GFP::lgg-1+rol-6(su1006)]; TJ356 nematodes: genotype zIs356

[0049] [daf-16p::daf-16a / b::GFP+rol-6(su1006)]; all the above nematodes were purchased from the CGC nematode bank.

[0050] The nematode culture medium is NGM culture medium. The preparation method of 1L NGM (Nematode Growth Meium) culture medium is as follows: 3g NaCl, 2.5g peptone, 17g agar powder, add 975mL distilled water, sterilize at 121℃ for 30min, keep constant temperature at 70℃ for 10min, and then add the following solutions that have been sterilized by high pressure: 25mL KPO4 buffer, 1mL cholesterol, 1mL MgSO4, and 1mL CaCl2.

[0051] Preparation of LB liquid culture medium (1 L): 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, 15 g of agar powder, adjust pH to 7.5, and add sterile water to 1 L.

[0052] Preparation of lysis solution: 100 mL of distilled water contains: 10 mL (2 g) of 5 mol / L sodium hydroxide and 10 mL of 10% sodium hypochlorite.

[0053] Example 4 Implementation steps of Caenorhabditis elegans

[0054] 1. Activation of E. coli OP50 and coating of drug-containing or drug-free plates

[0055] (1) Activation of Escherichia coli

[0056] Take a sterilized 15mL centrifuge tube in a sterile clean bench, take 10mL of sterilized LB liquid medium, and add 10μL of E. coli OP50. Place the 15mL centrifuge tube in a 37℃ constant temperature shaker overnight for 8h, so that the OD600 of E. coli is between 0.8-1.2, and store the centrifuge tube in a 4℃ refrigerator for use.

[0057] (2) Escherichia coli OP50 plate

[0058] The culture medium was prepared as described in Example 3. OP50 was taken out of the 4°C refrigerator and placed in a sterile clean bench. 200 μL of OP50 was pipetted into each plate and spread evenly. The inoculated plates were incubated at 37°C for 8 h.

[0059] (3) Add different concentrations of EMA to the drug-containing OP50 plate

[0060] Drug-free culture medium: spread the autoclaved culture medium onto culture dishes of the same size. After NGM solidifies, add 200 μL OP50 as food for nematodes to each culture dish, spread it evenly, and use it to culture nematodes after OP50 is blown dry. Drug-containing culture medium: add EMA diluted with OP50 to a final concentration of 5 μM, 10 μM, 20 μM, 40 μM, and 80 μM to the drug-free culture medium, mix thoroughly, and store in a 16°C constant temperature incubator for later use.

[0061] 2. Synchronization of nematodes

[0062] In the clean bench, take the nematodes that have grown to sexual maturity and are laying eggs. The sign is that nematode eggs can be seen everywhere on the surface of the NGM plate under the stereoscope. Wash the nematodes with M9 buffer into a sterile 1.5mL centrifuge tube and let it stand for 5 minutes. After the adult worms are precipitated, discard the supernatant, add 4mL of freshly prepared nematode lysis buffer, shake vigorously until the solution is clear, and then quickly transfer it to a 1.5mL centrifuge tube and centrifuge it at 5000rpm for 1min. After the centrifugation, pour out the supernatant, quickly add 1mL of M9 buffer to each tube, vortex for 3s, and centrifuge at 5000rpm for 2min. After the centrifugation, carefully discard the supernatant, add 1mL of M9 buffer to each tube, vortex for 3s, and centrifuge at 5000rpm for 3min. After the centrifugation, carefully discard the supernatant, transfer all the precipitates to a 1.5mL centrifuge tube, add 1mL of M9 buffer, vortex to mix, and place it in a constant temperature incubator at the corresponding temperature for 24-48h.

[0063] 3. Screening of EMA concentration

[0064] Collect the synchronized L4-stage Caenorhabditis elegans, screen the appropriate concentration range of EMA, dilute a series of concentrations, and add 200 μL of EMA at the above concentrations to the culture dishes containing nematode food OP50. Pick 30 nematodes from each NGM culture dish and observe the head swing frequency of the nematodes within 30 seconds.

[0065] The results showed that when the EMA concentration was ≥80μM, the behavioral ability of C. elegans decreased significantly after one day of treatment, and the reaction was slower than that of the blank group. When the EMA concentration was ≤5μM, the behavioral ability of C. elegans did not change significantly compared with the blank group, and there was no statistical significance. The remaining concentrations restored the motor neuron dysfunction of the nematode to varying degrees, increased the motor ability, and increased the average number of swings within 30s, which was concentration-dependent. Therefore, the final EMA concentrations were determined to be 10μM, 20μM, and 40μM. The experimental results are shown in Figure 2 .

[0066] 4. Effect of EMA on the chemotaxis of nematodes strain CL2355

[0067] The L4 transgenic nematodes of the same period, CL2355 and CL2122, were collected, and NGM containing EMA at a final concentration of 10 μM, 20 μM, and 40 μM was prepared. A blank control group was set up, and the nematodes were evenly dropped into each plate and cultured at 16°C for 36 hours, and then induced at 25°C for 36 hours, and the nematodes were collected. The nematodes of different treatment groups were placed in the center of the culture dish. After 3 minutes, 1 μL of inducer (1% benzaldehyde) and 1 μL of 1 mol / L sodium azide were dropped at one end of the culture dish, which was called the "odorant" end, and 1 μL of anhydrous ethanol and 1 μL of 1 mol / L sodium azide were dropped at the opposite end, which was called the "control" end. After the nematodes were allowed to crawl on their own at 23°C for 1 hour, the number of nematodes crawling to each end was recorded, and the chemotaxis index CI was calculated. CI = (the number of nematodes at the "odorant" end - the number of nematodes at the "control" end) / the total number of nematodes.

[0068] The experimental results showed that the transgenic nematode CL2355 strain had Aβ introduced into the nematode neurons, thereby simulating the symptoms of AD, while its control strain CL2122 did not have Aβ introduced, and the chemotaxis response of the nematodes occurred by stimulating the motor neurons. In the control group nematode CL2122, there was no significant difference in the chemotaxis index after administration of different concentrations of EMA (P>0.05), while in CL2355, different concentrations of EMA significantly increased the chemotaxis index of the nematodes (P<0.01). The results show that EMA can improve the chemotaxis disorder of AD nematodes in a dose-dependent manner, and further improve learning and memory abilities. The experimental results are as follows Figure 3 .

[0069] 5. Effect of EMA on the lifespan of N2 strain nematodes

[0070] Collect the synchronized L4 stage N2 strain nematodes, prepare NGM containing EMA at final concentrations of 10μM, 20μM, and 40μM, set up a blank control group, pick 50 nematodes and put them in different NGM culture dishes, and each group has three parallel groups. The synchronized L4 stage administration is recorded as day0, and the number of nematode deaths is counted every day, and the remaining nematodes are transferred to new culture dishes. Repeat the cycle until all nematodes die, stop counting, and draw a lifespan curve. The criterion for judging the death of nematodes is: touch the body of the nematode with platinum wire continuously, and if there is no reaction, it is judged to be dead.

[0071] The lifespan curve shows that the average lifespan of nematodes in the EMA administration groups of different concentrations is higher than that of the blank control group, and as the EMA administration concentration increases, the lifespan of nematodes gradually increases in a concentration-dependent manner. This indicates that EMA can effectively delay the aging of nematodes and prolong their lifespan. Figure 4 .

[0072] 6. Effects of EMA on the reproductive capacity of N2 strain nematodes

[0073] The synchronized L4 stage N2 strain nematodes were collected, and NGM containing EMA at final concentrations of 10μM, 20μM, and 40μM was prepared. A blank control group was set up, and 10 nematodes were picked and placed in different NGM culture dishes. The egg-laying amount of the nematodes was observed every day for 5 consecutive days, and the results were statistically analyzed.

[0074] Nematodes generally have reproductive capacity in the first 4 days of the L4 stage, and as their lifespan increases, their reproductive capacity gradually decreases until it is lost. From the experimental results, it can be seen that the total number of reproductive offspring of nematodes in the EMA treatment groups with different concentrations is higher than that in the blank control group, indicating that EMA has the potential to delay nematode aging and prolong its reproductive capacity. Figure 5 .

[0075] 7. Effect of EMA on the deposition of lipofuscin in N2 strain nematodes

[0076] Lipofuscin is also known as senile hormone. In nematodes, the spontaneous fluorescence intensity of the intestine can reflect the content of lipofuscin. Lipofuscin in nematodes increases with age and can be used as an effective biomarker of nematode aging. Synchronized L4-stage N2 nematodes were treated with 10μM, 20μM, and 40μM EMA for two days. A control was set up. A 1wt% concentration of agarose gel was prepared, and a small piece was placed on a slide. Ten nematodes were selected from each group, and then sodium azide was added for anesthesia. The spontaneous green fluorescence of the nematode intestine was observed by laser confocal microscopy. The experiment was repeated three times. ImageJ statistically analyzed the fluorescence intensity.

[0077] The experimental results show that as the drug concentration increases, the lipofuscin content in the nematode gradually decreases and is concentration-dependent. It is significantly lower than the blank group, which shows that EMA can significantly reduce the lipofuscin content in the nematode and delay the aging of the nematode. Figure 6 .

[0078] 8. Protective effect of EMA on nematodes oxidatively damaged by juglone

[0079] Collect the synchronous L4 stage nematodes, rinse the nematodes with M9 buffer, and prepare a medium containing 150μM fluorouracil. Place the collected nematodes in a juglone solution (final concentration of 2mM) for 4 hours. Pick nematodes onto the culture medium, 30 per plate, count every hour, and count the number of dead nematodes. Repeat the experiment three times until all nematodes die, and draw a nematode lifespan curve.

[0080] The experimental results show that according to the aging theory, the improvement of nematode's ability to resist oxidative stress may be accompanied by an extension of lifespan, including tolerance to oxidative damage. The nematode survival lifespan curve shows that the nematode's ability to resist oxidative stress in the EMA treatment groups of different concentrations is higher than that in the blank control group, and is concentration-dependent. Figure 7 .

[0081] 9. Effect of EMA on the Nuclear Translocation of DAF-16 in TJ356 C. elegans

[0082] A large number of L4 nematodes were collected and synchronized, and a blank control group was set up. The transgenic nematodes TJ356 were treated with different concentrations of 10μM, 20μM, and 40μM EMA for three days. The treated nematodes were transferred to a slide containing an agar sheet, and ten nematodes were selected from each group and anesthetized with sodium azide. The subcellular localization of green fluorescence in the nematodes was observed with a fluorescent inverted microscope, and the nematodes were classified into cytoplasm, intermediate body, and nucleus according to the fluorescence localization. TJ356 nematodes express the DAF-16::GFP gene, and the nuclear entry of transcriptional regulators affects the extension of lifespan. Therefore, this experiment aims to detect the effect of EMA on the nuclear entry of DAF-16 transcriptional regulators. Three parallel groups were set up for each experiment.

[0083] The experimental results showed that when nuclear translocation occurs in vivo, the DAF-16::GFP gene in the nematode will be transferred from the cytoplasm to the nucleus, and the uniform green fluorescence of the whole body will change to dot-like green fluorescence, reflecting the nuclear translocation of the nematode. Compared with the blank group, EMA treatment can significantly increase the nuclear translocation of DAF-16 in TJ356 nematodes in a concentration-dependent manner. Figure 8 .

[0084] 10. Effect of EMA on autophagy fluorescence in DA2123 nematodes

[0085] This study detected the degree of autophagy activation of transgenic nematodes DA2123 at different concentrations of 10μM, 20μM, and 40μM EMA. The nematodes stably expressed LGG-1::GFP in muscle cells. When autophagy occurred in the body, the LGG-1::GFP protein in the nematodes aggregated to form fluorescent spots, reflecting the occurrence of autophagy in the nematodes. The nematodes synchronized to the L4 stage were collected and added to NGM plates containing different concentrations of drugs or without drugs. After culturing for 2 days, 10 nematodes were picked and placed on a slide containing an agar pad, covered with a coverslip, and observed and photographed under an inverted microscope. The fluorescence intensity of the total number of LGG-1::GFP / Atg8 dots expressed in DA2123 nematodes was analyzed and measured using ImageJ software. Each group of experiments was repeated three times.

[0086] There is increasing evidence that the enhancement of autophagy plays a key role in preventing aging and aging-related diseases. The experimental results showed that EMA increased the number of fluorescent spots formed by aggregation in the nematode in a dose-dependent manner, indicating that EMA can induce autophagy in DA2123 nematodes. Fig. 9 .

[0087] 11. Effects of EMA on SKN-1 pathway and autophagy pathway-related genes

[0088] Collect the nematodes synchronized to the L4 stage and transfer them to culture dishes containing 10μM, 20μM, and 40μM EMA, respectively, and set up a blank control group. Treat at 25℃ for two days, wash three times with M9 and collect the nematodes in a 1.5ml centrifuge tube, extract the total RNA of the nematodes by the Trizol method, and then reverse transcribe according to the instructions of the corresponding kit to obtain cDNA. Use cDNA as a template for real-time fluorescence quantitative PCR reaction, and place it in a fluorescence quantitative PCR instrument. After preheating and denaturing at 95℃ for 5min, amplify at 95℃ for 10s, cool to 60℃, react for 30s, and perform 40 cycles in total. The melting curve temperature is 65-90℃. Detect changes in the transcription levels of genes such as the SKN-1 pathway, autophagy, and hsp-16.2. Set up 3 sub-wells for each sample, and the primer design is shown in Table 1

[0089] Table 1 Primer sequences of related genes

[0090]

[0091]

[0092] Caenorhabditis elegans is a classic model organism for aging research, and the study of aging-related gene expression is essential, and the induction of autophagy has been shown to be associated with life extension and healthy life span. In this study, EMA was used to verify the expression of mRNA of downstream genes of the SKN-1 pathway by PCR. The experimental results showed that the expression of genes related to the skn-1 signaling pathway was significantly upregulated in the EMA treatment groups compared with the blank group. At the same time, the mRNA expression of two key autophagy genes, lgg-1 and bec-1, was also significantly improved. HSF is an evolutionarily conserved heat shock transcription factor. The HSF in Caenorhabditis elegans and the human HSF-1 gene are highly homologous. It is reported that when HSF-1 is activated, its downstream target gene hsp-16.2 will also be activated. In this study, qRT-PCR was used to determine the expression of hsp-16.2 gene in nematodes. The experimental results showed that the expression of hsp-16.2 gene in nematodes in the drug-treated groups with different concentrations increased compared with the control group. The results are as follows Fig.10 , Fig.11 shown.

Claims

1. An application of EMA in the preparation of neuroprotective or anti-aging drugs.

2. The use according to claim 1, characterized in that: The EMA is extracted from Marchantia polyphylla.

3. The use according to claim 1, characterized in that: The EMA extraction method is as follows: fresh Marchantia polyphylla is extracted with methanol at room temperature to obtain a crude extract, the crude extract is dissolved in an aqueous solution, further extracted three times with an equal volume of ethyl acetate, the extract is placed on an MCI gel column, and gradient eluted with a methanol-water system in sequence to obtain four fractions; the fraction obtained by eluting with 80% methanol is further purified on RP-HPLC using 45% MeCN as an eluent to obtain compound EMA.

4. The use according to claim 3, characterized in that: 1.1 kg of Marchantia polyphylla was extracted with methanol three times, with 10 L of methanol used each time.

5. The use according to claim 3, characterized in that: The methanol-water system was 50%, 70%, 80%, and 100% methanol; the dosage of 45% MeCN was 2.0 mL / min.

6. The use according to claim 1, characterized in that: The neuroprotection is any of the following: 1) reduce inflammation in RAW264.7 cells; or 2) improve the basic slow response of C. elegans; or 3) Improve the chemotaxis disorder of Caenorhabditis elegans.

7. The use according to claim 1, characterized in that: The delayed aging is any one of the following: 1) reducing the level of lipofuscin deposition in Caenorhabditis elegans; or 2) extend the lifespan of C. elegans; or 3) improving the anti-oxidative stress capacity of Caenorhabditis elegans; or 4) increasing the expression of the autophagy gene LGG::GFP in nematodes; or 5) increase the nuclear translocation of DAF-16 in nematodes; or 6) Up-regulate the levels of SKN-1 and downstream related genes, and up-regulate the expression of LGG1 and BEC-1 genes.

8. The use according to claim 1, characterized in that: The concentration of EMA in the preparation of neuroprotective or anti-aging drugs is 5-80 μM.

9. The use according to claim 1, characterized in that: The neuroprotective drug is used to treat or restore Alzheimer's disease and Parkinson's disease neurodegenerative diseases caused by aging.

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