WO2023133984A1 - An anti-aging pharmaceutical composition and preparation method thereof - Google Patents

An anti-aging pharmaceutical composition and preparation method thereof Download PDF

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WO2023133984A1
WO2023133984A1 PCT/CN2022/078829 CN2022078829W WO2023133984A1 WO 2023133984 A1 WO2023133984 A1 WO 2023133984A1 CN 2022078829 W CN2022078829 W CN 2022078829W WO 2023133984 A1 WO2023133984 A1 WO 2023133984A1
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parts
pharmaceutical composition
nematodes
aging
lutein
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French (fr)
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Qingxiong MENG
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Meng Qingxiong
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/10—Laxatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/07—Retinol compounds, e.g. vitamin A
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
    • A61K31/385—Heterocyclic compounds having sulfur as a ring hetero atom having two or more sulfur atoms in the same ring
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/455—Nicotinic acids, e.g. niacin; Derivatives thereof, e.g. esters, amides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/525—Isoalloxazines, e.g. riboflavins, vitamin B2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/658—Medicinal preparations containing organic active ingredients o-phenolic cannabinoids, e.g. cannabidiol, cannabigerolic acid, cannabichromene or tetrahydrocannabinol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/716—Glucans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/716—Glucans
    • A61K31/722—Chitin, chitosan
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/06—Tripeptides
    • A61K38/063—Glutathione

Definitions

  • This application relates to the field of functional foods and health foods, and specifically relates to a pharmaceutical composition for delaying aging and a preparation method thereof.
  • Aging is a natural and inevitable life process. Based on cell aging, the bodys various tissues, organs, and physiological functions undergo irreversible degenerative changes with age. With the aging process, free radicals continue to accumulate and cause oxidative damage to cells and tissues.
  • Caenorhabditis elegans is a multicellular eukaryote with simple individual structure, short life span, clear genetic background, fast reproduction speed and easy to cultivate. It is relatively conservative in genes and signal pathways with humans, and it genes have 60%similarity with humans, and it is a classic model organism for studying aging.
  • the present invention is based on experiments to nematodes and the experiments can quickly test the anti-aging effect of the present invention.
  • the purpose of the present invention is to provide a pharmaceutical composition for delaying aging and a preparation method thereof.
  • the pharmaceutical composition comprises: ⁇ -glucan, chitosan, coenzyme Q10, CBD, glutathione, lipoic acid, lutein, nicotinamide, and vitamin B2.
  • the pharmaceutical composition has a scientific and reasonable formula and has no compatibility contraindications. After safety toxicology test, it has no obvious toxic effect on the observation indexes of male and female rats.
  • the pharmaceutical composition is simple to make and convenient to take. It not only considers the repair and rejuvenation of the complete respiratory chain of mitochondria, but also combines with super antioxidant and free radical scavenger astaxanthin.
  • the present invention is achieved through the following technical solutions.
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising in parts by weight, 20-25 parts of ⁇ -glucan, 10-15 parts of chitosan, 4-5 parts of Coenzyme Q10, 10-15 parts of CBD, 2-3 parts of glutathione , 2-3 parts of lipoic acid, 8-10 parts of lutein, 20-30 parts of nicotinamide, and 15-20 parts of vitamin B2.
  • the preferred pharmaceutical composition comprises: in parts by weight, 22 parts of ⁇ -glucan, 12 parts of chitosan, 4 parts of coenzyme Q10, 12 parts of CBD, 2 parts of glutathione, 2 parts of lipoic acid, 8 parts of lutein, 22 parts of niacinamide, and 16 parts of vitamin B2.
  • Glucan is a natural polysaccharide compound. In the fungus kingdom, ⁇ -glucan exists in large quantities, and it is a natural substance that cannot be artificially synthesized. Many pharmacological experiments and clinical reports have shown that ⁇ -glucan mainly regulates the stability of the intestinal flora to maintain human health. Its main functions are anti-tumor, immune regulation, protection of myocardium, promotion of wound healing, anti-coagulation, anti-virus, anti-bacteria, anti-oxidation and anti-aging.
  • Chitosan is the only alkaline cationic substance in nature. It has a variety of specific physical and chemical properties and biological properties, and is non-toxic, non-irritating, and naturally degradable. It has great clinical application value. Chitosan has good biocompatibility, safety and non-toxicity, and can maintain intestinal homeostasis by regulating the intestinal flora to reduce the burden on the body. Its main functions include immune regulation, anti-tumor, treatment of gastric ulcer, lowering blood lipid and blood sugar, immune adjuvant, drug carrier and anti-fatigue.
  • Coenzyme Q10 is a quinone ring compound, also known as ubiquinone 10.
  • ubiquinone 10 a quinone ring compound
  • Coenzyme Q10 is present in the main organs of the human body, such as the heart, liver, kidney, and pancreas. Studies have shown that Coenzyme Q10 can effectively delay cell senescence and restore the activity of senescent cells. Its main functions include anti-oxidation and scavenging free radicals, anti-tumor, improving human immunity, anti-inflammation, relieving fatigue and improving exercise capacity, preventing aging and anti-aging, and protecting the cardiovascular system.
  • Glutathione is present in all animal cells. In human cells, it is composed of glutamic acid, cysteine and glycine. It exists in its thiol-reduced form under normal circumstances and is the prosthetic group of glyceraldehyde phosphate dehydrogenase and the coenzyme of glyoxalase and triose phosphate dehydrogenase. It plays a direct or indirect role in many life activities, participates in the tricarboxylic acid cycle and sugar metabolism in the body, enables the human body to obtain high energy, and can also activate a variety of enzymes. Its main effects are anti-oxidation, detoxification, anti-convulsions, anti-thrombosis, anti-hypertension, anti-atherosclerosis, kidney protection, liver protection, and anti-inflammation.
  • Lipoic acid is a coenzyme that exists in mitochondria. Similar to vitamins, it can eliminate free radicals that accelerate aging and disease (the basis for improving sperm motility) . Lipoic acid enters cells after being absorbed by the intestine in the body. It has both fat-soluble and water-soluble properties. Therefore, it can pass through the whole body without hindrance and reach any cell part, providing the body with comprehensive performance. It is a fat-soluble and water-soluble universal anti -oxidant and can effectively remove the active oxygen in the oxidized cells to restore their activity.
  • Niacinamide is a component of coenzyme I and coenzyme II. When lacking, it can affect the normal respiration and metabolism of cells and cause pellagra. Nicotinamide is easily absorbed in the gastrointestinal tract, and after absorption, it is distributed to the body tissues, and is metabolized by the liver, and only a small amount is excreted in the urine in its original form. Niacinamide is a recognized skin anti-aging ingredient in the field of beauty and dermatology. Its most important effect in skin anti-aging is to reduce and prevent the dull and yellow skin produced in the early aging process of the skin. It can also repair the damaged stratum corneum lipid barrier and improve skin resistance.. Its main effects are anti-arrhythmia, improvement of vascular endothelial function, stimulation of ⁇ cell regeneration, anti-inflammatory and anti-oxidation, etc.
  • Lutein is a fat-soluble vitamin. Supplementing lutein can delay the development of myopia. Lutein has an antioxidant effect, can reduce facial pigmentation and reduce wrinkles. Secondly, lutein has the effect of scavenging free radicals and can help the human body to remove harmful substances in the body. It also has anti-ultraviolet and anti-radiation effects, so it has a certain protective effect on the eyes.. In addition, lutein also has anti-mutation, anti-tumor and anti-cancer effects.
  • Vitamin B2 is a B vitamin, which is a water-soluble vitamin. It participates in the bodys biological oxidation and energy metabolism, can increase the bodys protein utilization, promote growth and development, maintain the integrity of skin and cell membranes, and have the functions of protecting skin, hair follicles and sebaceous glands. In addition, it also has the functions of regulating the growth and metabolism of the cells, anti-oxidation and delaying cell senescence.
  • the raw materials used in the present invention are all obtained through commerce, ⁇ -glucan, chitosan, coenzyme Q10, CBD, glutathione, lipoic acid, lutein, nicotinamide, vitamin B2 can maintain human health after scientific formulation.
  • Glutathione, lipoic acid, lutein, nicotinamide, CBD and vitamin B2 all have antioxidant functions, which are beneficial to delay cell senescence and rejuvenate aging cells;
  • ⁇ -glucan and chitosan are conducive to the formation of a stable structure of intestinal microflora that is conducive to the survival of the human body.. Combining the two parts can can delay cell senescence while maintaining human health and effectively extend life span.
  • the pharmaceutical composition of the present invention may be in the form of oral preparations, tablets, capsules or granules.
  • the preparation method of the present invention is as follows: weighing the raw materials according to the above formula ratio, mixing the raw materials and diluting the mixture with distilled water, heating and stirring in a water bath at 40°C until the mixture is well mixed, and diluting 100 g of the mixture to a constant volume to 1000 mL to obtain the oral preparation of the present invention.
  • composition is applied to nematode experiments to determine the anti-aging effect .
  • Figure 1 is a survival curve diagram of the experimental group vs. the control group in the Examples;
  • Figure 2 is a histogram of the healthy lifespan of nematodes according to the Examples of the present invention; wherein, a is the pharyngeal pump exercise test chart, and b is the body exercise test chart.
  • Figure 3 is a histogram of survival of nematodes against heat stress in the Examples of the present invention (N2, 35°C liquid heat stress experiment) ;
  • Figure 4 is a histogram of survival against oxidative stress in nematodes in the Examples, wherein a is the survival curve of H 2 O 2 oxidative stress, and b is the histogram of survival agains juglone oxidative stress .
  • a pharmaceutical composition for delaying aging and a preparation method thereof, the steps are as follows:
  • composition formula in parts by weight, 22 parts of ⁇ -glucan, 12 parts of chitosan, 4 parts of coenzyme Q10, 12 parts of CBD, 2 parts of glutathione, 2 parts of lipoic acid, 8 parts of lutein , 22 parts of niacinamide, 16 parts of vitamin B2 .
  • the preparation method is as follows: Take 100g as an example, weigh 22g ⁇ -glucan and 12g chitosan according to the above formula, stir evenly at 40°C to obtain solution A; then weigh 4g coenzyme Q10, 12g CBD, 2g glutathione, 2g lipoic acid , 8g lutein , 22g nicotinamide , and 16g vitamin B2. After they are mixed well, slowly add the mixture to solution A while stirring in a 40°Cwater bath; slowly add 500-600mL distilled water to the mixture, stir well and make the volume constant to 1000mL, an oral preparation is thus obtained.
  • a pharmaceutical composition for delaying aging and a preparation method thereof, the steps are as follows:
  • composition formula in parts by weight, 20 parts of ⁇ -glucan, 13 parts of chitosan, 5 parts of coenzyme Q10, 11 parts of CBD, 3 parts of glutathione, 2 parts of lipoic acid, 9 parts of lutein , 21 parts of niacinamide, 16 parts of vitamin B2 .
  • the preparation method is as follows: Take 100g as an example, weigh 20g ⁇ -glucan and 13g chitosan according to the above formula, stir evenly at 40°C to obtain solution A; then weigh 5g coenzyme Q10, 11g CBD, 3g glutathione, 2g lipoic acid , 9g lutein , 21g nicotinamide , and 16g vitamin B2. After they are mixed well, slowly add the mixture to solution A while stirring in a 40°Cwater bath; slowly add 500-600mL distilled water to the mixture, stir well and make the volume constant to 1000mL, an oral preparation is thus obtained.
  • a pharmaceutical composition for delaying aging and a preparation method thereof, the steps are as follows:
  • composition formula in parts by weight, 21 parts of ⁇ -glucan, 12 parts of chitosan, 5 parts of coenzyme Q10, 12 parts of CBD, 3 parts of glutathione, 3 parts of lipoic acid, 8 parts of lutein, 22 parts of niacinamide, 15 parts of vitamin B2 .
  • the preparation method is as follows: Take 100g as an example, weigh 21g ⁇ -glucan and 12g chitosan according to the above formula, stir evenly at 40°C to obtain solution A; then weigh 5g coenzyme Q10, 12g CBD, 2g glutathione, 3g lipoic acid , 8g lutein , 22g nicotinamide , and 15g vitamin B2. After they are mixed well, slowly add the mixture to solution A while stirring in a 40°Cwater bath; slowly add 500-600mL distilled water to the mixture, stir well and make the volume constant to 1000mL, an oral preparation is thus obtained.
  • Reagents pentafluorouracil deoxynucleoside, juglone, agar, sodium chloride, yeast extract, tryptone, deionized water, KH 2 PO 4 , K 2 HPO 4 , peptone, MgSO 4 , NaOH, hypochlorous acid, water and chloral, etc.
  • N2 Caenorhabditis elegans was tested as experimental subjects.
  • the experimental subjects were divided into control group (0 ⁇ M Exa) and experimental groups.
  • the experimental groups included Example 1 (120 ⁇ M Exa 1) , Example 2 (120 ⁇ M Exa 2) and Example 3 (120 ⁇ M Exa 3) .
  • the control group was fed with E. coli OP50, and the experimental groups were fed with E. coli OP50 and the above-mentioned different formulations in the Examples (final concentration 40 ⁇ M) .
  • this study used autoclaved E. coli OP50 bacterial solution to be applied to the surface of NGM.
  • LB liquid medium 10g NaCl, 10g peptone, and 5g yeast powder are mixed, add water to make the volume of the mixture to 1L, and sterilize the mixture at 121°C for 20min to obtain the LB liquid medium.
  • S-complete medium comprising 977ml S-basal medium, 10ml 1M potassium citrate buffer, 10ml Trace metals solution, 3ml 1M NaOH, 3ml 1mol/L MgSO 4 ;
  • Bleach Lysis solution is prepared by 4ml deionized water, 1.5ml 5mol/L NaOH, and 2ml NaClO.
  • Oviposition method pick the adults in the peak period of egg production and put them on the NGM medium plate containing OP50 (6 cm diameter petri dish was dripped with 300 ⁇ L of bacterial solution at OD600 0.6) , and 5h after laying eggs in a biochemical incubator at 25°C, pick out the adults and continue to cultivate them in the incubator to get the nematodes of the same period.
  • the lifespan changes of N2 nematodes after the intervention of the examples are shown in Table 1. Compared with the control group (0 ⁇ M Exa) , the survival curve and average lifespan of nematodes were significantly extended after the intervention ( Figure 1 and Table 1) , indicating that the intervention of the examples can prolong the lifespan of N2 nematodes.
  • the average pharyngeal pump movement rate of the nematodes in the control group was 81 times/min
  • the average pharyngeal pump movement rate of the nematodes in the experimental groups was 113.69 times/min (P ⁇ 0.001)
  • the average pharyngeal pump movement rate of nematodes in the control group was 42.15 times/min
  • the average pharyngeal pump movement rate of the nematodes in the experimental groups was 75.35 times/min (P ⁇ 0.001) .
  • the average pharyngeal pump movement rate of the nematodes in the control group was 12.59 times/min, and the average pharyngeal pump movement rate of the nematodes in the experimental groups was 27.25 times/min (P ⁇ 0.05) ( Figure 2a) .
  • the motor ability of the nematode pharyngeal pump after the intervention of the example was significantly higher than that of the control group, that is, the intervention of the example significantly improved the motor function of the nematode pharyngeal pump in the middle age.
  • the average body swing rate of the control group was 50.25 times/min, and the average body swing rate of the nematodes in the experimental groups was 81.36 times/min (P ⁇ 0.01) .
  • the average body swing rate of nematodes in the control group was 22.33 times/min, and the average body swing rate of the nematodes in the experimental groups was 43.58 times/min (P ⁇ 0.01) .
  • the average body swing rate of nematodes in the control group was 13.36 times/min, and the average body swing rate of the nematodes in the experimental groups was 27.69 times/min (P ⁇ 0.05) ( Figure 2b) .
  • the body swinging ability of the nematodes after the intervention of the Example compositions was significantly higher than that of the control group, that is, the intervention of the Example compositions significantly improved the body swinging function of the nematodes in the middle-aged age.
  • 35°C liquid heat stress experiment After synchronization, the nematodes cultured to the end of L4 or early adult stage were transferred to the NGM of the experimental group and the control group. After 5 days of culture, add 1 mL of freshly prepared S-complete culture medium (including the experimental groups and the control group) to each well of the 12-well plate, put 10 worms in each well, and record the number of nematode deaths and survivals every 2 hours. There are three parallels in each group, and 40 worms in each parallel.
  • N2 nematodes was intervened with Examples to the 6th day. Transfer the nematodes on different groups of culture media to NGM culture medium, add 1 mL of freshly prepared S-complete culture medium with a mass concentration of 0.003%H 2 O 2 to each well of the 12-well plate, and put in each well 10 nematodes, the number of death and survival of nematodes was recorded every 2 hours. Each group has 3 boards and each board has 50 boards.
  • N2 nematodes was intervened with Examples to the 6th day. Transfer the nematodes on different groups of culture medium to NGM medium, take a 12-well plate, add 1 ml of freshly prepared S basal buffer containing 240 ⁇ M juglone to each well, and put 10 nematodes in each well. The number of death and survival of nematodes was recorded every 2 hours. Each group has 3 boards and each board has 50 boards..
  • Example compositions have a certain degree of influence on the overall lifespan of wild-type N2 nematodes within 6 hours under the oxidative stress caused by 240 ⁇ M juglone, but compared with the control group, the effect of Example 1 composition is not significant.
  • Example compositions 1-3 can resist the oxidative stress caused by 0.003%H 2 O 2 and 240 ⁇ M juglone to a certain extent.
  • composition of the present invention can be used alone to delay aging and prolong life of a subject.

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Abstract

The invention belongs to the field of functional foods and health foods, and specifically relates to a composition for anti-aging composite products and a preparation method thereof; the raw materials of the composition are: β-glucan, chitosan, coenzyme Q10, CBD, glutathione, lipoic acid, lutein, nicotinamide, vitamin B2. The formula of the composition is scientific and reasonable, has no contraindications, and is non-toxic and harmless. Glutathione, lipoic acid, lutein, nicotinamide, CBD and vitamin B2 are beneficial to rejuvenate aging cells; β-glucan and chitosan are beneficial to maintain a healthy intestinal microbiota, a combination of the ingredients can extend life. After a series of operations, the above-mentioned raw materials were made into finished products, and the finished products were applied to C. elegans, and it was found that the life of C. elegans was prolonged and promoted to resist various oxidative stresses. The above shows that the composition of present invention has the effects of resisting aging and prolonging life span.

Description

An anti-aging pharmaceutical composition and preparation method thereof
The present application claims the priority of Chinese Patent Application No. 202210029930.4, the disclosures of which are incorporated herein by reference.
Technical field
This application relates to the field of functional foods and health foods, and specifically relates to a pharmaceutical composition for delaying aging and a preparation method thereof.
Background technique
Aging is a natural and inevitable life process. Based on cell aging, the bodys various tissues, organs, and physiological functions undergo irreversible degenerative changes with age. With the aging process, free radicals continue to accumulate and cause oxidative damage to cells and tissues. However, there is currently no specific mechanism of aging. Carlos et al. proposed nine major characteristics of aging: genome instability, shortening of telomeres, changes in epigenetics, imbalance of protein dynamics, disturbance of nutritional perception, mitochondrial dysfunction, cell senescence, stem cell exhaustion, and changes in cell-to-cell communication. These nine major aging characteristics are not all harmful. For example, severe mitochondrial DNA mutations and telomere loss can damage the body; while slight mitochondrial dysfunction is beneficial to the body’s resistance. It can be seen that when the degree of damage is different, the lesion caused is also different. The occurrence of aging is not caused by a single factor, but by multiple complex factors. These complex factors can make certain changes through artificial regulation, and delaying the occurrence of aging is the purpose of regulating its occurrence.
Caenorhabditis elegans, abbreviated as nematode, is a multicellular eukaryote with simple individual structure, short life span, clear genetic background, fast reproduction speed and easy to cultivate. It is relatively conservative in genes and signal pathways with humans, and it genes have 60%similarity with humans, and it is a classic model organism for studying aging.
The present invention is based on experiments to nematodes and the experiments can quickly test the anti-aging effect of the present invention.
Summary of the Invention
The purpose of the present invention is to provide a pharmaceutical composition for delaying aging and a preparation method thereof. The pharmaceutical composition comprises: β-glucan, chitosan, coenzyme Q10, CBD, glutathione, lipoic acid, lutein, nicotinamide, and vitamin B2. The pharmaceutical composition has a scientific and reasonable formula and has no compatibility contraindications. After safety toxicology test, it has no obvious toxic effect on the observation indexes of male and female rats. The pharmaceutical composition is simple to make and convenient to take. It not only considers the repair and rejuvenation of the complete respiratory chain of mitochondria, but also combines with super antioxidant and free radical scavenger astaxanthin. The experiment with Caenorhabditis elegans proves that the life extension effect is significant. After used by a large population of subjects , it is found that the effects of restoring youth, such as improving sleep, improving skin quality, improving excretion, and whitening skin, are quite significant.
The present invention is achieved through the following technical solutions.
In one embodiment, the present application provides a pharmaceutical composition comprising in parts by weight, 20-25 parts of β-glucan, 10-15 parts of chitosan, 4-5 parts of Coenzyme Q10, 10-15 parts of CBD, 2-3 parts of glutathione , 2-3 parts of lipoic acid, 8-10 parts of lutein, 20-30 parts of nicotinamide, and 15-20 parts of vitamin B2.
In a further embodiment, the preferred pharmaceutical composition comprises: in parts by weight, 22 parts of β-glucan, 12 parts of chitosan, 4 parts of coenzyme Q10, 12 parts of CBD, 2 parts of glutathione, 2 parts of lipoic acid, 8 parts of lutein, 22 parts of niacinamide, and 16 parts of vitamin B2.
β-Glucan: Glucan is a natural polysaccharide compound. In the fungus kingdom, β-glucan exists in large quantities, and it is a natural substance that cannot be artificially synthesized. Many pharmacological experiments and clinical reports have shown that β-glucan mainly regulates the stability of the intestinal flora to maintain human health. Its main functions are anti-tumor, immune regulation, protection of myocardium, promotion of wound healing, anti-coagulation, anti-virus, anti-bacteria, anti-oxidation and anti-aging.
Chitosan: Chitosan is the only alkaline cationic substance in nature. It has a variety of specific physical and chemical properties and biological properties, and is non-toxic, non-irritating, and naturally degradable. It has great clinical application value. Chitosan has good biocompatibility, safety and non-toxicity, and can maintain intestinal homeostasis by regulating the intestinal flora to reduce the burden on the body. Its main functions include immune regulation, anti-tumor, treatment of gastric ulcer, lowering blood lipid and blood sugar, immune adjuvant, drug carrier and anti-fatigue.
Coenzyme Q10: Coenzyme Q10 is a quinone ring compound, also known as ubiquinone 10. For different sources of coenzyme Q10, the number of side chain isopentenyl units are different. Humans and mammals have 10 isopentenyl units, so they are called coenzyme Q10. Coenzyme Q10 is present in the main organs of the human body, such as the heart, liver, kidney, and pancreas. Studies have shown that Coenzyme Q10 can effectively delay cell senescence and restore the activity of senescent cells. Its main functions include anti-oxidation and scavenging free radicals, anti-tumor, improving human immunity, anti-inflammation, relieving fatigue and improving exercise capacity, preventing aging and anti-aging, and protecting the cardiovascular system.
Glutathione: Glutathione is present in all animal cells. In human cells, it is composed of glutamic acid, cysteine and glycine. It exists in its thiol-reduced form under normal circumstances and is the prosthetic group of glyceraldehyde phosphate dehydrogenase and the coenzyme of glyoxalase and triose phosphate dehydrogenase. It plays a direct or indirect role in many life activities, participates in the tricarboxylic acid cycle and sugar metabolism in the body, enables the human body to obtain high energy, and can also activate a variety of enzymes. Its main effects are anti-oxidation, detoxification, anti-convulsions, anti-thrombosis, anti-hypertension, anti-atherosclerosis, kidney protection, liver protection, and anti-inflammation.
Lipoic acid: Lipoic acid is a coenzyme that exists in mitochondria. Similar to vitamins, it can eliminate free radicals that accelerate aging and disease (the basis for improving sperm motility) . Lipoic acid enters cells after being absorbed by the intestine in the body. It has both fat-soluble and water-soluble properties. Therefore, it can pass through the whole body without hindrance and reach any cell part, providing the body with comprehensive performance. It is a fat-soluble and water-soluble universal anti -oxidant and can effectively remove the active oxygen in the oxidized cells to restore their activity.
Niacinamide: Niacinamide is a component of coenzyme I and coenzyme II. When  lacking, it can affect the normal respiration and metabolism of cells and cause pellagra. Nicotinamide is easily absorbed in the gastrointestinal tract, and after absorption, it is distributed to the body tissues, and is metabolized by the liver, and only a small amount is excreted in the urine in its original form. Niacinamide is a recognized skin anti-aging ingredient in the field of beauty and dermatology. Its most important effect in skin anti-aging is to reduce and prevent the dull and yellow skin produced in the early aging process of the skin. It can also repair the damaged stratum corneum lipid barrier and improve skin resistance.. Its main effects are anti-arrhythmia, improvement of vascular endothelial function, stimulation of β cell regeneration, anti-inflammatory and anti-oxidation, etc.
Lutein: Lutein is a fat-soluble vitamin. Supplementing lutein can delay the development of myopia. Lutein has an antioxidant effect, can reduce facial pigmentation and reduce wrinkles. Secondly, lutein has the effect of scavenging free radicals and can help the human body to remove harmful substances in the body. It also has anti-ultraviolet and anti-radiation effects, so it has a certain protective effect on the eyes.. In addition, lutein also has anti-mutation, anti-tumor and anti-cancer effects.
Vitamin B2: Vitamin B2 is a B vitamin, which is a water-soluble vitamin. It participates in the bodys biological oxidation and energy metabolism, can increase the bodys protein utilization, promote growth and development, maintain the integrity of skin and cell membranes, and have the functions of protecting skin, hair follicles and sebaceous glands. In addition, it also has the functions of regulating the growth and metabolism of the cells, anti-oxidation and delaying cell senescence.
The raw materials used in the present invention are all obtained through commerce, β-glucan, chitosan, coenzyme Q10, CBD, glutathione, lipoic acid, lutein, nicotinamide, vitamin B2 can maintain human health after scientific formulation. Glutathione, lipoic acid, lutein, nicotinamide, CBD and vitamin B2 all have antioxidant functions, which are beneficial to delay cell senescence and rejuvenate aging cells; β-glucan and chitosan are conducive to the formation of a stable structure of intestinal microflora that is conducive to the survival of the human body.. Combining the two parts can can delay cell senescence while maintaining human health and effectively extend life span.
The pharmaceutical composition of the present invention may be in the form of oral preparations, tablets, capsules or granules.
Taking oral preparations as an example, the preparation method of the present invention is as follows: weighing the raw materials according to the above formula ratio, mixing the  raw materials and diluting the mixture with distilled water, heating and stirring in a water bath at 40℃ until the mixture is well mixed, and diluting 100 g of the mixture to a constant volume to 1000 mL to obtain the oral preparation of the present invention.
Furthermore, this composition is applied to nematode experiments to determine the anti-aging effect .
Description of the drawings
Figure 1 is a survival curve diagram of the experimental group vs. the control group in the Examples;
Figure 2 is a histogram of the healthy lifespan of nematodes according to the Examples of the present invention; wherein, a is the pharyngeal pump exercise test chart, and b is the body exercise test chart.
Figure 3 is a histogram of survival of nematodes against heat stress in the Examples of the present invention (N2, 35℃ liquid heat stress experiment) ;
Figure 4 is a histogram of survival against oxidative stress in nematodes in the Examples, wherein a is the survival curve of H 2O 2 oxidative stress, and b is the histogram of survival agains juglone oxidative stress .
Detailed description of the Invention
The following are specific embodiments of the present invention, but the following examples are only preferred examples of the present invention, not all of them. Based on the examples, other examples obtained by those skilled in the art without creative work shall fall within the protection scope of the present invention.
The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.
Example 1
A pharmaceutical composition for delaying aging and a preparation method thereof, the steps are as follows:
Pharmaceutical composition formula: in parts by weight, 22 parts of β-glucan, 12 parts  of chitosan, 4 parts of coenzyme Q10, 12 parts of CBD, 2 parts of glutathione, 2 parts of lipoic acid, 8 parts of lutein , 22 parts of niacinamide, 16 parts of vitamin B2 .
The preparation method is as follows: Take 100g as an example, weigh 22g β-glucan and 12g chitosan according to the above formula, stir evenly at 40℃ to obtain solution A; then weigh 4g coenzyme Q10, 12g CBD, 2g glutathione, 2g lipoic acid , 8g lutein , 22g nicotinamide , and 16g vitamin B2. After they are mixed well, slowly add the mixture to solution A while stirring in a 40℃water bath; slowly add 500-600mL distilled water to the mixture, stir well and make the volume constant to 1000mL, an oral preparation is thus obtained.
Example 2
A pharmaceutical composition for delaying aging and a preparation method thereof, the steps are as follows:
Pharmaceutical composition formula: in parts by weight, 20 parts of β-glucan, 13 parts of chitosan, 5 parts of coenzyme Q10, 11 parts of CBD, 3 parts of glutathione, 2 parts of lipoic acid, 9 parts of lutein , 21 parts of niacinamide, 16 parts of vitamin B2 .
The preparation method is as follows: Take 100g as an example, weigh 20g β-glucan and 13g chitosan according to the above formula, stir evenly at 40℃ to obtain solution A; then weigh 5g coenzyme Q10, 11g CBD, 3g glutathione, 2g lipoic acid , 9g lutein , 21g nicotinamide , and 16g vitamin B2. After they are mixed well, slowly add the mixture to solution A while stirring in a 40℃water bath; slowly add 500-600mL distilled water to the mixture, stir well and make the volume constant to 1000mL, an oral preparation is thus obtained.
Example 3
A pharmaceutical composition for delaying aging and a preparation method thereof, the steps are as follows:
Pharmaceutical composition formula: in parts by weight, 21 parts of β-glucan, 12 parts  of chitosan, 5 parts of coenzyme Q10, 12 parts of CBD, 3 parts of glutathione, 3 parts of lipoic acid, 8 parts of lutein, 22 parts of niacinamide, 15 parts of vitamin B2 .
The preparation method is as follows: Take 100g as an example, weigh 21g β-glucan and 12g chitosan according to the above formula, stir evenly at 40℃ to obtain solution A; then weigh 5g coenzyme Q10, 12g CBD, 2g glutathione, 3g lipoic acid , 8g lutein , 22g nicotinamide , and 15g vitamin B2. After they are mixed well, slowly add the mixture to solution A while stirring in a 40℃water bath; slowly add 500-600mL distilled water to the mixture, stir well and make the volume constant to 1000mL, an oral preparation is thus obtained.
Experimental example A nematode model is used to prove that the formulation has anti-aging effects
1. Experimental materials
Experimental materials: Example 1, Example 2, Example 3
Experimental strains: Uracil-deficient Escherichia coli (E. coli) OP50; Caenorhabditis elegans wild-type N2 strain.
Reagents: pentafluorouracil deoxynucleoside, juglone, agar, sodium chloride, yeast extract, tryptone, deionized water, KH 2PO 4, K 2HPO 4, peptone, MgSO 4, NaOH, hypochlorous acid, water and chloral, etc.
Instruments: 20℃ constant temperature incubator, 37℃ constant temperature incubator, 37℃ constant temperature shaker, electronic balance, ordinary microscope, fluorescence microscope, ultra-clean table, oscillator, 4℃ refrigerator, pipette gun, microwave oven.
2. Experimental method
2.1 Experiment grouping and intervention methods
N2 Caenorhabditis elegans was tested as experimental subjects. The experimental subjects were divided into control group (0 μM Exa) and experimental groups. The experimental groups included Example 1 (120 μM Exa 1) , Example 2 (120 μM Exa 2) and  Example 3 (120 μM Exa 3) . The control group was fed with E. coli OP50, and the experimental groups were fed with E. coli OP50 and the above-mentioned different formulations in the Examples (final concentration 40 μM) . In order to prevent certain components in the E. coli OP50 metabolic formula from changing its biochemical properties, this study used autoclaved E. coli OP50 bacterial solution to be applied to the surface of NGM.
2.2 The preparation of the media
Standard nematode NGM medium: 3g NaCl, 2.5g peptone, and 17g agar are mixed, add water to make the volume to 1L, after sterilization, add 1ml 5mg/ml cholesterol solution, 1ml 1mol/L MgSO 4, 25ml 1mol/L PK buffer (pH=6) to the mixture to obtain the standard nematode NGM medium;
LB liquid medium: 10g NaCl, 10g peptone, and 5g yeast powder are mixed, add water to make the volume of the mixture to 1L, and sterilize the mixture at 121℃ for 20min to obtain the LB liquid medium.
M9 buffer solution (pH=6) : 119g KH 2PO 4, and 21.5g K 2HPO 4 are mixed, add water to make the volume of the mixture to 1L, and sterilize the mixture at 121℃ for 20 minutes to obtain the M9 buffer solution.
S-complete medium: comprising 977ml S-basal medium, 10ml 1M potassium citrate buffer, 10ml Trace metals solution, 3ml 1M NaOH, 3ml 1mol/L MgSO 4;
Bleach Lysis solution is prepared by 4ml deionized water, 1.5ml 5mol/L NaOH, and 2ml NaClO.
2.3 Strain culture
Pick a single OP50 colony and place it in 100ml LB liquid medium, culture the colony overnight at 37℃ with shaking at 200rpm in a shaking incubator until OD600 reaches 0.4-0.6, and store it in a refrigerator at 4℃ for later use.
2.4 Nematode culture and synchronization
(1) Use standard nematode NGM medium to cultivate nematodes. The normal culture  temperature was 20℃ in the incubator, and the culture humidity was controlled within 40%-60%. Generally only one vigorous Dauer-period nematode was picked for different nematode strains and put in the medium which had been added 200μl OP50 bacterial solution and cultured overnight and had a flat surface for expansion. After it had multiplied to a certain number, the C. elegans was synchronized 2 to 3 times for further experiment.
(2) Synchronize with Bleach lysing method. Wash the hermaphrodite nematodes in the egg-laying stage with M9 buffer from the NGM medium, put them in a 15ml centrifuge tube, centrifuge at 1700rpm, 21℃ for 1-2min, and discard the supernatant. After washing the dishes twice using the same method, add an equal volume of Bleach Lysis Solution and shake quickly for 3-5 minutes until the solution is clear and contains a large number of eggs under a stereo microscope. Add a certain volume of M9 buffer to terminate the lysis process. After centrifugation, discard the supernatant and leave the precipitate. Add 2ml M9 buffer to wash the precipitate, repeat the operation twice. Place it on a shaker at 20℃ and cultivate it overnight to make the eggs develop into L1 stage larvae.
Oviposition method: pick the adults in the peak period of egg production and put them on the NGM medium plate containing OP50 (6 cm diameter petri dish was dripped with 300 μL of bacterial solution at OD600 0.6) , and 5h after laying eggs in a biochemical incubator at 25℃, pick out the adults and continue to cultivate them in the incubator to get the nematodes of the same period.
2.5 Life observation
Transfer 50 synchronized L4 nematodes to NGM medium and cultivate them in a constant temperature and humidity incubator at 20℃, and count the number of nematode deaths, survivals and dropouts every 2 days (in order to ensure sufficient food for nematodes, it is needed to change the NGM medium once every 2 days) . Criteria for the death of nematodes: the nematode has no movement or swallowing action, no response after touch. Dropout criteria: the nematode escapes to the plate wall or cover and is dried to die; the eggs have hatched into bag-like insects in the body; drilled into agar; the life span is defined  as:the time span that the nematode is from the L4 larval stage (Age=0) to the time that it is counted as death, repeating three times.
2.6 Statistics
All statistical analysis uses SPSS 20.0 software and Prism 8 is used for drawing.
3. Result analysis
3.1 The impact of the examples on the average lifespan of N2 nematodes
The lifespan changes of N2 nematodes after the intervention of the examples are shown in Table 1. Compared with the control group (0 μM Exa) , the survival curve and average lifespan of nematodes were significantly extended after the intervention (Figure 1 and Table 1) , indicating that the intervention of the examples can prolong the lifespan of N2 nematodes.
Table 1 Statistical analysis of the lifespan of nematodes in each group
Figure PCTCN2022078829-appb-000001
(Note: *, compared with N2+0 μM Exa, P<0.05; **, compared with N2+0 μM Exa, P<0.01)
3.2 Effects of Examples on the Healthy Lifespan of N2 Nematodes
In the middle -aged age of N2 nematodes (8th, 10th, and 12th days after adulthood) , the number of swallows by the pharyngeal pump of each nematode within 1 min was recorded. In the same way, in the middle -aged age of N2 nematodes, the number of body swings of each nematode within 1 min was recorded. In the experimental group and the control group, each of the pharyngeal pump movement rate experiment and the body swing experiment were done in three parallel groups, each parallel group had 10 nematodes.
Through the N2 nematode pharyngeal pump movement rate experiment , it was found that after 8 days of intervention through the administration of the composition of the Examples, the average pharyngeal pump movement rate of the nematodes in the control group  was 81 times/min, and the average pharyngeal pump movement rate of the nematodes in the experimental groups was 113.69 times/min (P<0.001) . After 10 days of intervention, the average pharyngeal pump movement rate of nematodes in the control group was 42.15 times/min, and the average pharyngeal pump movement rate of the nematodes in the experimental groups was 75.35 times/min (P <0.001) . At 12 days of intervention, the average pharyngeal pump movement rate of the nematodes in the control group was 12.59 times/min, and the average pharyngeal pump movement rate of the nematodes in the experimental groups was 27.25 times/min (P <0.05) (Figure 2a) . The motor ability of the nematode pharyngeal pump after the intervention of the example was significantly higher than that of the control group, that is, the intervention of the example significantly improved the motor function of the nematode pharyngeal pump in the middle age.
Through the N2 nematode body swing rate experiment, it was found that after 8 days of intervention through the administration of the example, the average body swing rate of the control group was 50.25 times/min, and the average body swing rate of the nematodes in the experimental groups was 81.36 times/min (P<0.01) . After 10 days of intervention, the average body swing rate of nematodes in the control group was 22.33 times/min, and the average body swing rate of the nematodes in the experimental groups was 43.58 times/min (P<0.01) . After the intervention for 12 days, the average body swing rate of nematodes in the control group was 13.36 times/min, and the average body swing rate of the nematodes in the experimental groups was 27.69 times/min (P <0.05) (Figure 2b) . The body swinging ability of the nematodes after the intervention of the Example compositions was significantly higher than that of the control group, that is, the intervention of the Example compositions significantly improved the body swinging function of the nematodes in the middle-aged age.
3.3 Effect of Examples on N2 Nematode Heat Stress Resistance
35℃ liquid heat stress experiment: After synchronization, the nematodes cultured to the end of L4 or early adult stage were transferred to the NGM of the experimental group and the control group. After 5 days of culture, add 1 mL of freshly prepared S-complete culture  medium (including the experimental groups and the control group) to each well of the 12-well plate, put 10 worms in each well, and record the number of nematode deaths and survivals every 2 hours. There are three parallels in each group, and 40 worms in each parallel.
The results of the 35℃ liquid heat stress experiment showed that the average survival rate of nematodes in the control group after 8 hours of intervention was 38.25%, 58.23%in the group administered Example 1 composition (P <0.05) , and 57.69%in the group administered Example 2 composition (P <0.05) , 62.03%of the group administered Example 3 composition (P <0.05) . Under liquid heat stress conditions, the experimental group with Examples prolonged the lifespan of wild-type N2 nematodes (Figure 3) . In summary, it can be concluded that the examples significantly enhanced the resistance of nematodes to liquid heat stress.
3.4 Effects of Examples on N2 Nematode Antioxidant Stress
H 2O 2 oxidative stress: N2 nematodes was intervened with Examples to the 6th day. Transfer the nematodes on different groups of culture media to NGM culture medium, add 1 mL of freshly prepared S-complete culture medium with a mass concentration of 0.003%H 2O 2 to each well of the 12-well plate, and put in each well 10 nematodes, the number of death and survival of nematodes was recorded every 2 hours. Each group has 3 boards and each board has 50 boards.
Juglone oxidative stress: N2 nematodes was intervened with Examples to the 6th day. Transfer the nematodes on different groups of culture medium to NGM medium, take a 12-well plate, add 1 ml of freshly prepared S basal buffer containing 240 μM juglone to each well, and put 10 nematodes in each well. The number of death and survival of nematodes was recorded every 2 hours. Each group has 3 boards and each board has 50 boards..
The results of H 2O 2 oxidative stress showed (Figure 4a) that the median lifespan of nematodes in the control group was 18 hours, and the maximum lifespan was 30 hours. The maximum lifespan of the nematodes with Example 1 composition was 35 hours; the maximum lifespan of the nematodes with Example 2 composition was 34 hours; the  maximum lifespans of the nematodes with Example 3 composition was 35 hours. It shows that under the oxidative stress caused by 0.003%H 2O 2, the examples have a certain degree of influence on the maximum lifespan of N2 nematodes.
Juglone oxidative stress results showed (Figure 4b) that the average survival rate of nematodes in the control group after 6 hours of intervention was 26.58%, the average survival rate of nematodes with Example 1 composition was 29.63%after 6 hours of intervention, and the average survival rate of nematodes with Example 2 composition after 6 hours of intervention was 38.26% (P <0.05) , the average survival rate of nematodes with Example 3 composition after 6 hours of intervention was 39.65% (P <0.05) . It shows that the Example compositions have a certain degree of influence on the overall lifespan of wild-type N2 nematodes within 6 hours under the oxidative stress caused by 240 μM juglone, but compared with the control group, the effect of Example 1 composition is not significant.
In summary, Example compositions 1-3 can resist the oxidative stress caused by 0.003%H 2O 2 and 240 μM juglone to a certain extent.
The pharmaceutical composition of the present invention (including the composition of the examples) can be used alone to delay aging and prolong life of a subject.
The above are only the preferred embodiments of the present invention, and do not impose any formal restrictions on the present invention. Therefore, without departing from the content of the technical solution of the present invention, anything simple modification, or equivalent change made to the above embodiments based on the technical essence of the present invention is within the scope of the present invention.

Claims (6)

  1. A pharmaceutical composition for delaying aging, the pharmaceutical composition comprises: β-glucan, chitosan, coenzyme Q10, CBD, glutathione, lipoic acid, lutein, nicotinamide, and vitamin B2.
  2. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition comprises: in parts by weight, 20-25 parts of β-glucan, 10-15 parts of chitosan, 4-5 parts of coenzyme Q10, 10-15 parts of CBD 2-3 parts of glutathione, 2-3 parts of lipoic acid, 8-10 parts of lutein, 20-30 parts of nicotinamide, and 15-20 parts of vitamin B2.
  3. A pharmaceutical composition according to claim 1, wherein the pharmaceutical composition comprises: in parts by weight, 22 parts of β-glucan, 12 parts of chitosan, 4 parts of coenzyme Q10, 12 parts of CBD, 2 parts of glutathione, 2 parts of lipoic acid, 8 parts of lutein, 22 parts of nicotinamide, and 16 parts of vitamin B2.
  4. The pharmaceutical composition according to claims 1-3, wherein the delaying aging is delaying the aging of C. elegans; prolonging the lifespan of C. elegans; enhancing the resistance of C. elegans to heat stress; enhancing the resistance of C. elegans to various oxidative stresses.
  5. A pharmaceutical composition according to claims 1-4, wherein the pharmaceutical composition is in the form of oral preparations, tablets, capsules or granules.
  6. A method of preparing the pharmaceutical composition according to claims 1-5, which is an oral preparation and prepared by weighing β-glucan, chitosan, coenzyme Q10, CBD, glutathione, lipoic acid, lutein, nicotinamide, and vitamin B2 in the ratio of any of claims 1-5, mixing the components and diluting the mixture with distilled water, heating and stirring in a water bath at 40℃ until the mixture is well mixed, and diluting 100 g of the mixture to a constant volume to 1000 mL to obtain the oral preparation.
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