WO2023115628A1 - 一种组合物和制备方法及其用途 - Google Patents

一种组合物和制备方法及其用途 Download PDF

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WO2023115628A1
WO2023115628A1 PCT/CN2021/142986 CN2021142986W WO2023115628A1 WO 2023115628 A1 WO2023115628 A1 WO 2023115628A1 CN 2021142986 W CN2021142986 W CN 2021142986W WO 2023115628 A1 WO2023115628 A1 WO 2023115628A1
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weight
parts
astaxanthin
preparation
nicotinamide mononucleotide
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PCT/CN2021/142986
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English (en)
French (fr)
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赵玲玲
张华�
王莲婧
马鸿鑫
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健合香港有限公司
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Priority to AU2021480072A priority Critical patent/AU2021480072B2/en
Publication of WO2023115628A1 publication Critical patent/WO2023115628A1/zh

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/13—Nucleic acids or derivatives thereof
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/75—Rutaceae (Rue family)
    • A61K36/752—Citrus, e.g. lime, orange or lemon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • A61K8/35—Ketones, e.g. benzophenone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/60—Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9783—Angiosperms [Magnoliophyta]
    • A61K8/9789—Magnoliopsida [dicotyledons]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/20—Hypnotics; Sedatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations

Definitions

  • the invention relates to the technical field of medicine, in particular to a composition, a preparation method and an application thereof.
  • Aging refers to the phenomenon that the body's physiological and psychological adaptability to the environment is progressively reduced, and it gradually tends to die. Aging can be divided into two categories: physiological aging and pathological aging. The former refers to the physiological degeneration process that occurs after maturity, and the latter refers to senile changes caused by various external factors (including various diseases).
  • the physiological changes in the aging process of the human body are mainly reflected in the loss of body tissue cells and constituent substances, the slowdown of the body's metabolic rate, and the decline of body and organ functions.
  • the most direct manifestation of aging is skin wrinkles, skin loss of elasticity, dryness, sagging, and roughness.
  • skin aging is mainly caused by endogenous and exogenous factors in the human body. Endogenous aging is mainly caused by cell oxidation; exogenous aging is mainly caused by many factors such as environment and cell oxidation.
  • causesd by factors such as solar radiation, cigarette smoke or other pollution, while ultraviolet radiation is the main factor of photo
  • Fatigue is a subjective sense of discomfort, mainly manifested as lassitude, lack of energy or physical exhaustion. It will be noticed during long-term labor, strenuous exercise or long-term mental stress. This is a normal physiological protective response, prompting The body should recover its functions through rest, so as to avoid further damage to the body. Fatigue includes mental fatigue and physical fatigue. Mental fatigue is a subjective feeling of lack of motivation and alertness, manifested as dizziness, decreased concentration, difficulty thinking, forgetfulness, etc. Physical fatigue is a feeling of lack of energy or strength in muscles, often manifested as Tired easily after a certain amount of physical activity. With the improvement and improvement of people's material and cultural life, people pay more and more attention to their health.
  • “Poor sleep” has become the norm in this society. Some people are often unable to fall asleep or maintain a sleep state, which seriously leads to sleep insufficiency, which is called sleep onset and sleep maintenance disorders; A condition in which the frequency is too short (light sleep insomnia), early awakening, and insufficient or poor quality sleep. Insufficient sleep can cause adverse effects on human health.
  • the main hazards are: (1) Autonomic dysfunction, manifested as palpitation, sweating, elevated blood pressure, dizziness, headache, low learning and work efficiency, etc. (2) Endocrine dysfunction, facial acne, metabolic disorders of blood lipids and blood sugar may occur. (3) It can lead to mental anxiety and depression, manifested as inner nervousness, lack of security, restlessness, loss of interest, depression, self-blame, etc. (4) Significantly decreased immunity, which can lead to various diseases, such as neurasthenia, colds, and gastrointestinal diseases.
  • Whitening and brightening skin is an eternal topic in skin care. From ancient times to the present, Chinese people have been pursuing fair and smooth skin. The mechanism of melanin formation in human skin is relatively complicated. Genetics, living environment, etc. all have a direct impact on skin color. Asian skin color is mainly determined by the amount of melanin in the epidermis. Reducing the appearance of melanin can achieve fairness.
  • the formation process of skin melanin includes a series of complex physiological and biochemical processes such as the migration of melanocytes, the division and maturation of melanocytes, the formation of melanosomes, the transport of melanin granules, and the excretion of melanin.
  • tyrosine is the main raw material for producing melanin
  • tyrosinase is the main rate-limiting enzyme for the conversion of tyrosine into melanin, and copper
  • tyrosine produces melanin under the action of tyrosinase, and the second action is oxidation process, which must be combined with oxygen to convert into melanin.
  • the principle of whitening is mainly to reduce the formation of melanin by inhibiting tyrosinase, prevent the melanin that has been produced from transferring to the epidermis, decompose and restore the melanin that has been formed, and directly accelerate the shedding of epidermal cells.
  • the present invention provides a composition, a preparation method and its use.
  • the composition not only has the effect of anti-aging, but also has the effects of improving sleep, anti-fatigue, whitening, anti-wrinkle and firming.
  • the technical problem to be solved by the present invention is to provide a composition, preparation method and application thereof. , whitening, anti-oxidation and anti-wrinkle firming effects.
  • An anti-aging composition comprising the following components:
  • Beta-Nicotinamide Mononucleotide Blood Orange Powder Concentrate, and Astaxanthin.
  • the concentration of the ⁇ -nicotinamide mononucleotide is ⁇ 90% by mass
  • the concentration of ⁇ -nicotinamide mononucleotide is 99% by mass
  • the concentrated blood orange powder contains 2.8%-3.2% by mass of anthocyanins, 1.8%-2.2% by mass of hydroxycinnamic acid, and 8.5%-9.5% by mass of flavanones;
  • the concentration of the astaxanthin is 2.5-10% by mass
  • the astaxanthin is selected from at least one of Haematococcus pluvialis powder, astaxanthin oil and astaxanthin microcapsule powder.
  • a method for preparing the anti-aging composition comprising weighing ⁇ -nicotinamide mononucleotide, concentrated blood orange powder and astaxanthin, and mixing them uniformly.
  • An anti-aging preparation which uses the anti-aging composition as an active ingredient.
  • compositions are also included.
  • the anti-aging preparations include liquid preparations and solid preparations.
  • the formulation forms include injections, granules, powders, oral liquids, sprays, powder injections, sugar-coated tablets, film-coated tablets, enteric-coated tablets, orally disintegrating tablets, hard capsules, soft capsules medicaments, buccal preparations, granules, pills, dropping pills, pellets, ointments, elixirs or disintegrants.
  • the anti-aging composition or the anti-aging preparation has any of the following uses:
  • the type I collagen gene includes col1a1a gene and col1a1b gene;
  • the elastin gene includes eln1 gene and eln2 gene.
  • the food includes health food.
  • a composition provided by the present invention including the following components: ⁇ -nicotinamide mononucleotide, concentrated blood orange powder, and astaxanthin; the present invention found that ⁇ -nicotinamide mononucleotide , blood orange concentrated powder and astaxanthin have synergistic anti-aging, sleep improvement, treatment of cardiovascular and cerebrovascular diseases, recovery of nerve health, anti-fatigue, whitening, anti-oxidation and anti-wrinkle firming effects.
  • An anti-aging composition comprising the following components by weight: 2-20 parts by weight of ⁇ -nicotinamide mononucleotide, 2-20 parts by weight of concentrated blood orange powder, and 1 part by weight of astaxanthin -10 parts by weight; within the range of the above weight parts, ⁇ -nicotinamide mononucleotide, concentrated blood orange powder and astaxanthin have significant effects of synergistic anti-aging, improving sleep, anti-fatigue, whitening and anti-wrinkle firming .
  • An anti-aging composition comprising the following components by weight: 4-16 parts by weight of ⁇ -nicotinamide mononucleotide, 4-10 parts by weight of concentrated blood orange powder, and 2 parts by weight of astaxanthin -8 parts by weight; within the range of the above parts by weight, ⁇ -nicotinamide mononucleotide, concentrated blood orange powder and astaxanthin have synergistic anti-aging, sleep improvement, anti-fatigue, whitening and anti-wrinkle firming effects. significantly.
  • the ⁇ -nicotinamide mononucleotide, concentrated blood orange powder, and astaxanthin in the following examples are all commercially available products.
  • the concentration of ⁇ -nicotinamide mononucleotide (NMN) is not lower than 90% by mass, and the concentration of ⁇ -nicotinamide mononucleotide (NMN) in the following examples and experimental examples is 99% by mass; blood orange The concentrated powder contains 2.8%-3.2% by mass of anthocyanins, 1.8%-2.2% by mass of hydroxycinnamic acid, and 8.5%-9.5% by mass of flavanones;
  • the concentration of astaxanthin of the present invention is 2.5% by mass -10% astaxanthin
  • astaxanthin can be selected from Haematococcus pluvialis powder, astaxanthin oil and astaxanthin microcapsule powder, the astaxanthin in the following examples and experimental examples is astaxanthin content quality Percentage 2.5% astaxanthin microencapsulated powder
  • An anti-aging composition the formula is: ⁇ -nicotinamide mononucleotide 10g, blood orange concentrated powder 5g, and astaxanthin 4g.
  • the preparation method of the above-mentioned anti-aging composition is as follows: weighing the above-mentioned ⁇ -nicotinamide mononucleotide, concentrated blood orange powder and astaxanthin, and mixing them evenly.
  • An anti-aging composition the formula is: ⁇ -nicotinamide mononucleotide 16g, blood orange concentrated powder 20g, and astaxanthin 8g.
  • the preparation method of the above-mentioned anti-aging composition is as follows: weighing the above-mentioned ⁇ -nicotinamide mononucleotide, concentrated blood orange powder and astaxanthin, and mixing them evenly.
  • An anti-aging composition the formula of which is: 4g of ⁇ -nicotinamide mononucleotide, 10g of concentrated blood orange powder, and 10g of astaxanthin.
  • the preparation method of the above-mentioned anti-aging composition is as follows: weighing the above-mentioned ⁇ -nicotinamide mononucleotide, concentrated blood orange powder and astaxanthin, and mixing them evenly.
  • An anti-aging composition the formula is: ⁇ -nicotinamide mononucleotide 2g, blood orange concentrated powder 4g, and astaxanthin 8g.
  • the preparation method of the above-mentioned anti-aging composition is as follows: weighing the above-mentioned ⁇ -nicotinamide mononucleotide, concentrated blood orange powder and astaxanthin, and mixing them evenly.
  • An anti-aging composition the formula is: 20g of ⁇ -nicotinamide mononucleotide, 2g of concentrated blood orange powder, and 2g of astaxanthin.
  • the preparation method of the above-mentioned anti-aging composition is as follows: weighing the above-mentioned ⁇ -nicotinamide mononucleotide, concentrated blood orange powder and astaxanthin, and mixing them evenly.
  • An anti-aging composition the formula is: ⁇ -nicotinamide mononucleotide 16g, blood orange concentrated powder 20g, and astaxanthin 1g.
  • the preparation method of the above-mentioned anti-aging composition is as follows: weighing the above-mentioned ⁇ -nicotinamide mononucleotide, concentrated blood orange powder and astaxanthin, and mixing them evenly.
  • the above samples include: the anti-aging composition prepared in Examples 1-6, ⁇ -nicotinamide mononucleotide (NMN), concentrated blood orange powder and astaxanthin.
  • NNN ⁇ -nicotinamide mononucleotide
  • the wild-type AB strain zebrafish was reproduced by natural pair mating. Zebrafish aged 6 hours after fertilization (6hpf) were used for evaluation of NAD + content and NAD + /NADH ratio.
  • Dissecting microscope SZX7, OLYMPUS, Japan
  • CCD camera VertA1, Shanghai Tusen Vision Technology Co., Ltd., China
  • precision electronic balance CP214, OHAUS, USA
  • 6-well plate Nest Biotech, China
  • 96-well plate Nest Biotech, China
  • multifunctional microplate reader SPARK, TECAN, Switzerland
  • high-speed refrigerated centrifuge Heraeus Fresco17, ThermoFisher, Germany).
  • the NAD + content and the NAD + /NADH ratio in the zebrafish of the concentration group were used to evaluate the influence of the samples on the NAD + content and the NAD + /NADH ratio based on the statistical analysis results of this index.
  • Statistical results are expressed as mean ⁇ SE. SPSS26.0 software was used for statistical analysis, and p ⁇ 0.05 indicated that the difference was statistically significant.
  • the above samples include: the anti-aging composition prepared in Examples 1-6, ⁇ -nicotinamide mononucleotide (NMN), concentrated blood orange powder and astaxanthin.
  • NNN ⁇ -nicotinamide mononucleotide
  • Positive control Zhonghua Dieda Pills, dark brown pills, batch number 191112, Guangxi Wuzhou Pharmaceutical (Group) Co., Ltd., stored in a cool and dry place. Prepare a 20.0mg/mL mother solution with ultrapure water, and store in aliquots at -20°C.
  • Positive control salidroside, white powder, batch number 80515SE1158, Huazhong Haiwei (Beijing) Gene Technology Co., Ltd., stored at 4°C. Prepare 4.00mg/mL mother solution with standard dilution water, and prepare it for immediate use.
  • the wild-type AB strain zebrafish was reproduced by natural pair mating. Zebrafish aged 4dpf were used to improve the efficacy of hypoxic fatigue MTC assay and its efficacy evaluation.
  • Dissecting microscope SZX7, OLYMPUS, Japan
  • CCD camera VertA1, Shanghai Tusen Vision Technology Co., Ltd., China
  • behavior analyzer Zebra Lab 3.22.3.31, ViewPoint, France
  • multi-functional microplate reader SPARK, TECAN , Switzerland
  • precision electronic balance CP214, OHAUS, USA
  • 6-well plate Nest Biotech, China
  • 96-well plate Nest Biotech, China
  • white 96-well microplate plate Perkin Elmer, USA.
  • Cobalt chloride hexahydrate (lot number WXBB8070V, Vetec, China); ATP content assay kit (lot number 451306, Promega, USA).
  • the MTC of the samples on the hypoxic fatigue model zebrafish was 2000 ⁇ g/mL, and the formal experimental evaluation concentrations for improving hypoxic fatigue evaluation were all set at 62.5, 125, 250 and 500 ⁇ g/mL. See Table 3 for details.
  • examples 1-6 in terms of increasing the ATP content, compared with the model control group, examples 1-6, NMN (125ug/ml, 250ug/ml, 500ug/ml), astaxanthin (500ug/ml) have significant difference.
  • NMN500ug/ml concentration of NMN500ug/ml
  • embodiment 1 and embodiment 5 compare with NMN500ug/ml, effect is stronger and there is significant difference;
  • Other embodiment compares effect with NMN500ug/ml quite. It is proved that although the prescription only has a lower concentration of NMN, it can show the same or even more significant effect than the high concentration of NMN.
  • the above samples include: the anti-aging composition prepared in Examples 1-6, ⁇ -nicotinamide mononucleotide (NMN), concentrated blood orange powder and astaxanthin.
  • NNN ⁇ -nicotinamide mononucleotide
  • Positive control melatonin, white powder, batch number F1804064, Shanghai Aladdin Biochemical Technology Co., Ltd., stored in a cool and dark place. Prepare 50.0 mg/mL stock solution with DMSO and store at -20°C.
  • the wild-type AB strain zebrafish was reproduced by natural pair mating. Zebrafish aged 4 days post fertilization (4dpf) were used for the determination of the maximum detectable concentration (MTC) of sleep-improving efficacy and its efficacy evaluation.
  • MTC maximum detectable concentration
  • Absolute ethanol (batch number 20210107, Sinopharm Chemical Reagent Co., Ltd., China); FastQuant RT Kit (With gDNase) kit (Cat. No. KR106, TIANGEN, China); RNA-Quick Purification Kit (RNA Rapid Extraction Kit) (Cat. No. RN001 , YiShan Biotech, China); iTaq Universal SYBR Green Supermix (product number 1725124, Bio-rad, USA); pentylenetetrazolium (PTZ, batch number YH0171126, Shanghai Yihe Biotechnology Co., Ltd., China).
  • RNA of zebrafish in each group was extracted using the RNA Rapid Extraction Kit, and the concentration and purity of the total RNA were measured by a UV-visible spectrophotometer. 2.00 ⁇ g of total RNA from zebrafish samples was taken, and 20.0 ⁇ L of cDNA was synthesized according to the instructions of the cDNA first-strand synthesis kit, and the expression of ⁇ -actin and gabra1 genes was detected by q-PCR. Using ⁇ -actin as an internal reference for gene expression, the relative expression level of RNA of gabra1 gene was calculated. Statistical results are expressed as mean ⁇ SE. SPSS26.0 software was used for statistical analysis, and p ⁇ 0.05 indicated that the difference was statistically significant.
  • NMN 125, 250, 500ug/ml
  • blood orange 500ug/ml
  • astaxanthin 500ug/ml
  • embodiment 5 compares with NMN500ug/ml, and effect is stronger and has significant difference
  • Other embodiment compares effect quite with NMN500ug/ml. It is proved that although the prescription only has a lower concentration of NMN, it can show the same or even more significant effect than the high concentration of NMN.
  • the above samples include: the anti-aging composition prepared in Examples 1-6, ⁇ -nicotinamide mononucleotide (NMN), concentrated blood orange powder and astaxanthin.
  • NNN ⁇ -nicotinamide mononucleotide
  • the wild-type AB strain zebrafish was reproduced by natural pair mating. Zebrafish aged 6 hours post fertilization (6hpf) were used for MTC determination of whitening efficacy and its efficacy evaluation.
  • Pierce TM Coomassie (Bradford) protein detection kit (batch number WE317426, Thermo Fisher Scientific (China) Co., Ltd., USA); RIPA lysate (batch number 0205212120315, Shanghai Biyuntian Biotechnology Co., Ltd., China); PMSF (Lot No. ST505, Shanghai Biyuntian Biotechnology Co., Ltd., China); Levodopa (Lot No. I170522, Shanghai Aladdin Biochemical Technology Co., Ltd., China).
  • NMN 250ug/ml, 500ug/ml
  • blood orange 250ug/ml, 500ug/ml
  • astaxanthin 250ug/ml, 500ug/ml
  • the above samples include: the anti-aging composition prepared in Examples 1-6, ⁇ -nicotinamide mononucleotide (NMN), concentrated blood orange powder and astaxanthin.
  • NNN ⁇ -nicotinamide mononucleotide
  • Positive control verisol collagen, white powder, lot number 893660, Gelita, store in a cool place away from light. Prepare the 2.00mg/mL mother solution with standard dilution water, and prepare it for immediate use.
  • the wild-type AB strain zebrafish was reproduced by natural pair mating. Zebrafish aged 4dpf were used for anti-wrinkle and firming efficacy evaluation.
  • Absolute ethanol (batch number 20210107, Sinopharm Chemical Reagent Co., Ltd., China); FastQuant RT Kit (With gDNase) kit (Cat. No. KR106, TIANGEN, China); RNA-Quick Purification Kit (RNA Rapid Extraction Kit) (Cat. No. RN001 , YiShan Biotech, China); iTaq Universal SYBR Green Supermix (Product No. 1725124, Bio-rad, USA).
  • RNA Take 2.00 ⁇ g of total RNA from zebrafish samples, follow the instructions of cDNA first-strand synthesis kit, synthesize 20.0 ⁇ L cDNA, and detect the expression of ⁇ -actin, col1a1a and col1a1b genes by q-PCR. Using ⁇ -actin as an internal reference for gene expression, the relative RNA expression levels of col1a1a and col1a1b genes were calculated. Statistical results are expressed as mean ⁇ SE. SPSS26.0 software was used for statistical analysis, and p ⁇ 0.05 indicated that the difference was statistically significant.
  • RNA from zebrafish samples 2.00 ⁇ g of total RNA from zebrafish samples was taken, and 20.0 ⁇ L of cDNA was synthesized according to the instructions of the cDNA first-strand synthesis kit, and the expression of ⁇ -actin, eln1 and eln2 genes were detected by q-PCR. Using ⁇ -actin as an internal reference for gene expression, the relative RNA expression levels of eln1 and eln2 genes were calculated. Statistical results are expressed as mean ⁇ SE. SPSS26.0 software was used for statistical analysis, and p ⁇ 0.05 indicated that the difference was statistically significant.
  • zebrafish total RNA was extracted, and the concentration and A260/A280 ratio of RNA were measured with a UV-visible spectrophotometer (Table 9). The quality is good and can be used in subsequent q-PCR experiments. See Table 10 for primer sequences.
  • zebrafish total RNA was extracted, and the concentration and A260/A280 ratio of RNA were measured with a UV-visible spectrophotometer (Table 12).
  • the A260/A280 ratio was between 1.8-2.2, indicating that the zebrafish total RNA was extracted
  • the quality is good and can be used in subsequent q-PCR experiments. See Table 13 for primer sequences.
  • NMN 500ug/ml, blood orange 500ug/ml and astaxanthin 500ug/ml are equivalent to the examples in the expression of one or two genes, the other examples are the same as the single blood orange and astaxanthin.
  • NMN 500ug/ml, blood orange 500ug/ml and astaxanthin 500ug/ml are equivalent to the examples in the expression of one or two genes, the other examples are the same as the single blood orange and astaxanthin.
  • NMN 500ug/ml, blood orange 500ug/ml and astaxanthin 500ug/ml are equivalent to the examples in the expression of one or two genes
  • the other examples are the same as the single blood orange and astaxanthin.

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Abstract

涉及医药技术领域，提供的一种组合物，包括如下的组分：β-烟酰胺单核苷酸，血橙浓缩粉，和虾青素；在研究中发现β-烟酰胺单核苷酸、血橙浓缩粉和虾青素之间具有协同抗衰老、改善睡眠、治疗心脑血管疾病、恢复神经健康、抗疲劳、美白、抗氧化和抗皱紧致的作用。

Description

一种组合物和制备方法及其用途 技术领域
本发明涉及医药技术领域，具体涉及一种组合物和制备方法及其用途。
背景技术
衰老，指机体对环境的生理和心理适应能力进行性降低、逐渐趋向死亡的现象。衰老可分为两类：生理性衰老和病理性衰老。前者指成熟期后出现的生理性退化过程，后者是由于各种外来因素(包括各种疾病)所导致的老年性变化。人体衰老过程中的生理变化主要体现在机体组织细胞和构成物质的丧失，机体代谢率的减缓，机体和器官功能减退。衰老最为直接的表现就是皮肤出现皱纹、皮肤失去弹性，干燥，松弛，粗糙等。其中皮肤的老化现象主要是人体受到内源性及外源性因素引起的，内源性的衰老主要是由于细胞氧化而引起的；而外源性的衰老主要是由环境、细胞氧化等诸多因素引起的，如太阳辐射，香烟烟雾或其他污染因素，而紫外线辐射是光老化的主要因素。
疲劳是一种主观的不适感，主要表现为倦怠、精力不够或身体精疲力竭，在进行长时间劳动、剧烈运动或长时间精神压力下会察觉到，这是一种正常的生理性保护反应，提示机体应该通过休息使机能恢复，从而避免对身体的进一步伤害。疲劳包括脑力疲劳和体力疲劳，脑力疲劳是一种缺乏动机与警觉的主观感觉，表现为头脑昏沉、注意力下降、思考困难、健忘等；体力疲劳是感觉到肌肉缺乏能量或力量，常表现为进行一定的体力活动后容易疲劳。随着人们物质文化生活的改善和提高，人们对身体健康越来越重视，但是现代社会生活节奏越来越快，饮食不合理、睡眠不足、作息不正常已经成为常见现象。过度紧张，压力太大，运动不足、长久的情绪不良等，易导致身体免疫力下降，易疲劳。长期处于疲劳状态，会损害人体的体力、体能，导致身体失衡，使人难以从事或完成某些消耗体力较大或动作细腻、精巧的工作，从而使工作效率下降；另一方面，长期疲劳还会使人皮肤松弛，面色无华，呈现出未老先衰的征兆。
“睡眠不好”已经成了这个社会的常态。有的人经常无法入睡或者无法保持睡眠状态，严重导致睡眠不足，这称为入睡和维持睡眠障碍；有的人是经常不能获得正常睡眠为特征，由各种原因导致的入睡困难、睡眠深度或频度过短(浅睡性失眠)、早醒及睡眠时间不足或质量差的一种病症。睡眠不足，对人体健康可造成不利影响，主要危害有：(1)植物神经功能紊乱，表现为心慌、出汗、血压升高、头晕、头痛、学习和工作效率低下等。(2)内分泌功能紊乱，可出现面部痤疮，血脂、血糖的代谢紊乱。(3)可导致精神焦虑、抑郁，表现为内心紧张不安、缺乏安全感、坐立不安、兴趣丧失、情绪低落、自责等。(4)免疫力明显的感觉降低，由此会导致种种疾病发生，如神经衰弱、感冒、胃肠疾病等。
美白亮肤是皮肤护理永恒的话题，从古至今国人都在追求白皙滑嫩的肌肤。人体皮肤 黑色素形成的机理比较复杂，遗传基因、生活环境等都对皮肤颜色有直接影响，亚洲人皮肤颜色主要由表皮中黑色素数量决定，减少黑色素的呈现即可获得白皙效果。皮肤黑素的形成过程包括黑素细胞的迁移、黑素细胞的分裂成熟、黑素小体的形成、黑素颗粒的转运以及黑素的排泄等一系列复杂的生理生化过程。从生物化学反应的角度来看，黑素的形成必须有三种基本物质：酪氨酸为制造黑素的主要原料；酪氨酸酶是酪氨酸转变为黑素的主要限速酶，为铜及蛋白质的组合物；酪氨酸在酪氨酸酶的作用下产生黑素，次种作用为氧化过程，必须与氧结合才能转变为黑素。美白的原理主要就是通过抑制酪氨酸酶而减少黑色素形成，阻止已经生产的黑色素转移至表皮层，并分解还原已经形成的黑色素，还有就是直接加快表皮细胞的脱落。
随着人们生活水平的提高以及保养护理意识的增强，人们越来越注重抗衰老、抗疲劳、改善睡眠以及美白皮肤护理，为此，本发明提供一种组合物和制备方法及其用途，所述组合物不仅具有抗衰老的作用，还具有改善睡眠、抗疲劳、美白和抗皱紧致的作用。
发明内容
因此，本发明要解决的技术问题在于提供一种组合物和制备方法及其用途，所述组合物不仅具有抗衰老的作用，还具有改善睡眠、治疗心脑血管疾病、恢复神经健康、抗疲劳、美白、抗氧化和抗皱紧致的作用。
为此，本发明提供了如下的技术方案：
一种抗衰老组合物，包括如下的组分：
β-烟酰胺单核苷酸，血橙浓缩粉，和虾青素。
可选的，包括如下重量份的组分：
β-烟酰胺单核苷酸2-20重量份，血橙浓缩粉2-20重量份，和虾青素1-10重量份。
可选的，包括如下重量份的组分：
β-烟酰胺单核苷酸4-16重量份，血橙浓缩粉4-10重量份，和虾青素2-8重量份。
可选的，包括如下重量份的组分：
β-烟酰胺单核苷酸10重量份，血橙浓缩粉5重量份，和虾青素4重量份；或
β-烟酰胺单核苷酸16重量份，血橙浓缩粉20重量份，和虾青素8重量份；或
β-烟酰胺单核苷酸4重量份，血橙浓缩粉10重量份，和虾青素10重量份；或
β-烟酰胺单核苷酸2重量份，血橙浓缩粉4重量份，和虾青素8重量份；或
β-烟酰胺单核苷酸20重量份，血橙浓缩粉2重量份，和虾青素2重量份；或
β-烟酰胺单核苷酸16重量份，血橙浓缩粉20重量份，和虾青素1重量份。
可选的，所述β-烟酰胺单核苷酸的浓度为≥质量百分比90％；
可选的，所述β-烟酰胺单核苷酸的浓度为质量百分比99％；
可选的，所述血橙浓缩粉中含花青素质量百分比2.8％-3.2％，羟基肉桂酸质量百分比1.8％-2.2％，黄烷酮类质量百分比8.5％-9.5％；
可选的，所述虾青素的浓度为质量百分比2.5-10％；
可选的，所述虾青素选自雨生红球藻粉、虾青素油和虾青素微囊粉中的至少一种。
一种所述的抗衰老组合物制备方法，称取β-烟酰胺单核苷酸、血橙浓缩粉和虾青素，混合均匀。
一种抗衰老制剂，以所述的抗衰老组合物为活性成分。
可选的，还包括制剂允许的药物赋形剂或载体。
可选的，所述的抗衰老制剂形式包括液体制剂和固体制剂。
可选的，所述制剂形式包括注射液、冲剂、散剂、口服液、喷雾剂、粉针剂、糖衣片剂、薄膜衣片剂、肠溶衣片剂、口崩片、硬胶囊剂、软胶囊剂、口含剂、颗粒剂、丸剂、滴丸剂、微丸、膏剂、丹剂或崩解剂。
所述的抗衰老组合物或所述的抗衰老制剂具有如下任一项的用途：
(1)在制备抗衰老、改善睡眠、治疗心脑血管疾病、恢复神经健康、抗疲劳、美白、抗氧化或抗皱紧致的食品、药物或护肤品中的应用；
(2)在制备提高人体或动物的NAD +/NADH水平的食品、药物或护肤品中的应用；
(3)在制备提高人体或动物的NAD +水平的食品、药物或护肤品中的应用；
(4)在制备提高人体或动物的ATP含量水平的食品、药物或护肤品中的应用；
(5)在制备提高人体或动物的gabra1基因表达水平的食品、药物或护肤品中的应用；
(6)在制备抑制人体或动物的酪氨酸酶活性的食品、药物或护肤品中的应用；
(7)在制备提高人体或动物的I型胶原蛋白基因表达水平的食品、药物或护肤品中的应用；
(8)在制备提高人体或动物的弹性蛋白基因表达水平的食品、药物或护肤品中的应用；
可选的，I型胶原蛋白基因包括col1a1a基因和col1a1b基因；
可选的，弹性蛋白基因包括eln1基因和eln2基因。
可选的，所述食品包括保健食品。
本发明技术方案，具有如下优点：
1.本发明提供的一种组合物，包括如下的组分：β-烟酰胺单核苷酸，血橙浓缩粉，和虾青素；本发明在研究中发现β-烟酰胺单核苷酸、血橙浓缩粉和虾青素之间具有协同抗衰老、改善睡眠、治疗心脑血管疾病、恢复神经健康、抗疲劳、美白、抗氧化和抗皱紧致的作用。
2.本发明提供的一种抗衰老组合物，包括如下重量份的组分：β-烟酰胺单核苷酸2-20重量份，血橙浓缩粉2-20重量份，和虾青素1-10重量份；在上述重量份范围内，β-烟酰胺单核苷酸、血橙浓缩粉和虾青素之间具有协同抗衰老、改善睡眠、抗疲劳、美白和抗皱紧致的作用显著。
3.本发明提供的一种抗衰老组合物，包括如下重量份的组分：β-烟酰胺单核苷酸4-16重量份，血橙浓缩粉4-10重量份，和虾青素2-8重量份；在上述重量份范围内，β-烟酰胺单核苷酸、血橙浓缩粉和虾青素之间具有协同抗衰老、改善睡眠、抗疲劳、美白和抗皱紧致的作用更显著。
具体实施方式
提供下述实施例是为了更好地进一步理解本发明，并不局限于所述最佳实施方式，不对本发明的内容和保护范围构成限制，任何人在本发明的启示下或是将本发明与其他现有技术的特征进行组合而得出的任何与本发明相同或相近似的产品，均落在本发明的保护范围之内。
实施例中未注明具体实验步骤或条件者，按照本领域内的文献所描述的常规实验步骤的操作或条件即可进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规试剂产品。
下述实施例中的β-烟酰胺单核苷酸，血橙浓缩粉，和虾青素均市售产品。β-烟酰胺单核苷酸(NMN)浓度不低于质量百分比90％，在下述实施例中和实验例中的β-烟酰胺单核苷酸(NMN)浓度为质量百分比99％；血橙浓缩粉中含花青素质量百分比2.8％-3.2％，羟基肉桂酸质量百分比1.8％-2.2％，黄烷酮类质量百分比8.5％-9.5％；本发明的虾青素为浓度为质量百分比2.5-10％的虾青素，虾青素可以选自雨生红球藻粉、虾青素油和虾青素微囊粉，在下述实施例和实验例中的虾青素为虾青素含量质量百分比2.5％的虾青素微囊粉。β-烟酰胺单核苷酸(NMN)原料不限于化学合成法、发酵法和生物酶催化法制备。
实施例1
一种抗衰老组合物，配方为：β-烟酰胺单核苷酸10g，血橙浓缩粉5g，和虾青素4g。
上述抗衰老组合物的制备方法为：称取上述的β-烟酰胺单核苷酸、血橙浓缩粉和虾青素，混合均匀。
实施例2
一种抗衰老组合物，配方为：β-烟酰胺单核苷酸16g，血橙浓缩粉20g，和虾青素8g。
上述抗衰老组合物的制备方法为：称取上述的β-烟酰胺单核苷酸、血橙浓缩粉和虾青素，混合均匀。
实施例3
一种抗衰老组合物，配方为：β-烟酰胺单核苷酸4g，血橙浓缩粉10g，和虾青素10g。
上述抗衰老组合物的制备方法为：称取上述的β-烟酰胺单核苷酸、血橙浓缩粉和虾青素，混合均匀。
实施例4
一种抗衰老组合物，配方为：β-烟酰胺单核苷酸2g，血橙浓缩粉4g，和虾青素8g。
上述抗衰老组合物的制备方法为：称取上述的β-烟酰胺单核苷酸、血橙浓缩粉和虾青素，混合均匀。
实施例5
一种抗衰老组合物，配方为：β-烟酰胺单核苷酸20g，血橙浓缩粉2g，和虾青素2g。
上述抗衰老组合物的制备方法为：称取上述的β-烟酰胺单核苷酸、血橙浓缩粉和虾青素，混合均匀。
实施例6
一种抗衰老组合物，配方为：β-烟酰胺单核苷酸16g，血橙浓缩粉20g，和虾青素1g。
上述抗衰老组合物的制备方法为：称取上述的β-烟酰胺单核苷酸、血橙浓缩粉和虾青素，混合均匀。
实验例1 NAD +含量、NAD +/NADH比值评价
1.检测材料
1.1.样品配制信息
样品均用标准稀释水配制成2.00mg/mL母液，现配现用。
上述样品包括：实施例1-6制备的抗衰老组合物、β-烟酰胺单核苷酸(NMN)、血橙浓缩粉和虾青素。
1.2.实验动物
斑马鱼均饲养于28℃的养鱼用水中(水质：每1L反渗透水中加入200mg速溶海盐，电导率为450～550μS/cm；pH为6.5～8.5；硬度为50～100mg/L CaCO 3)，实验动物使用许可证号为：SYXK(浙)2012-0171。饲养管理符合国际AAALAC认证(认证编号：001458)的要求。
野生型AB品系斑马鱼，以自然成对交配繁殖方式进行。年龄为受精后6小时(6hpf)的斑马鱼用于NAD +含量、NAD +/NADH比值评价。
1.3.仪器、耗材与试剂
解剖显微镜(SZX7，OLYMPUS，Japan)；CCD相机(VertA1，上海土森视觉科技有限公司，China)；精密电子天平(CP214，OHAUS，USA)；6孔板(Nest Biotech，China)；96孔板(Nest Biotech，China)；多功能酶标仪(SPARK，TECAN，Switzerland)；高速冷冻离心机(Heraeus Fresco17，ThermoFisher，Germany)。
NAD(H)含量检测试剂盒(货号BC0315，Solarbio，China)；无水乙醇(批号20200302，国药集团化学试剂有限公司，China)；Quick Start Bradford 1x Dye Reagent(货号500-0205，Bio-rad，USA)。
2.检测方法
2.1.MTC测定
随机选取6hpf野生型AB品系斑马鱼于6孔板中，每孔(实验组)30尾。分别水溶给予样品(浓度见表1)，同时设置正常对照组，每孔容量为3mL。所有组别每天进行换液，28℃处理6天后，确定各样品对模型斑马鱼的MTC。
2.2.NAD +含量以及NAD +/NADH比值检测
随机选取6hpf野生型AB品系斑马鱼于6孔板中，每孔(实验组)均处理30尾斑马鱼。分别水溶给予样品(浓度见表2)，同时设置正常对照组，每孔容量为3mL。平行设 置三次实验。所有组别每天进行换液，28℃处理6天后，按照NAD(H)含量检测试剂盒说明书收集斑马鱼样本，利用多功能酶标仪进行NAD +水平检测和分析NAD +/NADH比值，分析各浓度组斑马鱼体内NAD +含量和NAD +/NADH比值，以该指标的统计学分析结果评价样品对NAD +含量和NAD +/NADH比值的影响。统计学处理结果采用mean±SE表示。用SPSS26.0软件进行统计学分析，p<0.05表明差异具有统计学意义。
3.检测结果
3.1.MTC
详见表1。
表1.样品抗衰老功效浓度摸索实验结果(n＝30)
Figure PCTCN2021142986-appb-000001
3.2.NAD +含量和NAD +/NADH比值检测
详见表2。
表2.各组NAD +含量和NAD +/NADH比值检测结果(n＝30)
Figure PCTCN2021142986-appb-000002
注：与正常对照组比较，*P＜0.05，**P＜0.01，***P＜0.001。NMN、血橙和虾青素同样引起了显著相差异，因此将引起显著性差异的组别同实施例1-6进行了比较，不同字母表示具有显著性差异(P＜0.05)。
由上述表2可知，在提高NAD+/NADH比值方面，与正常对照组比较，实施例1-6的组方和NMN能够显著提高NAD+/NADH比值，具有显著差异(***P＜0.001)，而血橙和虾青素无显著差异。实施例1-6的组方和NMN比较，实施例1-6的组方与NMN组具有显著的差异，实施例1-6的组方提高NAD+/NADH比值更显著。
实验例2：改善缺氧疲劳功效
1.检测材料
1.1.样品配制信息
样品均用标准稀释水配制成2.00mg/mL母液，现配现用。
上述样品包括：实施例1-6制备的抗衰老组合物、β-烟酰胺单核苷酸(NMN)、血橙浓缩粉和虾青素。
阳性对照：中华跌打丸，黑褐色丸剂，批号191112，广西梧州制药(集团)股份有限公司，阴凉干燥储存。用超纯水配制成20.0mg/mL母液，-20℃分装储存。
阳性对照：红景天苷，白色粉末，批号80515SE1158，华中海威(北京)基因科技有限公司，4℃储存。用标准稀释水配制成4.00mg/mL母液，现配现用。
1.2.实验动物
斑马鱼均饲养于28℃的养鱼用水中(水质：每1L反渗透水中加入200mg速溶海盐，电导率为450～550μS/cm；pH为6.5～8.5；硬度为50～100mg/L CaCO 3)，实验动物使用许可证号为：SYXK(浙)2012-0171。饲养管理符合国际AAALAC认证(认证编号：001458)的要求。
野生型AB品系斑马鱼，以自然成对交配繁殖方式进行。年龄为4dpf的斑马鱼用于改善缺氧疲劳功效MTC测定及其功效评价。
1.3.仪器、耗材与试剂
解剖显微镜(SZX7，OLYMPUS，Japan)；CCD相机(VertA1，上海土森视觉科技有限公司，China)；行为分析仪(Zebra Lab 3.22.3.31，ViewPoint，France)；多功能酶标仪(SPARK，TECAN，Switzerland)；精密电子天平(CP214，OHAUS，USA)；6孔板(Nest Biotech，China)；96孔板(Nest Biotech，China)；白色96孔酶标板(Perkin Elmer，USA)。
六水合氯化钴(批号WXBB8070V，Vetec，China)；ATP含量测定试剂盒(批号451306，Promega，USA)。
2.检测方法
2.1.MTC测定
随机选取4dpf野生型AB品系斑马鱼于6孔板中，每孔30尾。分别水溶给予(浓度见表3)，同时设置正常对照组和模型对照组，每孔容量为3mL。28℃处理24h后，除正常对照组外，其余各实验组均水溶给予六水合氯化钴(1mg/mL)建立斑马鱼缺氧疲劳模型。28℃继续处理2.5h后，确定样品对模型斑马鱼的MTC。
2.2.改善缺氧疲劳功效评价
随机选取4dpf野生型AB品系斑马鱼于6孔板中，每孔30尾。分别水溶给予样品(浓度见表4)，阳性对照：红景天苷4000μg/mL浓度，同时设置正常对照组和模型对照组，每孔容量为3mL。平行设置两次实验。28℃处理24h后，除正常对照组外，其余各实验组均水溶给予六水合氯化钴(1mg/mL)建立斑马鱼缺氧疲劳模型。1)28℃继续处理1.5h后，每个实验组随机选取10尾斑马鱼，利用行为分析仪测定斑马鱼的总运动距离。2)28℃继续处理2.5h后，每个实验组随机选取10尾斑马鱼，按照ATP含量测定试剂盒说明书收集斑马鱼样本，利用多功能酶标仪进行ATP含量检测。以上述指标的统计学分析结果评价样品对缺氧疲劳斑马鱼运动功能的改善功效。统计学处理结果采用mean±SE表示。用SPSS26.0软件进行统计学分析，p<0.05表明差异具有统计学意义。
3.检测结果
3.1.MTC
在本实验条件下，样品对缺氧疲劳模型斑马鱼的MTC均为2000μg/mL，改善缺氧疲劳评价正式实验评价浓度均设置为62.5、125、250和500μg/mL。详见表3。
表3.样品改善缺氧疲劳功效浓度摸索实验结果(n＝30)
Figure PCTCN2021142986-appb-000003
3.2.改善缺氧疲劳功效评价
详见表4。
表4.各组ATP含量(光信号强度)检测结果(n＝30)
Figure PCTCN2021142986-appb-000004
Figure PCTCN2021142986-appb-000005
注：与模型对照组比较，*P＜0.05，**P＜0.01，***P＜0.001。NMN和虾青素同样引起了显著相差异，因此将引起显著性差异的组别同实施例1-6进行了比较，不同字母具有显著性差异(P＜0.05)。
由上述表4可知，在提高ATP含量方面，与模型对照组比较，实施例1-6、NMN(125ug/ml、250ug/ml、500ug/ml)、虾青素(500ug/ml)具有显著性差异。组间比较时，除了NMN500ug/ml浓度，其它浓度同实施例比较均具有显著性差异。其中实施例1和实施例5同NMN500ug/ml比较，效果更强且有显著性差异；其它实施例同NMN500ug/ml相比效果相当。证明组方虽然只有较低浓度的NMN，但可以表现同高浓度NMN相当甚至更显著的效果。
实验例3：改善睡眠功效
1.检测材料
1.1.样品配制信息
样品均用标准稀释水配制成2.00mg/mL母液，现配现用。
上述样品包括：实施例1-6制备的抗衰老组合物、β-烟酰胺单核苷酸(NMN)、血橙浓缩粉和虾青素。
阳性对照：褪黑素，白色粉末，批号F1804064，上海阿拉丁生化科技股份有限公司，阴凉避光储存。用DMSO配制成50.0mg/mL母液，-20℃储存。
1.2.实验动物
斑马鱼均饲养于28℃的养鱼用水中(水质：每1L反渗透水中加入200mg速溶海盐， 电导率为450～550μS/cm；pH为6.5～8.5；硬度为50～100mg/L CaCO 3)，实验动物使用许可证号为：SYXK(浙)2012-0171。饲养管理符合国际AAALAC认证(认证编号：001458)的要求。
野生型AB品系斑马鱼，以自然成对交配繁殖方式进行。年龄为受精后4天(4dpf)的斑马鱼用于改善睡眠功效最大检测浓度(MTC)测定及其功效评价。
1.3.仪器、耗材与试剂
解剖显微镜(SZX7，OLYMPUS，Japan)；CCD相机(VertA1，上海土森视觉科技有限公司，China)；精密电子天平(CP214，OHAUS，USA)；6孔板(Nest Biotech，China)；普通PCR扩增仪(T100，BIO-RAD，Singapore)；荧光定量PCR仪(CFX Connect，BIO-RAD，Singapore)；高速冷冻离心机(Heraeus Fresco17，ThermoFisher，Germany)；紫外-可见光分光光度计(Nanodrop2000，Thermo，Austria)；微孔板迷你离心机(BE-6100，海门市其林贝尔仪器制造有限公司，China)；低位裙边96孔板(透明)(HSP9601，Bio-rad，USA)；光学粘性封膜B(MSB1001，Bio-rad，USA)。
无水乙醇(批号20210107，国药集团化学试剂有限公司，China)；FastQuant RT Kit(With gDNase)试剂盒(货号KR106，TIANGEN，China)；RNA-Quick Purification Kit(RNA快速提取试剂盒)(货号RN001，YiShan Biotech，China)；iTaq Universal SYBR Green Supermix(货号1725124，Bio-rad，USA)；戊四唑(PTZ，批号YH0171126，上海依赫生物科技有限公司，China)。
2.检测方法
2.1.MTC测定
随机选取4dpf野生型AB品系斑马鱼于6孔板中，每孔(实验组)30尾。分别水溶给予样品(浓度见表5)，同时设置正常对照组和模型对照组，每孔容量为3mL。28℃处理24h后，除正常对照组外，其余各实验组均水溶给予PTZ(5mM)建立斑马鱼失眠模型。28℃继续处理1h后，确定样品对模型斑马鱼的MTC。
2.2.改善睡眠功效评价
随机选取4dpf野生型AB品系斑马鱼于6孔板中，每孔30尾。分别水溶给予样品(浓度见表6)，阳性对照褪黑素125μg/mL浓度，同时设置正常对照组和模型对照组，每孔容量为3mL。平行设置三次实验。28℃处理24h后，除正常对照组外，其余各实验组均水溶给予PTZ(5mM)建立斑马鱼失眠模型。28℃继续处理1h后，使用RNA快速提取试剂盒提取各组斑马鱼总RNA，利用紫外-可见光分光光度计对总RNA浓度和纯度进行测定。取2.00μg斑马鱼样品总RNA，按照cDNA第一链合成试剂盒说明操作，合成20.0μL cDNA，通过q-PCR检测β-actin和gabra1基因的表达。用β-actin作为基因表达的内参， 计算gabra1基因的RNA相对表达量。统计学处理结果采用mean±SE表示。用SPSS26.0软件进行统计学分析，p<0.05表明差异具有统计学意义。
3.检测结果
3.1.MTC
详见表5。
表5.样品改善睡眠功效浓度摸索实验结果(n＝30)
Figure PCTCN2021142986-appb-000006
3.2.改善睡眠功效评价
详见表6。
表6.各组gabra1基因相对表达量检测结果(n＝30)
Figure PCTCN2021142986-appb-000007
Figure PCTCN2021142986-appb-000008
注：与模型对照组比较，*P＜0.05，**P＜0.01，***P＜0.001。NMN、血橙和虾青素同样引起了显著相差异，因此将引起显著性差异的组别同实施例1-6进行了比较，不同字母具有显著性差异(P＜0.05)。
由上述表6可知，在提高gabra1基因相对表达量方面，与模型对照组比较，实施例1-6、NMN(125、250、500ug/ml)、血橙(500ug/ml)、虾青素(500ug/ml)具有显著的差异。组间比较时，除了NMN500ug/ml浓度，其它浓度同实施例比较均具有显著性差异。其中实施例5同NMN500ug/ml比较，效果更强且有显著性差异；其它实施例同NMN500ug/ml相比效果相当。证明组方虽然只有较低浓度的NMN，但可以表现同高浓度NMN相当甚至更显著的效果。
实验例4：美白功效
1.检测材料
1.1.样品配制信息
样品均用标准稀释水配制成2.00mg/mL母液，现配现用。
上述样品包括：实施例1-6制备的抗衰老组合物、β-烟酰胺单核苷酸(NMN)、血橙浓缩粉和虾青素。
阳性对照：熊果苷，白色粉末，批号C12020957，上海麦克林生化科技有限公司，-20℃储存。用DMSO配制成300mg/mL母液，-20℃储存。
1.2.实验动物
斑马鱼均饲养于28℃的养鱼用水中(水质：每1L反渗透水中加入200mg速溶海盐，电导率为450～550μS/cm；pH为6.5～8.5；硬度为50～100mg/L CaCO 3)，实验动物使用许可证号为：SYXK(浙)2012-0171。饲养管理符合国际AAALAC认证(认证编号：001458)的要求。
野生型AB品系斑马鱼，以自然成对交配繁殖方式进行。年龄为受精后6小时(6hpf)的斑马鱼用于美白功效MTC测定及其功效评价。
1.3.仪器、耗材与试剂
解剖显微镜(SZX7，OLYMPUS，Japan)；CCD相机(VertA1，上海土森视觉科技有限公司，China)；精密电子天平(CP214，OHAUS，USA)；6孔板(Nest Biotech，China)；高速冷冻离心机(Heraeus Fresco17，ThermoFisher，Germany)；多功能酶标仪(SPARK，TECAN，Switzerland)。
Pierce TM考马斯(Bradford)蛋白检测试剂盒(批号WE317426，赛默飞世尔科技(中国)有限公司，USA)；RIPA裂解液(批号0205212120315，上海碧云天生物技术有限公司，China)；PMSF(批号ST505，上海碧云天生物技术有限公司，China)；左旋多巴(批号I170522，上海阿拉丁生化科技股份有限公司，China)。
2.检测方法
2.1.MTC测定
随机选取6hpf野生型AB品系斑马鱼于6孔板中，每孔30尾。分别水溶给予样品(浓度见表7)，同时设置正常对照组，每孔容量为3mL。每天换液。28℃处理4天后，确定样品对正常斑马鱼的MTC。
2.2.美白功效评价
随机选取6hpf野生型AB品系斑马鱼于6孔板中，每孔30尾。分别水溶给予样品(浓度见表8)，阳性对照熊果苷3000μg/mL浓度，同时设置正常对照组，每孔容量为3mL。每天换液。平行设置三次实验。28℃处理4天后，采用多巴胺氧化法，在475nm波长处测定吸光度值，以该指标的统计学分析结果评价样品对斑马鱼酪氨酸酶活性的抑制功效。统计学处理结果采用mean±SE表示。用SPSS26.0软件进行统计学分析，p<0.05表明差异具有统计学意义。
3.检测结果
3.1.MTC
在本实验条件下，美白功效评价正式实验评价浓度均设置为125、250和500μg/mL，详见表7。
表7.美白功效浓度摸索实验结果(n＝30)
Figure PCTCN2021142986-appb-000009
3.2.美白功效评价
详细见表8。
表8.各组美白功效检测结果(n＝30)
Figure PCTCN2021142986-appb-000010
Figure PCTCN2021142986-appb-000011
注：与正常对照组比较，*P＜0.05，**P＜0.01，***P＜0.001。对比例同样引起了显著相差异，因此同实施例1-6进行了比较，不同字母具有显著性差异(P＜0.05)。
由上述表8可知，在提高美白方面，在提高ATP含量方面，与正常对照组比较，实施例1-6、NMN(250ug/ml、500ug/ml)、血橙(250ug/ml、500ug/ml)、虾青素(250ug/ml、500ug/ml)具有显著性差异。组间比较时，除了NMN、血橙500ug/ml浓度，其它浓度同实施例比较均具有显著性差异。实施例同NMN以及血橙500ug/ml相比效果相当。证明组方虽然只有较低浓度的NMN或血橙，但可以表现同高浓度NMN或血橙相当甚至更显著的效果。
实验例5：抗皱紧致功效
1.检测材料
1.1.样品配制信息
样品均用标准稀释水配制成2.00mg/mL母液，现配现用。
上述样品包括：实施例1-6制备的抗衰老组合物、β-烟酰胺单核苷酸(NMN)、血橙浓缩粉和虾青素。
阳性对照：verisol胶原蛋白，白色粉末，批号893660，嘉利达，阴凉避光储存。用标准稀释水配制成2.00mg/mL母液，现配现用。
1.2.实验动物
斑马鱼均饲养于28℃的养鱼用水中(水质：每1L反渗透水中加入200mg速溶海盐，电导率为450～550μS/cm；pH为6.5～8.5；硬度为50～100mg/L CaCO 3)，实验动物使用许可证号为：SYXK(浙)2012-0171。饲养管理符合国际AAALAC认证(认证编号：001458)的要求。
野生型AB品系斑马鱼，以自然成对交配繁殖方式进行。年龄为4dpf的斑马鱼用于抗皱紧致功效评价。
1.3.仪器、耗材与试剂
解剖显微镜(SZX7，OLYMPUS，Japan)；CCD相机(VertA1，上海土森视觉科技有限公司，China)；精密电子天平(CP214，OHAUS，USA)；6孔板(Nest Biotech，China)；普通PCR扩增仪(T100，BIO-RAD，Singapore)；荧光定量PCR仪(CFX Connect，BIO-RAD， Singapore)；高速冷冻离心机(Heraeus Fresco17，ThermoFisher，Germany)；紫外-可见光分光光度计(Nanodrop2000，Thermo，Austria)；微孔板迷你离心机(BE-6100，海门市其林贝尔仪器制造有限公司，China)；低位裙边96孔板(透明)(HSP9601，Bio-rad，USA)；光学粘性封膜B(MSB1001，Bio-rad，USA)。
无水乙醇(批号20210107，国药集团化学试剂有限公司，China)；FastQuant RT Kit(With gDNase)试剂盒(货号KR106，TIANGEN，China)；RNA-Quick Purification Kit(RNA快速提取试剂盒)(货号RN001，YiShan Biotech，China)；iTaq Universal SYBR Green Supermix(货号1725124，Bio-rad，USA)。
2.检测方法
2.1.抗皱紧致(I型胶原蛋白基因)功效评价
随机选取4dpf野生型AB品系斑马鱼于6孔板中，每孔30尾。分别水溶给予样品(浓度见表9)，阳性对照verisol胶原蛋白250μg/mL浓度，同时设置正常对照组，每孔容量为3mL。平行设置三次实验。28℃处理24h后，使用RNA快速提取试剂盒提取各组斑马鱼总RNA，利用紫外-可见光分光光度计对总RNA浓度和纯度进行测定。取2.00μg斑马鱼样品总RNA，按照cDNA第一链合成试剂盒说明操作，合成20.0μL cDNA，通过q-PCR检测β-actin、col1a1a和col1a1b基因的表达。用β-actin作为基因表达的内参，计算col1a1a和col1a1b基因的RNA相对表达量。统计学处理结果采用mean±SE表示。用SPSS26.0软件进行统计学分析，p<0.05表明差异具有统计学意义。
2.2.抗皱紧致(弹性蛋白基因)功效评价
随机选取4dpf野生型AB品系斑马鱼于6孔板中，每孔30尾。分别水溶给予样品(浓度见表12)，阳性对照verisol胶原蛋白250μg/mL浓度，同时设置正常对照组，每孔容量为3mL。平行设置三次实验。28℃处理24h后，使用RNA快速提取试剂盒提取各组斑马鱼总RNA，利用紫外-可见光分光光度计对总RNA浓度和纯度进行测定。取2.00μg斑马鱼样品总RNA，按照cDNA第一链合成试剂盒说明操作，合成20.0μL cDNA，通过q-PCR检测β-actin、eln1和eln2基因的表达。用β-actin作为基因表达的内参，计算eln1和eln2基因的RNA相对表达量。统计学处理结果采用mean±SE表示。用SPSS26.0软件进行统计学分析，p<0.05表明差异具有统计学意义。
3.检测结果
3.1.抗皱紧致(I型胶原蛋白基因)功效评价
3.1.1.RNA提取结果及引物序列信息
在实验终点，提取斑马鱼总RNA，用紫外-可见光分光光度计测定RNA的浓度及A260/A280比值(表9)，A260/A280比值均在1.8-2.2之间，表明提取得到斑马鱼总RNA质量较好，可用于后续q-PCR实验。引物序列见表10。
表9.总RNA的浓度及A260/A280比值(n＝30)
Figure PCTCN2021142986-appb-000012
表10.引物序列信息
Figure PCTCN2021142986-appb-000013
3.1.2 col1a1a基因相对表达量和col1a1b基因相对表达量
详细见表11。
表11.各组col1a1a/col1a1b基因相对表达量检测结果(n＝30)
Figure PCTCN2021142986-appb-000014
注：与正常对照组比较，*P＜0.05，**P＜0.01，***P＜0.001。对比例同样引起了显著相差异，因此同实施例1-6进行了比较，不同字母具有显著性差异(P＜0.05)。
由上述表11可知，除去血橙500ug/ml以及虾青素500ug/ml在其中一个基因的表达中同实施例相当以外，其它实施例同单独的血橙、虾青素和NMN相比均具有更优的效果，且具有显著性差异。证明组方虽然只有较低浓度的血橙或虾青素，但可以表现同高浓度血橙或虾青素相当甚至更显著的效果。
3.2.抗皱紧致(弹性蛋白基因)功效评价
3.2.1.RNA提取结果及引物序列信息
在实验终点，提取斑马鱼总RNA，用紫外-可见光分光光度计测定RNA的浓度及A260/A280比值(表12)，A260/A280比值均在1.8-2.2之间，表明提取得到斑马鱼总RNA质量较好，可用于后续q-PCR实验。引物序列见表13。
表12.总RNA的浓度及A260/A280比值(n＝30)
Figure PCTCN2021142986-appb-000015
Figure PCTCN2021142986-appb-000016
表13.引物序列信息
Figure PCTCN2021142986-appb-000017
3.2.2 eln1基因相对表达量和eln2基因相对表达量
详细见表14。
表14.各组eln1/eln2基因相对表达量检测结果(n＝30)
Figure PCTCN2021142986-appb-000018
注：与正常对照组比较，*P＜0.05，**P＜0.01，***P＜0.001。对比例同样引起了显著相差异，因此同实施例1-6进行了比较，不同字母具有显著性差异(P＜0.05)。
由上述表14可知，除去NMN500ug/ml,血橙500ug/ml以及虾青素500ug/ml在其中一个或两个基因的表达中同实施例相当以外，其它实施例同单独的血橙、虾青素和NMN相比均具有更优的效果，且具有显著性差异。证明组方虽然只有较低浓度的NMN，血橙或虾青素，但可以表现同高浓度NMN，血橙或虾青素相当甚至更显著的效果。
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。

Claims (10)

  1. 一种组合物，其特征在于，包括如下的组分：
    β-烟酰胺单核苷酸，血橙浓缩粉，和虾青素。
  2. 根据权利要求1所述的组合物，其特征在于，包括如下重量份的组分：
    β-烟酰胺单核苷酸2-20重量份，血橙浓缩粉2-20重量份，和虾青素1-10重量份。
  3. 根据权利要求1或2所述的组合物，其特征在于，包括如下重量份的组分：
    β-烟酰胺单核苷酸4-16重量份，血橙浓缩粉4-10重量份，和虾青素2-8重量份。
  4. 根据权利要求1-3任一项所述的组合物，其特征在于，包括如下重量份的组分：
    β-烟酰胺单核苷酸10重量份，血橙浓缩粉5重量份，和虾青素4重量份；或
    β-烟酰胺单核苷酸16重量份，血橙浓缩粉20重量份，和虾青素8重量份；或
    β-烟酰胺单核苷酸4重量份，血橙浓缩粉10重量份，和虾青素10重量份；或
    β-烟酰胺单核苷酸2重量份，血橙浓缩粉4重量份，和虾青素8重量份；或
    β-烟酰胺单核苷酸20重量份，血橙浓缩粉2重量份，和虾青素2重量份；或
    β-烟酰胺单核苷酸16重量份，血橙浓缩粉20重量份，和虾青素1重量份。
  5. 根据权利要求1-4任一项所述的组合物，其特征在于，所述β-烟酰胺单核苷酸的浓度为≥质量百分比90％；
    可选的，所述β-烟酰胺单核苷酸的浓度为质量百分比99％；
    可选的，所述血橙浓缩粉中含花青素质量百分比2.8％-3.2％，羟基肉桂酸质量百分比1.8％-2.2％，黄烷酮类质量百分比8.5％-9.5％；
    可选的，所述虾青素的浓度为质量百分比2.5-10％；
    可选的，所述虾青素选自雨生红球藻粉、虾青素油和虾青素微囊粉中的至少一种。
  6. 一种权利要求1-5任一项所述的组合物制备方法，其特征在于，称取β-烟酰胺单核苷酸、血橙浓缩粉和虾青素，混合均匀。
  7. 一种制剂，其特征在于，以权利要求1-5任一项所述的抗衰老组合物为活性成分。
  8. 根据权利要求7所述的制剂，其特征在于，还包括制剂允许的药物赋形剂或载体。
  9. 根据权利要求7或8所述的制剂，其特征在于，所述的抗衰老制剂形式包括液体制剂和固体制剂；
    可选的，所述制剂形式包括注射液、冲剂、散剂、口服液、喷雾剂、粉针剂、糖衣片剂、薄膜衣片剂、肠溶衣片剂、口崩片、硬胶囊剂、软胶囊剂、口含剂、颗粒剂、丸剂、滴丸剂、微丸、膏剂、丹剂或崩解剂。
  10. 权利要求1-5任一项所述的组合物或权利要求7-9任一项所述的制剂具有如下任 一项的用途：
    (1)在制备抗衰老、改善睡眠、治疗心脑血管疾病、恢复神经健康、抗疲劳、美白、抗氧化或抗皱紧致的食品、药物或护肤品中的应用；
    (2)在制备提高人体或动物的NAD +/NADH水平的食品、药物或护肤品中的应用；
    (3)在制备提高人体或动物的NAD +水平的食品、药物或护肤品中的应用；
    (4)在制备提高人体或动物的A4TP含量水平的食品、药物或护肤品中的应用；
    (5)在制备提高人体或动物的gabra1基因表达水平的食品、药物或护肤品中的应用；
    (6)在制备抑制人体或动物的酪氨酸酶活性的食品、药物或护肤品中的应用；
    (7)在制备提高人体或动物的I型胶原蛋白基因表达水平的食品、药物或护肤品中的应用；
    (8)在制备提高人体或动物的弹性蛋白基因表达水平的食品、药物或护肤品中的应用；
    可选的，I型胶原蛋白基因包括col1a1a基因和col1a1b基因；
    可选的，弹性蛋白基因包括eln1基因和eln2基因；
    可选的，所述食品包括保健食品。
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