KR20200091662A - Composition for lifespan extension, anti-ageing, or preventing, treating or improving of diseases caused by lipofuscin accumulation comprising Damaurone D or salt thereof as an active ingredient - Google Patents

Abstract

The present invention relates to a composition that can be used for prolonging the lifespan of humans or animals or delaying aging by containing damauron D or a salt thereof as an active ingredient, and a composition that can be used for preventing, treating, or improving diseases caused by accumulation of lipofuscin. The composition according to the present invention contains damauron D or a salt thereof as an active ingredient, thereby increasing resistance to heat, osmotic pressure and oxidative stress, and controlling aging-related factors to prevent developmental disorders. Ultimately, it is possible to prolong the lifespan of humans and other animals, or delay aging. In addition, the composition according to the present invention can inhibit the production of lipofuscin in the body, so that diseases caused by lipofuscin accumulation can be effectively prevented, treated or improved.

Description

Composition for lifespan extension, anti-ageing, or preventing, treating or improving of the disease caused by prolongation of life, delay of aging, or accumulation of lipofuscin, containing daumaron D or a salt thereof as an active ingredient diseases caused by lipofuscin accumulation comprising Damaurone D or salt thereof as an active ingredient}

The present invention is a composition that can be used for the purpose of prolonging the life of a human or animal or delaying aging by containing daumaron D or a salt thereof as an active ingredient, and preventing, treating, or treating diseases caused by lipofuscin accumulation. It relates to a composition that can be used for improvement purposes.

Aging can be defined as the accumulation of irreversible damage to cells or organs that increases the risk of disease or death. The precise mechanism of aging has not been clearly identified, as aging-related factors interact in a complex way. Nevertheless, research on aging has been actively and extensively conducted, and many people have focused on developing medicines and dietary supplements that delay and prevent aging and degenerative diseases. Previous research has shown that many natural products, such as blueberries, green tea, and onions, can be candidates for anti-aging drugs or functional foods.

Rosa × damascene Damask rose, a hybrid rose, has been used as a raw material for cosmetics, spices, and traditional medicine for the treatment of cramps, menstruation, and dyspepsia. Various pharmacological effects of this plant have been reported, including antioxidant, antibacterial, anticancer, antidepressant, anti-inflammatory and analgesic. Recently, an isoprenylated aurone compound called Damaurone D was isolated from the flower of this plant. Likewise, this daumaron D also has a unique therapeutic potential due to the presence of the chromane structure, the preferred structure in pharmaceutical chemistry (see FIG. 1). Although anti-inflammatory activity has been reported in vitro and in vivo through pharmacological studies on this plant-derived chemical, its effect on longevity has not been reported so far.

The present inventors tried to develop a material that is effective in prolonging life and delaying aging from natural products using a pretty nematode animal model, and studied using various natural products. As a result of this, it is confirmed that the above-described component of the damask rose, Damauron D, has an excellent effect on prolonging life and delaying aging in a real animal model, and can also effectively suppress the production of lipofuscin and complete the present invention. Became.

Curr Opin Neurol 1994; 7: 283-286. Gerontologist 2007; 47: 150-158. Planta Med 2009; 75: 216. J Agric Food Chem 2011; 59: 5927-5934. Fitoterapia 2003; 74: 394-396. Neuropsychiatr Dis Treat 2015; 11: 625-635. Iran J Pharm Res 2010; 9: 163-168. Food Chem Toxicol 2009; 47: 361-367. Asian Pac J Trop Dis 2014; 7: S294-S300. Helv Chim Acta 2014; 97: 414-419. Chem Pharm Bull 2015; 63: 907-912. Arch Pharm Res 2018; 41: 314-323. WormBook 2007; 12: 1-12. Nature 2007; 447: 536-537. Pharmacol Biochem Behav 2006; 85: 620-628.

The main object of the present invention is to provide a composition that is less likely to cause side effects by containing components derived from natural products, and can exhibit a life-span or delayed aging effect in humans or other animals.

Another object of the present invention is to provide a composition capable of effectively preventing, treating or improving a disease caused by lipofussin accumulation.

According to one aspect of the present invention, the present invention provides a food composition for prolonging life or delaying aging in humans containing Damaurone D or a salt thereof as an active ingredient.

According to another aspect of the present invention, the present invention provides a feed composition for prolonging life or delaying aging of livestock containing Damaurone D or a salt thereof as an active ingredient.

According to another aspect of the present invention, the present invention provides a pharmaceutical composition for the prevention or treatment of diseases caused by accumulation of lipofuscin containing Damaurone D or a salt thereof as an active ingredient.

According to another aspect of the present invention, the present invention provides a food composition for preventing or improving a disease caused by accumulation of lipofuscin containing Damaurone D or a salt thereof as an active ingredient.

According to another aspect of the present invention, the present invention provides a feed composition for preventing or improving a disease caused by accumulation of lipofuscin containing Damaurone D or a salt thereof as an active ingredient.

The composition of the present invention increases the resistance to heat, osmotic pressure, and oxidative stress by containing Tamaron D or a salt thereof as an active ingredient, and regulates aging-related factors to ultimately reduce the lifespan of humans and other animals without causing developmental disorders. It can prolong or delay aging. In addition, the composition of the present invention can inhibit the production of lipofuscin in the body, thereby effectively preventing, treating or improving diseases caused by lipofussin accumulation.

Figure 1 shows the chemical structure of the active ingredient of Daumaron D of the composition of the present invention.
Figure 2 shows the results of investigating the effects of dauron D on the lifespan of a pretty nematode under normal and stress conditions. (a), survival graph of wild-type nematode nematodes according to treatment by concentration of Damauron D under normal conditions; (b), survival graph of wild-type nematode nematodes according to treatment by concentration of Damauron D under heat stress conditions; (c), survival graph of osr-1 mutation according to treatment by concentration of daumaron D under osmotic stress conditions; (d), life expectancy graph of wild-type nematode nematodes according to treatment by concentration of Damauron D under normal conditions (calculated from the survival graph of (a)); (e), survival graph of wild-type nematode nematodes according to treatment by concentration of Damauron D under osmotic stress conditions; (f) and (g), HSP-16.2::GFP expression pattern of HSP-16.2::GFP transforming mutants following treatment of daumaron D under normal and heat stress conditions; *, P<0.05 means significant compared to the control group; **, P<0.01 means significant compared to the control group; ***, P<0.001 means that it is significant compared to the control group.
Figure 3 shows the results of examining the antioxidant power of Damauron D in the nematode nematode. (a), survival graph of wild-type nematode nematodes according to treatment by concentration of Damauron D under oxidative stress conditions; (b), intracellular ROS level of wild-type nematode nematodes according to treatment by concentration of dauron D; (c) and (d), antioxidant enzymes of nematode lysate (SOD, (c); catalase, (d)) activity; (e) and (f), SOD-3::GFP expression pattern of SOD-3::GFP transformed mutants following treatment with dauron D; **, P<0.01 means significant compared to the control group; ***, P<0.001 means that it is significant compared to the control group.
Figure 4 shows the results of examining the effect of dauron D on aging-related factors. (a), the number of pharynx pumping of nematodes per minute according to the treatment by concentration of daumaron D; (b), body length according to the adult period of nematodes according to treatment by concentration of Damaron D; (c) and (d), lipofuscin production in nematodes following treatment by concentration of daumaron D; (e), body movements according to the adult period following the treatment of damauron D; *, P<0.05 means significant compared to the control group; **, P<0.01 means significant compared to the control group; ***, P<0.001 means that it is significant compared to the control group.
Figure 5 shows the results of examining the genes associated with the effect of extending the life of Damaron D. (a), survival graph of daf-2 knockout nematodes following treatment with dauron D; (b), survival graph of age-1 knockout nematodes following treatment with daumaron D; (c), survival graph of daf-16 knockout nematodes following treatment with dauron D; (d), daf-16::GFP expression pattern of daf-16::GFP transformed mutants following treatment with dauron D.

The present invention was devised based on the newly revealed physiological activity of Damaurone D.

The structure of dauron D used as an active ingredient in the present invention is shown in FIG. 1, obtained by a method such as purification from a damask rose, or a method proposed by Han et al. ( Chem Pharm Bull 2015; 63: 907-912) It can be obtained by synthesis.

According to the present invention, Damauron D was found to actually prolong life in experiments using the Caenorhabditis elegans animal model, and was investigated to increase survival rates under heat, osmotic and oxidative stress conditions. The effect of extending the life of Damauron D seems to be exerted through the regulation of aging-related factors including antioxidant, food intake and growth. In addition, it has been shown that Damaron D can not only extend lifespan, but also improve athletic performance and reduce lipofuscin accumulation, thereby delaying aging. In addition, it was investigated that Damauron D exhibits the above longevity effect through the regulation of daf-16, an important transcription factor of longevity.

The pretty little nematode is a famous animal model that is widely used to study the aging and lifespan of animals, and these animals are highly conserved with genes related to human longevity. Therefore, the physiological activity of the above-described daumaron D, which was investigated using this pretty little nematode animal model, could be exerted in higher animals or humans.

Based on the newly revealed physiological activity of the above-mentioned daumaron D, in the present invention, a food composition for prolonging or delaying aging of a human being containing daumaron D or a salt thereof as an active ingredient, and a feed for prolonging or delaying aging of livestock Provided is a composition.

The present invention also prevents, treats or ameliorates pharmaceutical compositions, food compositions and feeds for diseases caused by lipofussin accumulation that contains daumaron D or a salt thereof as an active ingredient based on the newly revealed activity of inhibiting lipofuscin production of daumaron D. Provided is a composition.

Lipofucin is well-known as an aging pigment, so it is not only used as an indicator of aging, but also the accumulation of this lipofucin is well known as a major risk factor for macular degeneration, degenerative diseases of the eyes, and Starkart's disease. In addition, lipofusinosis is a disease caused by abnormal accumulation of lipofuscin and is deeply related to neurodegenerative disorders. As investigated in the present invention, it is possible to effectively prevent, treat, or improve the disease caused by accumulation of lipofuscin as described above, since daumaron D can inhibit the production of such lipofuscin.

The content of the active ingredient of Damauron D or a salt thereof in the pharmaceutical composition of the present invention can be applied in various ranges depending on the use or the method of administration, but is typically 0.1 of Damauron D or a salt thereof based on the total weight of the pharmaceutical composition of the present invention. To 90% by weight.

The pharmaceutical composition of the present invention can be administered orally or parenterally during clinical administration, and when administered parenterally, intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine epidural injection, intracranial injection, or It can be administered by intrathoracic injection and may be used in the form of a generic pharmaceutical formulation.

The pharmaceutical composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormonal therapy, chemotherapy, and biological response modifiers.

The daily dosage of the pharmaceutical composition of the present invention may be about 0.1 to 100 mg/kg per day based on Damaron D contained in the composition, and may be administered once or several times a day, but the patient's weight, age, gender, The range will vary depending on health condition, diet, administration time, administration method, excretion rate and disease severity.

In actual clinical administration, it can be administered in various parenteral formulations. When formulated, it may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc., which are usually used. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. As a non-aqueous solvent and a suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerogelatin may be used.

The pharmaceutical composition of the present invention may contain one or more active ingredients exhibiting the same or similar function in addition to daumaron D or a salt thereof.

In the food composition of the present invention, the food products include meat products, fish meat products, tofu, jelly, porridge, noodles, noodles such as ramen or noodles, soy sauce, miso, red pepper paste, seasoned foods such as red pepper paste, sauces, cookies, fermented milk or cheese, etc. Dairy products, kimchi or pickled foods such as pickles, fruits, vegetables, soy milk, fermented beverages, etc. may be beverages, and may be prepared by adding the dauron D or a salt thereof during the process of manufacturing these foods. At this time, the amount of addition of Damaron D or a salt thereof may be appropriately selected in consideration of the type, shape, purpose, and manufacturing method of the food, for example, 0.0001 to 10% by weight based on the final food weight, preferably 0.001 to It may be 5% by weight, more preferably 0.01 to 1% by weight.

In the feed composition of the present invention, the feed may be prepared in the form of blended feed, pellet form, and silage. The blended feed may be prepared by mixing several types of general feed with the dauron D or a salt thereof. The feed in the form of pellets may be prepared by formulating the blended feed with a pellet, and silage may be prepared by fermenting a cultivar feed and then adding the dauron D or a salt thereof. At this time, the amount of the addition of Damaron D or a salt thereof may be appropriately selected in consideration of the type, form, purpose, and manufacturing method of the feed, and the amount of addition as in the food may be used.

Hereinafter, the present invention will be described in more detail through examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

[Example]

1. Method

1-1. Synthesis of Tamaron D

Damauron D (hereinafter abbreviated as'DaD') was synthesized in 5 steps without protection-deprotection, and the structure was confirmed through 2D-NMR analysis including HSQC and HMBC. The specific procedure is the same as that of Han et al. ( Chem Pharm Bull 2015; 63: 907-912).

The synthesized DaD was stored at -20°C until use, dissolved in DMSO, diluted with M9 buffer, and brought to an appropriate concentration.

1-2. Nematode culture and treatment of daumaron D

Caenorhabditis elegans strains and Escherichia coli including Bristol N2 (wild-type), CF1553 (muIs84), CL2070 (dvls70), AM1 (rm1), DR1572 (e1368), GR1307 (mgDf50), TJ1052 (hx546), TJ356 (zIs356) OP50 was obtained from CGC (Caenorhabditis Genetic Center; University of Minnesota, Minneapolis, MN). Nematodes were incubated with E. coli OP50 (OD600 ≒ 0.7) from agar plates (nematode growth medium) NGM (Genetics 1974; 77: 71-94). DaD plates were prepared by adding DaD stock solution to sterile NGM plates (at 50°C). The final DMSO concentration was maintained at 0.1% (v/v) under all conditions. In addition, FUDR was contained in the plate at a concentration of 20 μM to block the generation of progeny. Nematodes with integrated age were obtained by embryo separation. L1 nematodes were transferred to NGM plates (with or without DaD) and incubated for several days or weeks at 20°C. More than 80 subjects were used for each experiment.

1-3. Life time analysis and stress tolerance analysis

Lifespan analysis was performed by a slightly modified method in the method of Lu L et al. ( J Biosci Bioeng 2017; 124: 1-7). Test nematodes were considered dead when they did not respond to stabbing with a platinum wire tip. All nematodes were transferred to fresh NGM plates every 3 days. All stress tolerance analyzes were performed on day 4 of adulthood. For thermal tolerance analysis, nematodes were incubated at 36° C. and then survival rates were recorded ( Free Radic Res 2017; 51: 529-544). Resistance to osmotic pressure and oxidative stress was measured by placing nematodes on NGM agar plates containing 500 mM NaCl or 200 mM paraquat ( Biochem Biophys Res Commun 2009; 390: 1402-1407, J Med Chem 2012; 55: 4169- 4177).

1-4. Measurement of antioxidant enzyme activity

To assess enzyme activity, nematode lysates were prepared. Wild type nematodes were collected on day 4 of adult with M9 buffer and washed 3 times. The collected nematodes were then resuspended in homogenization buffer (10 mM Tris-HCl, 150 mM NaCl, 0.1 mM EDTA and pH 7.5) and homogenized on ice.

SOD activity was measured by analyzing the bleaching of formazan with a spectrophotometer using the enzymatic reaction between xanthine and xanthine oxidase. Catalase activity was calculated with a spectrophotometer, such as Aebi H et al. ( Methods Enzymol 1984; 105: 121-126).

1-5. Intracellular ROS analysis

The nematode's intracellular reactive oxygen species (ROS) was measured using a molecular probe H ₂ DCF-DA ( J Biosci Bioeng 2017; 124: 1-7). On the 4th day of adulthood, nematodes were cultured in NGM agar plates containing 60 mM paraquat for 2 hours. Nematodes were then transferred to wells of a 96-well plate containing 50 μl of M9 buffer. Immediately after the final concentration was adjusted to 25 μM by adding 50 μM H ₂ DCF-DA solution to 50 μM, the basal fluorescence was quantified using a microplate fluorescence reader (Glomax-Multi+; Promega, WI, USA) at 490 nm and 510-570 nm emission. Did.

1-6. Measurement of aging-related factors

The nematodes were transferred to fresh medium for pharyngeal pumping analysis and pharyngeal contraction was measured under a microscope for 1 minute ( Proc Natl Acad Sci USA 2004; 101: 8084-8089). For analysis of growth changes, nematodes were photographed and the length of each nematode was analyzed with Nikon software (NIS-Elements; Nikon, Tokyo, Japan) ( Front Aging Neurosci 2015; 7: 220). To confirm the movement, the nematodes were transferred to a normal NGM plate and the movement distance was measured using a microscope for 1 minute ( Proc Natl Acad Sci USA 2004; 101: 8084-8089). To determine lipofuscin levels in the intestine, 15 nematodes were mounted on a 2% agarose pad on the 15th day of adulthood and fluorescence intensity was quantified ( Free Radic Res 2017; 51: 529-544).

1-7. Fluorescence microscopy analysis and visualization

Prior to microscopic observation, nematodes were anesthetized with sodium azide (2%, w/v) and placed on an agarose gel pad (2%, w/v). Fluorescence signals from nematodes were directly observed under a fluorescence microscope (SMZ1500; Nikon, Japan). The fluorescence intensity was analyzed by taking pictures using Nikon software (NIS-Elements, Nikon, Japan) and determining the average pixel intensity using Image J software.

1-8. Statistical analysis

Data obtained from life analysis were plotted using Kaplan-Meier analysis and statistical significance was analyzed by log-rank test. Other data are expressed as mean±standard deviation. Statistical significance for the difference between the control and treatment groups was analyzed by one-way ANOVA.

2. Results

2-1. Effects on the Lifespan of Pretty Little Nematodes under Normal and Stress Conditions

To determine the effect of Damauron D (hereinafter abbreviated as'DaD') on lifespan, lifespan analysis was performed using wild type N2 nematodes under normal culture conditions. As a result of this, as shown in FIG. 2A, DaD was found to prolong the nematode life in a concentration-dependent manner. At 5, 10, and 15 μM, the average lifespan extensions were 7.49%, 12.60%, and 16.70%, respectively (see d in FIG. 2 ). The maximum life was increased up to 7 days in the presence of DaD (15 μM). In addition, DaD (15 μM) was shown to improve nematode survival rates by about 24.68% and 43.22%, respectively, under heat and osmotic stress conditions (see b and e in FIG. 2).

In addition, the osr-1 null mutant was used to investigate whether osr-1 is required for the protective effect of DaD under osmotic stress conditions. As shown in FIG. There were no significant differences. This means that the osr-1 gene is involved in enhancing osmotic resistance by DaD .

In order to investigate whether DaD influences HSP-16.2 expression, as a result of performing an experiment to quantify the fluorescence signal of HSP-16.2::GFP transforming mutant, DaD does not regulate the expression of HSP-16.2 under normal conditions. Turned out to be unable. However, when heat stimulation was applied to nematodes, the fluorescence signal was enhanced, and the expression of this condition gene was further up-regulated by DaD (see f and g in FIG. 2). These results indicate that the increased expression of hsp-16.2 gene by DaD can enhance heat resistance.

2-2. Antioxidant activity

As a result of investigating the effect of DaD on paraquat-induced oxidative stress conditions, treatment with DaD was found to increase nematode survival at all concentrations (see FIG. 3A ). As a result of quantifying the intracellular ROS level in nematodes using fluorescent probe H ₂ DCF-DA to investigate whether the protective effect of DaD is due to a decrease in ROS formation, nematodes administered DaD are dose-dependent compared to the control group. As a result, it was found that the ROS level in the cells was significantly decreased (see b in FIG. 3).

As a result of investigating the effect on the enzymatic antioxidant using nematode lysate, it was found that the superoxide dismutase (SOD) and catalase activity were significantly increased by DaD (see c and d in FIG. 3).

In addition, as a result of investigating the effect of DaD on SOD-3 expression through a method of quantifying a fluorescence signal from the SOD-3::GFP transformed mutant, DaD treatment as shown in FIGS. It was found to increase the expression of about 10.42% compared to the control.

2-3. Aging-related factors and their effect on health life

In order to evaluate the effect of DaD on dietary restriction (DR), as a result of measuring the number of pharyngeal pumping on the 4th day of adulthood, the nematode administered DaD, as shown in FIG. 4A, has a pumping frequency of 9.54% (at 5 μM) compared to the control group. , 16.42% (at 10 μM) and 21.95% (at 15 μM).

In addition, as a result of examining the length of the nematode at two different time points (first and fourth days of adult) to investigate the effects of DaD on the growth and development of nematodes, DaD induced growth restriction only on the fourth day of adulthood. It means that DaD does not cause developmental disorder (see b in Fig. 4).

In addition, the experiment showed that DaD can reduce the fluorescence intensity of lip nematode intestinal lipofuscin (33.32% at 15 μM) compared to the control group (see c and d in FIG. 4 ).

As a result of investigating the effect of DaD on the aging-dependent decrease in motility, an important parameter for measuring the life expectancy, the treatment of DaD significantly recovered the decrease in motility until the 10th day of adulthood compared to the control group (FIG. 4). E).

2-4. Target gene of longevity effect by Tamaron D

As a result of life analysis using daf-2 , age-1 , and daf-16 null mutants to investigate the target gene of DaD, it was found that the life extension effect due to DaD was extremely limited in these mutants (Fig. 5, a to c). These results indicate that these genes are required for longevity effects by DaD.

In addition, as a result of experiments using daf-16::GFP transformed mutants to investigate whether DaD regulates long-lived important transcription factor daf-16 , the treatment of DaD as shown in FIG. It has been shown to accelerate nuclear transfer.

Claims (5)

Food composition for prolonging life or delaying aging in humans containing Damaurone D or a salt thereof as an active ingredient. Damaurone (Damaurone) D or a salt thereof as an active ingredient in the life of the livestock or delayed aging feed composition. Damaurone D or a pharmaceutical composition for the prevention or treatment of diseases caused by the accumulation of lipofuscin (lipofuscin) containing the salt as an active ingredient. Food composition for preventing or improving diseases caused by accumulation of lipofuscin containing Damaurone D or a salt thereof as an active ingredient. Feed composition for the prevention or improvement of diseases caused by accumulation of lipofuscin containing Damaurone D or a salt thereof as an active ingredient.

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