Application of lupinus luteus White ketone

Abstract

The invention belongs to the technical field of medicines, and particularly provides an application of lupinus luteus White ketone. Experiments prove that lupinone has positive influence on the life of caenorhabditis elegans and related parameters (brown lipid, oxidation resistance, life and the like) of senescence physiological indexes of caenorhabditis elegans; it is prompted that lupinus hupehensis White ketone has the effects of prolonging the life and resisting aging, can play a role in delaying aging by improving the antioxidant capacity of nematodes and improving the removal level of active oxygen, and can be used for preparing the medicine for prolonging the life and delaying aging.

Description

Technical Field

[0001] The invention belongs to the technical field of medicines, and particularly relates to an application of yellow lupin white ketone. Background Art

[0002] Aging is a complex, conservative and irreversible degenerative physiological process. During the aging process, the body's physiological functions continue to decline, the overall performance continues to degenerate, and it is closely related to human diseases.

[0003] Caenorhabditis elegans (hereinafter referred to as nematodes) is a classic model organism that participates in processes such as aging, metabolism, and apoptosis. It has a high degree of homology with human genes and a short life cycle. After hermaphroditic individuals lay eggs, they go through four larval stages (L1-L4). When the population is crowded or food is insufficient, C.elegans will enter another larval stage called dauer larvae. Dauer larvae can resist adversity and do not age. They are often used in the field of aging and aging-related disease research. During the aging process, nematodes show a decline in motor and cognitive abilities, immune aging, reduced food intake, and the accumulation of harmful metabolites such as brown lipids.

[0004] The following are the commonly used drugs for prolonging life or delaying aging:

[0005] (1) Metformin: As a first-line drug for the treatment of type 2 diabetes, metformin is one of the most widely used oral hypoglycemic drugs in the world due to its excellent performance in lowering blood sugar, controlling weight, protecting cardiovascular health, and safety. In recent years, metformin has attracted attention for its anti-aging effects. Studies have shown that it can extend the lifespan of a variety of organisms and improve overall healthspan.

[0006] (2) NAD+ precursors (such as nicotinamide riboside and nicotinamide mononucleotide): NAD+ is a key coenzyme for intracellular energy metabolism, and its level decreases with age. Supplementing NAD+ precursors can increase intracellular NAD+ levels, activate longevity-related genes, promote DNA repair and metabolic regulation, and thus slow down the aging process.

[0007] (3) Rapamycin and trametinib: These two drugs inhibit the mTORC1 and Ras signaling pathways, promote the autophagy process, and remove damaged proteins and organelles in cells, thereby delaying aging. When used in combination, they can significantly prolong the lifespan of mice and improve the health status of elderly mice.

[0008] (4) Probiotics: They can regulate the intestinal microbiota, enhance the host’s immune function, and reduce chronic inflammation, thereby delaying the aging process.

[0009] (5) GLP-1 receptor agonists: They stimulate insulin secretion, inhibit glucagon release, delay gastric emptying, and increase satiety by simulating the effects of incretin, thereby achieving the effects of lowering blood sugar and losing weight. Studies have found that GLP-1 inhibitors also have anti-aging effects, can improve mitochondrial function, protect the cardiovascular system, and delay cognitive decline.

[0010] (6) Spermidine: A polyamine compound that exists naturally in all biological cells. It delays aging and prolongs life by promoting the autophagy process.

[0011] (7) Drugs that eliminate senescent cells (such as Senolytics): They slow down the aging process by selectively killing senescent cells that stop dividing but do not die.

[0012] (8) Anti-inflammatory drugs: They may play an important role in anti-aging by reducing chronic inflammation.

[0013] The shortcomings and challenges of these drugs are as follows:

[0014] (1) Safety issues: Although some anti-aging drugs, such as rapamycin and trametinib, are FDA-approved drugs, their long-term safety and potential side effects still require further study.

[0015] (2) Uncertainty of effects: Although some drugs have shown significant anti-aging effects in animal models, their effects and long-term effects in humans are still unclear.

[0016] (3) Individual differences: Different individuals may have different responses to drugs, which increases the difficulty of drug development and application.

[0017] (4) Mechanism complexity: Aging is a complex process involving multiple factors and pathways, and it may be difficult to fully intervene with a single drug.

[0018] Lupiwighteone (Lup) is a natural product with anti-tumor activity, which is mainly isolated from the seeds and pods of white lupine, the leaves of licorice and the roots of broad bean, and belongs to the isoflavones. Isoflavones belong to flavonoids, which have a variety of biological and clinical effects, and have good preventive and therapeutic effects on many diseases, such as protecting cardiovascular, anti-cancer, anti-oxidation, and improving female aging syndrome. There is no literature report on the effect of lupiwighteone on neurodegenerative diseases and aging. Summary of the invention

[0019] In view of the above problems, the purpose of the present invention is to provide a use of yellow lupin white ketone in the treatment of neuroprotection or anti-aging drugs. The pharmacological effect of yellow lupin white ketone is obvious, and it has a significant effect of restoring Caenorhabditis elegans neuronal damage and prolonging the lifespan of Caenorhabditis elegans, and it is safe and has no toxic side effects on Caenorhabditis elegans.

[0020] The structural formula of yellow lupin white ketone is shown below:

[0021] Its preparation route is shown below:

[0022]

[0023] The present invention uses experimental means to investigate the protective effect of yellow lupin white ketone on naturally aged Caenorhabditis elegans. Yellow lupin white ketone can dose-dependently prolong the average lifespan of wild-type (N2) nematodes, enhance the oxidative stress resistance of N2 nematodes, downregulate the accumulation level of brown lipids in N2 nematodes, and improve the ROS scavenging ability, thereby achieving an antioxidant effect. This lays a pharmacological foundation for yellow lupin white ketone to become a drug for preventing or prolonging lifespan or delaying aging.

[0024] Based on the above new properties of yellow lupin white ketone, the present invention determines the new use of using it to prepare drugs for delaying aging and prolonging life, which has good therapeutic effects and good prospect value. The details are as follows:

[0025] (a) Increased head shaking level of healthy nematode N2;

[0026] (b) Extended the lifespan of healthy nematode N2;

[0027] (c) Increased egg production of healthy nematode N2;

[0028] (d) Improved the anti-oxidative stress capacity of healthy nematode N2;

[0029] (e) Reduced autofluorescence accumulation in healthy nematodes N2;

[0030] (f) Increased nuclear translocation of daf-16 transgenic nematode TJ356;

[0031] (g) Reduced the fluorescence intensity in the healthy nematode N2 nematode induced by H2O2 (25 mM);

[0032] The concentration range of yellow lupin white ketone for preparing drugs for extending life span or delaying aging is 0.75 μM-3.0 μM.

[0033] The medicine can be in any dosage form of capsules, tablets, granules, powders, oral liquids, and pills.

[0034] The present invention has the following beneficial effects:

[0035] (1) The present invention uses the model organism Caenorhabditis elegans as a model to simulate natural aging and study the effects of the flavonoid compound Lup on related indicators accompanying the aging process, thereby revealing that Lup may improve and regulate aging-related diseases.

[0036] (2) The present invention screens out the optimal concentration of Lup for the study of neuroprotection or anti-aging effects based on nematode egg-laying, head shaking and lifespan experiments, verifies the neuroprotective effect of Lup in nematodes through head shaking experiments and chemotaxis experiments; verifies the anti-stress effect of Lup in nematodes through oxidative stress experiments; verifies the potential pathway of Lup's action through nuclear translocation experiments; verifies through the determination of ROS that Lup clears excessive ROS in nematodes, improves the oxidative damage state, and thus prolongs the lifespan of nematodes, providing a strong theoretical basis for the research and development of Lup in neuroprotection and / or anti-aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Effects of yellow lupin white ketone on the head wiggling level of N2 nematodes.

[0038] Figure 2 Extension of the average lifespan of N2 nematodes by yellow lupin white ketone.

[0039] Figure 3 Effects of yellow lupin white ketone on the reproductive capacity of N2 nematodes.

[0040] Figure 4 Effects of yellow lupin white ketone on the accumulation level of brown lipids in N2 nematodes.

[0041] Figure 5 Effects of lupinus whitnerone on the antioxidant capacity of N2 nematodes.

[0042] Figure 6 Effects of lupinus whitnerone on ROS levels in N2 nematodes.

[0043] Figure 7 Effects of yellow lupin white ketone on nuclear translocation in TJ356 nematodes. DETAILED DESCRIPTION

[0044] 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.

[0045] Example 1 Preparation process of yellow lupin white ketone

[0046] First, 8 g of genistein was dissolved in 150 mL of DMF, 7 mL of acetic anhydride was added, and the mixture was refluxed for 3 hours, and then detected by TLC. After the reaction, a white solid was filtered out, washed with water, and then dried to obtain product a; 1 g of product a was dissolved in 50 mL of dry DMF, 3.5 g of potassium carbonate and 1.6 mL of bromoisoprene were added, and the mixture was reacted at room temperature for 1 hour, and then detected by TLC. After the reaction, the solvent was evaporated under reduced pressure, washed with petroleum ether, and then recrystallized with acetone 3 times, and dried to obtain product b; 0.5 g of product b was dissolved in 40 mL of N,N-diethylaniline was refluxed for 1 hour and then detected by TLC. After the reaction was completed, the reaction solution was extracted with ethyl acetate, then washed with saturated ammonium chloride solution until neutral, washed with water, dried, and the solution was poured out and the solvent was evaporated under reduced pressure to obtain a white solid. Methanol was added to dissolve the white solid, and excess sodium hydroxide was added. The mixture was stirred at room temperature for 2 hours and then detected by TLC. After the reaction was completed, the solvent was evaporated to obtain a brown oily substance. Petroleum ether: ethyl acetate = 30:1 was purified by column to obtain a light yellow solid yellow lupine white ketone.

[0047] Example 2 Effect of Yellow Lupin Whitetone on Caenorhabditis elegans

[0048] 1. Biomaterials

[0049] (1) Caenorhabditis elegans strains N2 and TJ356 were purchased from Caenorhabditis Genetics Center (CGC).

[0050] (2) Escherichia coli OP50 (uracil leakage mutant) as food for Caenorhabditis elegans.

[0051] 2. Reagents

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 3. Implementation steps

[0056] (1) Cultivation of Escherichia coli OP50

[0057] In the clean bench, take 10 μL of frozen E. coli OP50 bacterial solution, add it to 100 mL of LB liquid culture medium that has been cooled to room temperature after wet heat sterilization, and culture it overnight in a 37°C shaker at 180 rpm. Take it out and store it at 4°C, which can be used for one month.

[0058] (2) Preparation of NGM growth medium for nematodes

[0059] After NGM cools and solidifies, light an alcohol lamp, add 200 μL of OP50 as food for nematodes to each dish next to the alcohol lamp, and spread it evenly. After OP50 is blown dry, place it in a 37°C constant temperature incubator for overnight incubation, take it out and place it in a constant temperature incubator below 15°C for later use.

[0060] (3) Preparation of drug-containing nematode NGM culture medium

[0061] After wet heat sterilization, the temperature of NGM dropped to about 70°C. Add MgSO4 solution, CaCl2 solution and 5μg / mL cholesterol solution with a corresponding final concentration of 1mM in the clean bench, mix well, and then add 0.75μM, 1.5μM, and 3.0μM yellow lupine white ketone, mix well and pour into the glass culture dish. After standing for NGM to cool and solidify, add 450μL OP50 to each dish as food for nematodes and spread it evenly. After OP50 is blown dry, place it in a 37°C constant temperature incubator and culture it overnight, take it out and place it in a constant temperature incubator below 15°C for storage.

[0062] (4) Synchronization of nematodes

[0063] 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 15mL 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 for culture.

[0064] (5) Cryopreservation of nematodes

[0065] Take the starved Dauer stage nematodes from three culture dishes, wash them with S Buffer, and divide them into 3 cryopreservation tubes. Wash them three times, add an equal volume of cryopreservation solution to each cryopreservation tube to a final volume of 0.5 mL, and mix them evenly. Label the name of the C. elegans strain and the freezing date on the cryopreservation tube, put it in a cryopreservation box, place it at -80℃ for at least 24 hours, and then transfer it to liquid nitrogen for storage.

[0066] (6) Nematode resuscitation culture

[0067] Take out the cryopreserved tube of the target nematode from liquid nitrogen, thaw it quickly and pour it on NGM solid medium, blow dry it and place it in a constant temperature incubator. When the revived nematodes are found, pick them into new NGM solid medium for large-scale culture under a sterile environment.

[0068] (7) Basic slow response experiment of N2 strain nematodes

[0069] The N2 strain nematodes were synchronized and then cultured at 16°C. The L1 larvae were transferred to the NGM medium of each group. The N2 strain nematodes were divided into a blank group (without drug) and groups with different drug concentrations. They were cultured at 16°C until adulthood, and 15 nematodes were selected from each group to measure the number of head swings of each group within 30 seconds.

[0070] The experimental results showed that when the Lup concentration was greater than 3 μM, the behavior of the nematodes decreased significantly after 1 day of exposure, and the reaction was slower than that of the blank group. When the Lup concentration was less than 0.75 μM, the behavior of the nematodes 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 nematodes to varying degrees, increased the motor ability, and increased the average number of swings within 30 seconds, and were concentration-dependent. Therefore, the final Lup concentrations were determined to be 0.75 μM, 1.5 μM, and 3.0 μM. The experimental results are shown in Figure 1 .

[0071] (8) N2 life test

[0072] A control group and a gradient concentration group of yellow lupin white ketone were set up, and the concentrations of yellow lupin white ketone were 0.75μM, 1.5μM, and 3.0μM, respectively. Pick the N2 nematodes synchronized to the L4 stage and put them into the culture medium of each group, each group containing 60 worms, and place them in an incubator at 20℃. The survival time of the nematodes was counted from the date of transfer, and the day of transfer was recorded as the 0th day of the life span experiment. Check the nematodes every other day, record the number of dead nematodes, and eliminate accidental deaths, escapes, and bag-like nematodes. The criterion for judging the death of nematodes is that the platinum wire touches the worm body lightly without any reaction, which can be judged as death.

[0073] The lifespan curve shows that the average lifespan of nematodes in the Lup administration groups of different concentrations is higher than that in the blank control group, and as the Lup administration concentration increases, the lifespan of nematodes gradually increases in a concentration-dependent manner. This indicates that Lup can effectively delay the aging of nematodes and prolong their lifespan. Figure 2 .

[0074] (9) Analysis of N2 nematode egg-laying experiment

[0075] Randomly select one synchronized wild-type N2 nematode at the L4 stage and place it in the drug-treated group and the control group, with 5 culture dishes in each group. This was recorded as the first day of egg laying. Every 24 hours, the nematodes were selected and placed in new culture medium. The number of eggs (egg laying amount) was recorded every day for 5 consecutive days. The experiment was repeated 3 times. The data were sorted and graphed.

[0076] 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 Lup treatment groups with different concentrations is higher than that in the blank control group, indicating that Lup has the potential to delay nematode aging and prolong its reproductive capacity. Figure 3 .

[0077] (10) Determination of brown lipid accumulation in nematodes

[0078] Endogenous fluorescent particles in Caenorhabditis elegans include brown lipids, and the accumulation of brown lipids (oxidative byproducts of lysosomal degradation) is a widely recognized marker of aging. Synchronized nematodes, grown to the L4 stage, were treated with 0.75μM, 1.5μM, and 3.0μM yellow lupine wytrol for 10 days, with a control set, and then fixed on an agarose pad, anesthetized with sodium azide, and the autofluorescence of the nematode intestine was observed under a laser confocal microscope, and the fluorescence intensity was statistically analyzed by ImageJ.

[0079] 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 Lup can significantly reduce the lipofuscin content in the nematode and delay the aging of the nematode. Figure 4 .

[0080] (11) Oxidative stress measurement

[0081] L4 stage N2 nematodes were incubated on NGM plates with or without yellow lupine whittone at 20°C for 3 days. For the oxidative stress assay, N2 nematodes were placed on fresh NGM plates containing 25 mM H2O2, and the survival rate of N2 nematodes was recorded every hour until all worms died.

[0082] 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 Lup treatment groups at different concentrations is higher than that in the blank control group, and is concentration-dependent. Figure 5 .

[0083] (12) Detection of ROS levels

[0084] L4 stage N2 nematodes were incubated with or without yellow lupin white ketone at 20°C for 3d and then placed on new NGM plates coated with H2O2 (25mM) for 5h. Thirty treated N2 nematodes were randomly selected and transferred to a black 96-well plate containing 50μM H2DCF-DA and incubated at 20°C for 2.5h in the dark. Fluorescence microscopy (IX73, Olympus, Japan) was used to quantify ROS levels.

[0085] The experimental results showed that H2O2 (25 mM) significantly enhanced the fluorescence intensity of the worms, indicating that the intracellular ROS level increased, but the fluorescence intensity increased by H2O2 was gradually reduced by yellow lupine white ketone. Figure 6 .

[0086] (13) Fluorescence analysis of nuclear translocation of daf-16 in TJ356 nematodes

[0087] This experiment uses TJ356 transgenic nematodes expressing DAF-16::GFP green fluorescence in vivo. A large number of L2 stage nematodes were obtained by synchronization. After 3 days of drug administration, the subcellular localization of green fluorescence in the nematodes was observed under a microscope, and the nematodes were classified into: cytoplasm, intermediate body, and nucleus according to the fluorescence localization. The nuclear entry of transcriptional regulatory factors affects the extension of life span. Therefore, this experiment aims to detect the effect of Lup on the nuclear entry of daf-16 transcriptional regulatory factors. The specific operation is as follows: L4 stage nematodes are rinsed with M9 buffer and added to NGM plates with different drug concentrations or without drugs. After culturing for 2 days, 2-3 adult nematodes are picked and placed on a slide with an agar pad (5% agar solution is dropped in the center of the slide, flattened with another slide, and the upper slide is removed when used), and the coverslip is covered. The nuclear translocation of daf-16 in the nematodes is observed under an inverted microscope. Three parallel groups are set up for each experiment.

[0088] The experimental results show that TJ356 nematodes express the DAF-16::GFP gene. When nuclear translocation occurs in the body, the daf-16 gene in the nematode will transfer 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. Lup can significantly increase the nuclear translocation of daf-16 in TJ356 nematodes in a concentration-dependent manner. The experimental results are as follows Figure 7 。

Claims

1. An application of yellow lupin white ketone, characterized in that, The yellow lupin white ketone is used for preparing medicine for prolonging life or delaying aging.

2. The use of yellow lupin white ketone as claimed in claim 1, characterized in that The structural formula of the yellow lupin white ketone is shown below:

3. The use of yellow lupin white ketone as claimed in claim 1, characterized in that The yellow lupine white ketone increases the head swing level of the healthy nematode N2, prolongs the life span of the healthy nematode N2, increases the egg-laying amount of the healthy nematode N2, improves the anti-oxidative stress ability of the healthy nematode N2, reduces the spontaneous fluorescence accumulation in the healthy nematode N2 nematode, increases the nuclear translocation of the daf-16 transgenic nematode TJ356, and reduces the fluorescence intensity in the healthy nematode N2 nematode induced by H2O2.

4. The use of yellow lupin white ketone as claimed in claim 1, characterized in that The method for preparing the yellow lupin white ketone comprises the following steps: using genistein as a raw material and preparing the yellow lupin white ketone through four-step reactions.

5. The use of yellow lupin white ketone as claimed in claim 1, characterized in that The concentration range of the yellow lupin white ketone used for preparing drugs for prolonging life or delaying aging is 0.75 μM-3.0 μM.

6. The use of yellow lupin white ketone as claimed in claim 1, characterized in that The medicine is in any dosage form of capsule, tablet, granule, powder, oral liquid or pill.

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