CN113303476B - Application of rubidium salt and rubidium salt solution in preparation of product with prolonged service life and/or anti-aging - Google Patents

Abstract

The invention discloses the use of a rubidium salt and a rubidium salt solution for the preparation of a product with an extended life and/or an anti-ageing property. The invention confirms the correlation between rubidium salt and aging through a phenotype experiment, the rubidium salt can activate DAF-16 through an AMPK pathway to prolong the life of the caenorhabditis elegans, enhance the capabilities of resisting heat stress, ultraviolet radiation and oxidation stress, does not influence the normal vital signs and the fertility of the caenorhabditis elegans, and has no toxic or side effect on the safety of the caenorhabditis elegans. Therefore, the product has good anti-aging effect and can be applied to the preparation of products with anti-aging and life prolonging effects.

Description

Application of rubidium salt and rubidium salt solution in the preparation of life-extending and/or anti-aging products

technical field

The invention relates to the technical field of biomedicine, in particular to the application of rubidium salt and rubidium salt solution in preparing life-extending and/or anti-aging products.

Background technique

Aging is an inevitable part of the life process and can be delayed. Aging is characterized by a decreased ability to respond to stress, increased homeostasis imbalances, and an increased risk of aging-related diseases, including cancer and heart disease. Aging is defined as "the progressive deterioration of physiological function, age-related loss of vitality and increased vulnerability". Aging is a major factor leading to a substantial increase in the incidence of chronic diseases such as dementia, cardiovascular disease, stroke, chronic respiratory disease, and cancer. As a result, these diseases are also known as geriatric diseases and impose a huge burden on the health care system.

Lithium, sodium, potassium, rubidium, and cesium belong to the first main group of elements. Their elemental chemical properties are active, and their ionic chemical properties are stable. Sodium and potassium ions are the material basis for the functioning of various human systems, especially the nervous system. The living system has specialized sodium, potassium, chloride and other ion channels to maintain the operation of the living system.

Because of the difficulty of extraction, rubidium salts have relatively little research in the field of health. In 2019, ZhengxiaoOuyang et al. reported (Frontiers In Pharmacology, 2019, (10): 1–12.) that RbCl has an anti-osteoporotic effect, and the mechanism is still unclear. It is of great value and significance to conduct in-depth research on the elements of the first main group to expand their applications in the field of biomedicine and health.

SUMMARY OF THE INVENTION

The purpose of the present invention is to overcome the defects or deficiencies of the prior art, and to provide the application of rubidium salt and rubidium salt solution in the preparation of life-extending and/or anti-aging products. The present invention confirms the correlation between rubidium salts and aging through phenotypic experiments, and rubidium salts can activate DAF-16 through AMPK pathway to prolong the lifespan of Caenorhabditis elegans, and enhance anti-heat stress, anti-ultraviolet radiation and anti-oxidative responses. It does not affect the normal vital signs and fecundity of Caenorhabditis elegans, and is safe and has no toxic and side effects to the nematodes. Therefore, it can have a good anti-aging effect, and can be used in the preparation of anti-aging and life-extending products.

In order to realize the above-mentioned purpose of the present invention, the present invention provides the following technical solutions:

Use of rubidium salts and rubidium salt solutions in the preparation of life-extending and/or anti-aging products.

Caenorhabditis elegans is the main model organism for antiaging research. Based on this model, more than 800 genes were found to be associated with the regulation of lifespan in C. elegans, and compounds with anti-aging effects (eg, metformin, rapamycin) were discovered.

The hermaphroditic adult worm in C. elegans is 1 mm long, has 959 cells, and is also a post-mitotic model, with mitotic activity only in the gonads. This simplified functionality facilitates exploration of aging-related cellular degeneration. It can be passed from one generation to another in three days, allowing for faster genetic studies. C. elegans has larger propagules and can be cultured at different temperatures, so quantitative experiments on senescence can be performed and automated molecular target screening systems can be created.

The inventors of the present invention confirmed the correlation between rubidium salts and aging through phenotypic experiments. Rubidium salts can activate DAF-16 through the AMPK pathway to prolong the lifespan of Caenorhabditis elegans, enhance resistance to heat stress, anti-ultraviolet radiation, The ability to resist oxidative stress does not affect the normal vital signs and fecundity of Caenorhabditis elegans, and is safe and non-toxic to nematodes. Therefore, it can have a good anti-aging effect, and can be used in the preparation of anti-aging and life-extending products.

Specifically, rubidium salts can resist aging and prolong lifespan by at least the following ways:

(a) Enhance the ability to resist ultraviolet radiation;

(b) enhancing the ability to resist oxidative stress;

(c) Enhanced resistance to heat stress;

(d) prolong life;

(e) Activation of DAF-16 through the AMPK pathway enhances resistance and prolongs lifespan.

It should be understood that the solvent in the rubidium salt solution is water.

The prolongation of lifespan referred to in the present invention refers to prolonging the maximum length of the lifespan of an organism or delaying the time of death.

Preferably, the rubidium salt is a salt formed by a rubidium ion Rb ⁺ and a stable inorganic acid anion or an organic acid anion.

More preferably, the inorganic acid anion is one or more of chloride ion Cl ^- , sulfate SO ₄ ^2- , nitrate NO ₃ ^- , carbonate CO ₃ ^2- or acetate CH ₃ COO ^- .

More preferably, the organic acid anion is one or more of citrate, gallate or tannin.

Preferably, the drug concentration of the rubidium salt in the life-extending and/or anti-aging product is 1 μmol to 100 mmol.

Preferably, the life-extending and/or anti-aging product includes rubidium salt or rubidium salt solution, and one or more pharmaceutically acceptable excipients.

More preferably, the auxiliary materials are diluents, fillers, binders, wetting agents, absorption enhancers, surfactants, lubricants or stabilizers.

Preferably, the lifespan extension and/or anti-aging product is food, health product, feed or additives thereof.

It should be understood that the foods, health products, feeds or their additives also contain common ingredients in the technical field that are appropriately selected by those skilled in the art according to the dosage form or the purpose of use, and can be used together with other raw materials.

Preferably, the formulation form of the life-extending and/or anti-aging product is tablet, granule, capsule, powder, liquid or jelly.

Compared with the prior art, the present invention has the following advantages and effects:

The present invention confirms the correlation between rubidium salts and aging through phenotypic experiments, and rubidium salts can activate DAF-16 through AMPK pathway to prolong the lifespan of Caenorhabditis elegans, and enhance anti-heat stress, anti-ultraviolet radiation and anti-oxidative responses. It does not affect the normal vital signs and fecundity of Caenorhabditis elegans, and is safe and has no toxic and side effects to the nematodes. Therefore, it can have a good anti-aging effect, and can be used in the preparation of anti-aging and life-extending products.

Description of drawings

Fig. 1 The effect of rubidium chloride on the lifespan of wild-type C. elegans;

Fig. 2 The effect of rubidium chloride on the lifespan of C. elegans CF1038;

Figure 3 The effect of rubidium chloride on the lifespan of C. elegans RB754;

Fig. 4 The effect of rubidium chloride on the lifespan of C. elegans DA1113;

Fig. 5 The effect of rubidium chloride on the lifespan of C. elegans DR1572;

Figure 6 Rubidium sulfate has no effect on the fecundity of C. elegans;

Figure 7 The effect of rubidium acetate on the average lifespan of Caenorhabditis elegans heat stress (35°C);

Figure 8 Protective effect of rubidium acetate on heat stress (35°C) in C. elegans;

Fig. 9 The effect of rubidium nitrate on the average lifespan of C. elegans UV radiation;

Figure 10 The protective effect of rubidium nitrate on C. elegans ultraviolet radiation;

Figure 11 Protective effect of rubidium carbonate on oxidative stress in C. elegans;

Fig. 12 The effect of rubidium chloride on nuclear translocation of TJ356 C. elegans daf16;

Figure 13 Rubidium chloride activates nuclear translocation of TJ356 C. elegans daf16.

Detailed ways

The present invention is further explained below with reference to the embodiments and the accompanying drawings, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

Unless otherwise specified, the reagents and materials used in the present invention are commercially available.

The application of embodiment 1 rubidium chloride in prolonging the lifespan of Caenorhabditis elegans

(1) Strain preparation: inoculate Escherichia coli OP50 into LB liquid medium (4g peptone, 4g NaCl, 2g yeast extract, 4mL pH 8.0 1MTirs-HCl buffer, add ultrapure water to 400mL, high pressure Steam sterilization), shake culture overnight and place in a 4°C refrigerator for later use.

(2) Preparation of NGM medium: add 1.2g NaCl, 6.8g agar, 1g peptone to a 500mL conical flask, add ultrapure water to 400mL, and sterilize with high pressure steam. When the solution is cooled to about 60℃, add 400μl 1MCaCl ₂ , 400μl 5mg/mL cholesterol, 400μl 1MMgSO4, 10mL 1M KHPO ₄ buffer solution on the ultra-clean bench, stir evenly, pour into a petri dish (35mm), let stand and solidify and place at 4℃ Refrigerator spare.

(3) Seed plate: Pipette 200 μl of OP50 bacterial droplets into the center of the NGM medium with a pipette on the ultra-clean bench, shake slightly to spread the bacterial solution and let it stand (when setting the experimental group, mix the rubidium salt solution into the bacterial solution Make rubidium salt final concentrations of 100 μM and 10 mM). After the bacterial solution was dried, it was placed in a 4°C refrigerator for later use.

(4) Nematode transfer: The nematodes were picked on the NGM medium with platinum wire under a stereo microscope and cultured in a constant temperature incubator at 20°C.

(5) Nematode passage: Cut a small piece from the NGM medium that can be passaged with a knife on the ultra-clean bench, put it face down on the fresh NGM medium, and put it in a constant temperature incubator for 20 minutes after being lightly compacted. ℃ culture.

(6) Nematode synchronization: The anti-aging activity of rubidium salts was studied using N2 wild-type C. elegans (WT). In order to obtain nematodes in the same growth period, the nematodes were synchronized. Newly passaged nematodes grow to adult stages in about 2 days. Wash the surface of NGM medium with 1 mL of ultrapure water on a clean bench, transfer to a 5 mL centrifuge tube, and wash twice. A rinse solution (0.5 mL of 0.5 M sodium hydroxide, 1 mL of 4% sodium hypochlorite, and ultrapure water to 5 mL) was added. After vortexing for 10s, let stand for 2min, repeat about 5 times, until the nematodes are basically dissolved under the naked eye. Centrifuge at 800 rpm for 3 min. Discard the supernatant, add 5 mL of ultrapure water, and centrifuge at 800 rpm for 3 min. The supernatant was discarded, and the pellet was mixed by pipetting and dropped onto fresh NGM medium. After the liquid was evaporated, it was incubated in a constant temperature incubator at 20°C.

(7) Lifespan experiment: using WT wild type, CF1038(daf-16(mu86)I), DA1113(eat-2(ad1113)II), DR1572(daf-2(e1368)III), RB754(aak-2( ok524)X) and other strains for lifespan experiments. Synchronized nematodes were placed on NGM medium (blank control group (referred to as WT), 100 μM rubidium chloride, and 10 mM rubidium chloride), and this time was recorded as Day 0 (Day 0), in a constant temperature incubator for 20 Cultivated for 3 days. Adult nematodes were transferred to fresh NGM medium in corresponding groups (3 medium per group, 80 nematodes per medium). After that, the survival and death of nematodes were observed and counted every other day, and the nematodes were transferred to fresh NGM medium until all the nematodes died (when the number of nematodes surviving on the medium dropped to a certain number, the same group of nematodes on the same medium). The criteria for judging death were: C. elegans did not respond to strong light or tapping the dish, and the pharyngeal muscles did not move under the high-power microscope. Finally, the head of the nematode was hit with a pick needle. If there was still no response, it could be judged as dead.

(8) Experimental results: As shown in Figure 1 and Table 1, adding rubidium chloride (100 μM and 10 mM) to the nematode diet can significantly prolong the average lifespan of wild-type nematodes (P<0.0001). Compared with the control group, the average lifetime of 100μM rubidium chloride can be increased by 12%, and the maximum lifetime can be extended by 6%. Compared with the control group, the average lifetime of 10mM rubidium chloride can be increased by 18%, and the maximum lifetime can be extended by 6%. The average lifespan of nematodes increased with the increase of rubidium chloride concentration in a certain concentration-dependent manner. It is shown that rubidium chloride can prolong the lifespan of wild-type C. elegans and has an anti-aging effect.

As shown in Figures 2 to 5 and Tables 2 to 5, in the CF1038(daf-16(mu86)I) and RB754(aak-2(ok524)X) strains, rubidium chloride could not prolong the lifespan of nematodes, indicating that chlorination Rubidium may activate DAF-16 via the AMPK pathway to prolong lifespan in C. elegans.

Table 1 Effects of different concentrations of rubidium chloride on the lifespan of wild-type nematodes

*** represents P<0.001 compared to the WT group.

Table 2 Effects of different concentrations of rubidium chloride on the lifespan of CF1038 nematodes

*** represents P<0.001 compared to the WT group.

Table 3 Effects of different concentrations of rubidium chloride on the lifespan of RB754 nematodes

*represents P<0.05 compared to the WT group.

Table 4 Effects of different concentrations of rubidium chloride on the lifespan of DA1113 nematodes

*** represents P<0.001 compared to the WT group.

^### represents P<0.001 compared to the DA1113 group.

Table 5 Effects of different concentrations of rubidium chloride on the lifespan of DR1572 nematodes

*** represents P<0.001 compared to the WT group.

^# represents P<0.05 compared with the DR1572 group.

Embodiment 2 Rubidium sulfate has no effect on the fecundity of Caenorhabditis elegans

Nematode strain preparation, NGM medium preparation, seed plate, nematode transfer, nematode passage, and nematode synchronization methods are as described in Example 1. Synchronized nematodes were placed on NGM medium (WT, 100 μM Rb ₂ SO ₄ , 10 mM Rb ₂ SO ₄ ), which was recorded as Day 0, and cultured in a constant temperature incubator at 20° C. for 3 days. Adult nematodes were transferred to the corresponding fresh NGM medium (4 medium per group, 1 nematode per medium). Culture in a constant temperature incubator at 20°C, count the number of eggs laid by the nematodes every day, and transfer the nematodes to fresh NGM medium until the nematodes stop laying eggs.

Experimental results: In C. elegans, a decrease in nematode fertility is often observed along with an increase in lifespan. To investigate the changes in the number of eggs laid by adult nematodes after feeding with rubidium sulfate (100 μM and 10 mM). As shown in Figure 6, compared with the control group, the number of eggs laid by C. elegans did not change significantly after feeding with rubidium sulfate (100 μM and 10 mM), indicating that rubidium sulfate had no effect on the fecundity of the nematodes.

Example 3 Rubidium acetate enhances heat stress resistance in Caenorhabditis elegans

The preparation of strains, the preparation of NGM medium, the seed plate, the transfer of nematodes, the passage of nematodes, and the synchronization of nematodes were as described in Example 1. Synchronized nematodes were placed on NGM medium (WT, 100 μM CH ₃ COORb, 10 mM CH ₃ COORb), which was recorded as Day 0, and cultured in a constant temperature incubator at 20° C. for 3 days. Adult nematodes were transferred to the corresponding groups of fresh NGM medium (3 medium per group, 50 nematodes per medium) and continued to culture until Day 6. The medium was transferred to a constant temperature incubator at 35°C, and the survival and death of nematodes were observed and counted every hour.

Experimental results: While the compounds prolong the lifespan of the nematodes, they can often also enhance the worms' resistance to various environmental stresses. To investigate the changes in the tolerance of nematodes to high temperature (35°C) environment after feeding with rubidium acetate (100 μM and 10 mM). As shown in Figures 7-8 and Table 6, compared with the control group, the average lifetime of 100μM rubidium acetate can be increased by 14% and the maximum lifetime by 7% (P<0.001); the average lifetime of 10mM rubidium salt can be increased by 17% (from 8.5 ±0.5h to 10.3±0.7h), and the maximum lifespan increased by 7% (P<0.001). It is shown that rubidium acetate can significantly enhance the resistance of nematodes to acute heat stress.

Table 6 Protective effects of different concentrations of rubidium acetate on nematode heat stress (35°C)

**** represents P<0.0001 compared to the WT group.

Example 4 Rubidium nitrate enhances resistance to ultraviolet radiation in C. elegans

The preparation of strains, the preparation of NGM medium, the seed plate, the transfer of nematodes, the passage of nematodes, and the synchronization of nematodes were as described in Example 1. Ultraviolet radiation is also a kind of environmental stress. Synchronized nematodes were placed on NGM medium (blank control group (referred to as WT), 100 μM RbNO ₃ , 10 mM RbNO ₃ ), and this time was recorded as Day 0, Incubate at 20°C for 3 days in a constant temperature incubator. Adult nematodes were transferred to the corresponding groups of fresh NGM medium (3 medium per group, 80 nematodes per medium) and continued to culture until Day 6. The nematodes were transferred to unplated blank NGM medium and subjected to UV irradiation (300 mj/cm ² ). After irradiation, the nematodes were transferred to fresh NGM medium again, and the survival and death of nematodes were observed and counted every day.

Experimental results: As shown in Figures 9-10 and Table 7, in three independent repeated experiments, after feeding rubidium nitrate (100 μM and 10 mM), compared with the control group, the average lifespan of 100 μM rubidium nitrate can be increased by 4%, and the maximum lifespan can be increased 11%; 10mM rubidium salt can increase the average lifetime by 9% and the maximum lifetime by 11% (P<0.05). It was shown that rubidium nitrate can enhance the resistance of C. elegans to UV irradiation.

Table 7 Protective effects of different concentrations of rubidium nitrate on nematodes UV radiation

* represents P<0.05 compared with the WT group.

Example 5 Rubidium carbonate enhances resistance to oxidative stress in Caenorhabditis elegans

The preparation of strains, the preparation of NGM medium, the seed plate, the transfer of nematodes, the passage of nematodes, and the synchronization of nematodes were as described in Example 1. Synchronized nematodes were placed on NGM medium (blank control group (referred to as WT group), 10mM Rb ₂ CO ₃ ), recorded as Day 0, and cultured in a constant temperature incubator at 20°C for 3 days . Adult nematodes were transferred to the corresponding fresh NGM medium (50 nematodes per medium), and the culture was continued until Day 6. The nematodes were transferred to NGM medium containing 400 μM juglone, and continued to be cultured in a constant temperature incubator at 20°C for 24 h, and the survival and death of nematodes were observed and counted, and the experiment was repeated three times.

Experimental results: As shown in Figure 11, in three independent repeated experiments, after feeding with rubidium nitrate (10 mM), compared with the control group, the average survival rate of 10 mM rubidium nitrate for 24 hours can be increased by 16.2% (P<0.05). It was shown that rubidium nitrate can enhance the resistance of C. elegans to oxidative stress.

Example 6 Rubidium chloride extends C. elegans lifespan through DAF16 nuclear translocation

The preparation of strains, the preparation of NGM medium, the seed plate, the transfer of nematodes, the passage of nematodes, and the synchronization of nematodes were as described in Example 1. The translocation of DAF-16 from the cytoplasm to the nucleus is a critical step in its transcription factor activity. Activation of DAF16 is associated with stress resistance. The effect of rubidium salts on nuclear translocation of daf16 was observed using transgenic TJ356 nematodes (zIs356 IV[daf-16::GFP+pRF6]). Synchronized nematodes were placed on NGM medium (blank control group (denoted as TJ356), 10mMRbCl (denoted as TJ356/10mMRbCl)), recorded as Day 0 at this time, and cultivated in a constant temperature incubator at 20 °C 3 days. Pick 2-3 adult nematodes and place them on a slide with an agar pad (take 5% agar solution and drop it in the center of the slide, flatten it with another slide, and remove the top slide when it is used) ), covered with a cover glass, and observed the expression of DAF-16 in nematodes under a laser confocal microscope (20X, 488 nm). More than 10 nematodes were observed in each group at a time, and the experiment was repeated three times.

Experimental results: As shown in Figures 12-13, after feeding with rubidium chloride (10 mM), compared with the control group, the number of nematodes with 10 mM rubidium chloride nuclear translocation increased by 63.84% (P<0.001). It is shown that rubidium chloride can activate DAF16 to enhance the resistance of nematodes and prolong lifespan.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit the protection scope of the present invention. For those of ordinary skill in the art, on the basis of the above descriptions and ideas, the Variations or changes in other different forms are not required and cannot be exhaustive of all implementations here. Any modifications, equivalent replacements and improvements made within the spirit and principle of the present invention shall be included within the protection scope of the claims of the present invention.

Claims (9)

1. Use of a rubidium salt or a rubidium salt solution for the preparation of a product activating DAF-16 via the AMPK pathway.

2. The use according to claim 1, wherein said rubidium salt is rubidium ion Rb ⁺ Salts with stable inorganic or organic acid anions.

3. Use according to claim 2, characterized in that the mineral acid anion is chloride Cl ^- Sulfate radical SO ₄ ^2- Nitrate radical NO ₃ ^- Or carbonate CO ₃ ^2- One or more of them.

4. The use of claim 2, wherein the organic acid anion is one or more of acetate, citrate, gallate and tannin.

5. The use according to claim 1, wherein the rubidium salt concentration of said activated DAF-16 product is between 1 μmol and 100mmol.

6. The use according to claim 1, wherein said activated DAF-16 product comprises a rubidium salt or a rubidium salt solution, and further comprises one or more pharmaceutically acceptable adjuvants.

7. Use according to claim 6, wherein the excipient is a diluent, filler, binder, wetting agent, absorption enhancer, surfactant, lubricant or stabilizer.

8. The use according to claim 1, wherein the activated DAF-16 product is a general food, nutraceutical, feed or feed additive.

9. The use according to claim 1, wherein said activated DAF-16 product is formulated in the form of tablets, granules, capsules, powders, liquids or jellies.

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