WO2022022225A1 - 具有抗衰老作用的多肽rv3和rv4及其应用 - Google Patents
具有抗衰老作用的多肽rv3和rv4及其应用 Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- 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
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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Definitions
- the invention belongs to the field of biological medicine, and more particularly, relates to a polypeptide with anti-aging activity and its application.
- Aging is a process in which the body goes through a period of sexual maturity, the self-renewal and repair capabilities of cells are weakened, the structure and function of tissues and organs degenerate, and eventually die. Risk increases. With the aging of the global population, various degenerative diseases of the elderly and the huge medical expenses incurred have become more and more serious social problems. Maintaining the healthy state of the elderly population is a key link in reducing the social and economic burden brought about by population aging. Therefore, it is of great significance to explore measures and methods for intervening in aging and aging-related diseases that are simple, economical, effective, safe and suitable for promotion.
- Aging-related diseases generally refer to diseases whose incidence increases with aging.
- aging-related diseases are mainly considered to be cardiovascular disease, tumor, rheumatism, osteoporosis, cataract, type 2 diabetes, hypertension and Alzheimer's disease.
- cardiovascular disease mainly considered to be cardiovascular disease, tumor, rheumatism, osteoporosis, cataract, type 2 diabetes, hypertension and Alzheimer's disease.
- aging-related diseases are homologous to the aging process, and the aging process itself is the basic risk factor for many aging-related diseases.
- Social responsibility has also prompted biologists to realize that prolonging lifespan alone cannot alleviate the heavy socioeconomic burden brought about by aging, and only prolonging healthy lifespan has practical significance.
- C. elegans As a classic model animal for studying aging, C. elegans has the following advantages: firstly, its relatively short lifespan, only 2-3 weeks under standard laboratory conditions, makes lifespan survival analysis possible; secondly, in controlled It is easy to obtain a large number of genetically identical animals under environmental conditions; third, the transparency of the worm body facilitates the direct observation of how cells and tissues change with age; Research to aid in anti-aging.
- a major advantage of the C. elegans model is the ease with which genetic methods can be used to provide biological information, allowing the identification of a large number of genetic mutations that can alter lifespan.
- C. elegans lifespan exhibits remarkable plasticity and may be affected by environmental, nutritional, and genetic mutations, and even under controlled conditions, individual lifespan may vary, revealing stochastic factors in aging. Therefore, C. elegans can be used as a model animal to study the anti-aging activity of drugs.
- anti-aging drugs By far the most common form of anti-aging is the use of anti-aging drugs.
- Most of the anti-aging drugs used clinically are synthetic drugs, such as vitamin E, which can promote cell division and inhibit the generation of oxygen free radicals, procaine preparations can prolong cell lifespan, and amidopyrone can delay brain aging, etc.
- Oxidative stress delaying the decline of body function caused by aging, thereby prolonging the lifespan of nematodes
- Metformin as an activator of adenylate-activated protein kinase (AMPK)
- AMPK adenylate-activated protein kinase
- PAL-12 A six-membered peptide
- resveratrol analogs and other drugs are also favored for their anti-aging effects.
- the present invention provides a polypeptide with anti-aging activity and application thereof.
- the polypeptide of the present invention can effectively prolong the lifespan of Caenorhabditis elegans, has good anti-aging effect and has great development prospects.
- the polypeptide with anti-aging activity is characterized in that X 1 X 2 is AF or KA.
- Said polypeptide with anti-aging activity is characterized in that its amino acid sequence is TAFAA (named RV3) or TKAAA (named RV4).
- one or more pharmaceutically acceptable adjuvants can also be added to the above-mentioned medicines, and the adjuvants include conventional diluents, fillers, adhesives, wetting agents, absorption enhancers, surface active agents in the pharmaceutical field. agents, lubricants and stabilizers.
- the medicine of the present invention can be made into various forms such as injection, freeze-dried powder injection, tablet or granule.
- the medicines in the above-mentioned various dosage forms can be prepared according to the conventional methods in the pharmaceutical field.
- the polypeptide of the present invention has a novel structure, the basic unit of which is composed of natural amino acids, and is easy to synthesize, separate and purify;
- the polypeptide of the present invention has good safety, weak adverse reactions and toxic side effects, does not affect the growth and development of Caenorhabditis elegans, and does not affect the reproductive ability of the nematodes;
- the anti-aging effect of the polypeptide involved in the present invention performs well in the Caenorhabditis elegans model, which is embodied in significantly improving the motor behavior of the nematode, delaying the decline of the motor ability in the aging process of the nematode, prolonging half the survival days of the nematode, increasing the Stress stress capacity of nematodes and prolong lifespan of nematodes.
- FIG. 1 Survival curves of experiments on the effect of polypeptides on C. elegans lifespan. It can be seen from the figure that the RV4 polypeptide group has the effect of significantly prolonging the lifespan of C. elegans.
- Figure 2 Results of the effect of polypeptides on the body length and width of Caenorhabditis elegans. Among them, picture A is the result of body length, and picture B is the result of body width. The results are expressed as Mean ⁇ SEM. Compared with the blank group, n.s. means no significant difference, *P ⁇ 0.05 means significant difference, **P ⁇ 0.01 means very significant difference. There was no significant difference between the polypeptide group and the blank group, indicating that the polypeptides RV3 and RV4 did not affect the normal growth and development of C. elegans, and the polypeptides had good safety.
- Figure 3 Results of the effect of polypeptides on the number of broods in Caenorhabditis elegans. The results are expressed as Mean ⁇ SEM. Compared with the blank group, n.s. means no significant difference, *P ⁇ 0.05 means significant difference, **P ⁇ 0.01 means very significant difference. There was no significant difference between the polypeptide group and the blank group, indicating that the polypeptides RV3 and RV4 did not affect the normal reproductive ability of C. elegans, and the polypeptides had good safety.
- Figure 4 Results of the effect of polypeptides on the motor behavior of Caenorhabditis elegans.
- picture A is the result of head swing
- picture B is the result of body bending
- picture C is the result of pharyngeal pump frequency.
- the results are expressed as Mean ⁇ SEM.
- n.s. means no significant difference
- *P ⁇ 0.05 means significant difference
- **P ⁇ 0.01 means very significant difference.
- RV4 polypeptide group had significant differences, indicating that the polypeptide RV4 could enhance the muscle movement of C. elegans and improve the locomotor behavior of C. elegans.
- FIG. 5 Survival curves of acute heat stress experiments in C. elegans. Picture A is the survival curve of Day4 acute heat stress experimental nematodes, and picture B is the survival curve of Day8 acute heat stress experimental nematodes. It can be seen from the figure that both the RV3 and RV4 peptide groups can significantly improve the ability of nematodes to acute heat stress, indicating that the peptide drug can prolong the life cycle of nematodes in a thermal environment.
- Figure 6 Motility rating results of acute heat stress experiments in C. elegans.
- picture A is the exercise rating result of Day4 acute heat stress experimental nematodes
- B picture is the exercise ability rating result of Day8 acute heat stress experimental nematodes.
- the proportion of A-grade nematodes in the RV3 and RV4 peptide groups at Day4 is higher than that in the blank group
- the proportion of C-grade nematodes in the RV3 and RV4 peptide groups at Day8 is lower than that of the blank group, which further shows that
- the polypeptide drug can enhance the ability of Caenorhabditis elegans acute heat stress, that is, the ability of motor behavior under heat environment.
- Fig. 7 The detection results of the acute oxidative stress experiment of Caenorhabditis elegans. Picture A is the detection result of the acute oxidative stress test on Day4, and picture B is the detection result of the acute oxidative stress test on Day8. The results are expressed as Mean ⁇ SEM. Compared with the blank group, *P ⁇ 0.05 is a significant difference, and **P ⁇ 0.01 is a very significant difference. It can be seen from the figure that the survival rate of nematodes in the RV4 polypeptide group is significantly higher than that in the blank group, which indicates that the polypeptide drug can prolong the life cycle of nematodes in an oxidative environment.
- Figure 8 Motility rating results of acute oxidative stress experiments in C. elegans.
- picture A shows the exercise rating results of the nematodes in the acute oxidative stress experiment on Day 4
- picture B shows the exercise ability grading results in the nematodes in the acute oxidative stress experiment on Day 8.
- the proportion of nematodes in the RV3 and RV4 peptide groups in grade A is higher than that of the blank group, which further indicates that the peptide drug can enhance the ability of C. elegans acute oxidative stress;
- the Day4 acute oxidative stress experiment there was no difference in exercise between the peptide group and the blank group.
- Figure 9 Results of motility rating of C. elegans at different ages.
- picture A is the grading result of the nematode motility at the time point of Day4
- picture B is the grading result of the movement ability of the nematodes at the time point of Day8
- picture C is the grading result of the movement ability of the nematodes at the time point of Day12.
- the proportion of C-level nematodes in the RV3 and RV4 peptide groups on Day8 and Day12 was significantly lower than that in the blank group, which means that the peptides can delay the muscle aging of C. elegans. Increases the mobility of the nematode, thereby extending the lifespan of the nematode.
- Figure 10 The results of motility testing of C. elegans at different ages. Among them, picture A is the test result of nematode motility at the time point of Day4, picture B is the test result of the movement ability of nematodes at the time point of Day8, and picture C is the test result of the movement ability of nematodes at the time point of Day12. The results are expressed as Mean ⁇ SEM. Compared with the blank group, *P ⁇ 0.05 is a significant difference, and **P ⁇ 0.01 is a very significant difference.
- RV4 polypeptide can delay the muscle aging of C. elegans, increase The motility of nematodes can delay the decline of nematode motility, thereby prolonging the lifespan of nematodes.
- Polypeptide RV3 TAFAA and peptide RV4: TKAAA were synthesized by Jiangsu Synthetic Polypeptide Drug Discovery and Evaluation Engineering Center with purities of 97.30% and 93.85%, respectively.
- Escherichia coli OP50 culture conditions: shaking incubator, 220rpm, 37°C
- RV3 Thr-Ala-Phe-Ala-Ala, 10 nM
- RV4 Thr-Lys-Ala-Ala-Ala, 10 nM
- Synchronization of Caenorhabditis elegans Select an NGM petri dish with a moderate nematode density, pick L4 larvae with a picking needle, transfer them to a blank NGM petri dish, pick about 20 larvae in total, and wait until they develop into adults and give birth After the first batch of eggs, all the adults were picked away. After about 12 hours, the eggs hatched into larvae, and the synchronized L1-stage larvae were obtained.
- Preliminary preparation for the C. elegans lifespan experiment Rinse the NGM medium with 1 ml of M9 buffer, collect the buffer containing the L1 stage larvae in an EP tube, place in a chromatographic cabinet at 4°C for 5 minutes, and centrifuge at 1500 x g/4°C for 3 minutes. Discard the supernatant, resuspend in 100 ⁇ l of buffer, and count the nematodes under the microscope, then dilute the nematode concentration to 30 worms/10 ⁇ l; Observe and count to ensure that the number of nematodes is about 30, and then place the NGM petri dish in a constant temperature incubator for cultivation.
- Caenorhabditis elegans lifespan experiment After the L1 stage nematodes developed to the L4 stage, 100 ⁇ l of 15.6 ⁇ g/ml 5-FUDR was added to inhibit the oviposition of C. elegans; when the nematodes developed into adults, the drug was started and divided into control groups , 10nM administration group, this time was recorded as the first day of the lifespan experiment; after that, daily administration, observed and recorded the number of nematodes that survived, died and unexpectedly died, and observed and recorded the state of nematodes until the last nematode died. The criteria for judging death were: C.
- the number of days of death in the RV4 polypeptide group was relatively delayed, and the longest lifespan of nematodes was 33 days, 5 days longer than that in the blank group; the number of days of death in the RV3 group was relatively delayed compared with the blank group, and the nematodes had a longer lifespan than the blank group.
- the longest lifespan was 30 days, 2 days longer than the blank group, see Table 1 and Figure 1 for details.
- the half of the death days of the RV4 administration group had a very significant difference, indicating that the RV4 polypeptide has the effect of significantly prolonging the lifespan of C. elegans, which can prolong the lifespan by 4 days; No significant difference. See Table 2 and Figure 1 for details, and the experimental results are statistically significant.
- the detection method was as follows: 20 Caenorhabditis elegans were picked from each group, picked on a new NGM solid medium, and then 50 ⁇ l of NaN 3 was added dropwise at a concentration of 0.4M, after most of the worms are rigid, take pictures and record them under an inverted microscope, and then use the ruler tool of Photoshop to measure the body length and width of C. elegans. One-way ANOVAY statistical analysis was performed on the data, and the data results were expressed as Mean ⁇ SEM. Compared with the Control group, *P ⁇ 0.05 was a significant difference, and **P ⁇ 0.01 was a very significant difference.
- Reproductive ability detection of Caenorhabditis elegans Collect the larvae at L1 stage after synchronization, spread them on NGM solid medium inoculated with Escherichia coli OP50, and divide them into Control group and 10nM administration group, which are placed in a constant temperature and humidity incubator Cultured to L4 stage larvae.
- Detection of motor behavior of C. elegans larvae at L1 stage after synchronization were plated on NGM solid medium inoculated with Escherichia coli OP50, divided into Control group and 10nM administration group, placed in a constant temperature and humidity incubator Culture for 48h. Then, the following three indicators of motor behavior were detected: head swing frequency, 20 nematodes were picked from each group to a new clean medium, and after 1 hour of adaptation and recovery, an appropriate amount of M9 buffer was added and observed under an inverted microscope.
- RV4 polypeptide group Compared with the blank group, the RV4 polypeptide group had significant difference. This means that RV4 polypeptide can significantly enhance the muscle movement of Caenorhabditis elegans and improve the motor behavior of nematodes; RV3 polypeptide has no significant difference compared with the blank group. See Table 5 and Figure 4 for details, and the experimental results are statistically significant.
- Acute heat stress experiment of C. elegans Take C. elegans on the 4th day of continuous administration, and then place them in a 35°C constant temperature incubator for stress stress experiments. Record the life and death of the nematodes every 4 hours until the last one The nematodes died, and the criteria for judging death were the same as in the lifespan experiment; at the same time, at the 24th hour after the start of the experiment, the motor behavior of the surviving C. elegans was rated, and the mobility was divided into three grades, A, B, and C.
- a nematode that can crawl autonomously is defined as A, a nematode that starts to move only after the pick needle touches the head is defined as C, and a state between the two is defined as B; take the nematodes on the 8th day of continuous administration and place them at 35°C In a constant temperature incubator, the life and death of the nematodes were recorded every 2 hours until the last death, and the criteria for judging death and rating criteria for mobility were the same as the heat stress experiment on the fourth day above. Kaplan-Meier statistical analysis was performed on the data, and the data results were expressed as Median ⁇ SE. Compared with the Control group, *P ⁇ 0.05 was a significant difference, and **P ⁇ 0.01 was a very significant difference.
- Acute oxidative stress experiment of C. elegans Take C. elegans on the 4th and 8th day of continuous administration, soak in 30mM H2O2 for 4h, and then transfer to NGM solid medium for recovery after 24h , record the number of survival and death of C. elegans under a microscope, and rate the motor behavior of the surviving nematodes. The criteria for judging death and mobility are the same as those in the acute heat stress experiment.
- One-way ANOVAY statistical analysis was performed on the data, and the data results were expressed as Mean ⁇ SEM. Compared with the Control group, *P ⁇ 0.05 was a significant difference, and **P ⁇ 0.01 was a very significant difference.
- a nematode that can crawl autonomously is defined as A
- C a nematode that starts to move only after the pick needle touches the head
- B a state between the two
- a nematode that can crawl autonomously is defined as A
- C a nematode that starts to move only after the pick needle touches the head
- B a state between the two
- a nematode that can crawl autonomously is defined as A
- C a nematode that starts to move only after the pick needle touches the head
- B a state between the two
- a nematode that can crawl autonomously is defined as A
- C a nematode that starts to move only after the pick needle touches the head
- B a state between the two
- C. elegans exercise ability aging experiment take C. elegans on the 4th, 8th and 12th day of continuous administration, respectively, to detect the nematode's motor behavior, including head swing frequency and pharyngeal pump frequency, the detection method is the same Kinetic force detection in Example 4.
- the motility of the surviving nematodes was rated, and the rating standard was the same as the motility rating in Example 5.
- One-way ANOVAY statistical analysis was performed on the data, and the data results were expressed as Mean ⁇ SEM. Compared with the Control group, *P ⁇ 0.05 was a significant difference, and **P ⁇ 0.01 was a very significant difference.
- C C. elegans that can crawl autonomously is defined as A
- C nematodes that start to move only after the pick needle touches the head is defined as C
- B B
- the results of the nematode motility rating on Day8 and Day12 showed that in the RV3 and RV4 peptide groups, the proportion of nematodes in grade C was lower than that in the blank group; in the RV4 peptide group, the proportion of nematodes in grade A was higher than that in the blank group.
- the polypeptide can delay the muscle aging of C. elegans, increase the motor behavior of the nematodes, thereby prolonging the lifespan of the nematodes, as shown in Table 16 and Fig. 9 for details.
- the test results of nematode motility on Day4, Day8 and Day12 showed that the number of head swings between the RV4 polypeptide group and the blank group was significantly different, and the effect on the frequency of the pharyngeal pump was significantly different on the fourth day.
- the frequency of pharyngeal pump was not significantly higher than that of the blank group, which means that the polypeptide can delay the muscle aging of C. elegans, increase the motor behavior of C.
- the lifespan of nematodes; RV3 polypeptide group and blank group had no significant difference. See Table 17 and Figure 10 for details, and the experimental results are statistically significant.
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Abstract
Description
Claims (6)
- 一种具有抗衰老活性的多肽,其特征在于:多肽的氨基酸序列为X-TX 1X 2AA或其药学上可以接受的盐;其中X=H、乙酰基或丙酰基;X 1和X 2:任意氨基酸。
- 根据权利要求1所述的一种具有抗衰老活性的多肽,其特征在于X 1X 2为AF或者KA。
- 根据权利要求1或2中所述的一种具有抗衰老活性的多肽,其特征在于其氨基酸序列为TAFAA或TKAAA。
- 根据权利要求1至3任意一项所述的多肽在制备抗衰老药物或保健品中的应用。
- 根据权利要求4所述的应用,其特征在于所述的多肽中还加入一种或多种药学上可接受的辅料,所述辅料包括药学领域常规的稀释剂、填充剂、粘合剂、湿润剂、吸收促进剂、表面活性剂、润滑剂和稳定剂。
- 根据权利要求5所述的应用,其特征在于所述的多肽制成注射剂、冻干粉针剂、片剂或颗粒剂。
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JP2022521563A JP7367205B2 (ja) | 2020-07-27 | 2021-07-02 | 抗老化作用を有するポリペプチドrv3及びrv4、並びにその使用 |
EP21850783.8A EP4190798A4 (en) | 2020-07-27 | 2021-07-02 | POLYPEPTIDES RV3 AND RV4 WITH ANTI-AGING FUNCTION AND APPLICATIONS THEREOF |
US17/767,397 US20240076313A1 (en) | 2020-07-27 | 2021-07-02 | Polypeptides rv3 and rv4 having anti-aging function and applications thereof |
AU2021319252A AU2021319252A1 (en) | 2020-07-27 | 2021-07-02 | Polypeptides RV3 and RV4 having anti-aging function and applications thereof |
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WO2004031211A2 (en) * | 2002-10-03 | 2004-04-15 | Epimmune Inc. | Hla binding peptides and their uses |
CN104402975A (zh) * | 2014-12-11 | 2015-03-11 | 太原锦波生物医药科技有限公司 | 抗衰老短肽及其制备方法 |
CN109206480A (zh) * | 2017-07-03 | 2019-01-15 | 大连理工大学 | 一类促胶原分泌超短肽及其应用 |
CN109232714A (zh) * | 2017-07-03 | 2019-01-18 | 大连理工大学 | 一类用于促胶原分泌的超短肽及其应用 |
CN111704651A (zh) * | 2020-07-27 | 2020-09-25 | 南京安吉生物科技有限公司 | 具有抗衰老作用的多肽rv3和rv4及其应用 |
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AU8305491A (en) * | 1990-08-01 | 1992-03-02 | Cytel Corporation | Novel immunosuppressant peptides |
CN1329160A (zh) * | 2000-06-21 | 2002-01-02 | 上海博德基因开发有限公司 | 一种新的多肽——人线粒体天冬氨酸转氨酶91.85和编码这种多肽的多核苷酸 |
WO2010063124A1 (en) * | 2008-12-05 | 2010-06-10 | Angiochem Inc. | Peptide therapeutic conjugates and uses thereof |
CN104256347B (zh) * | 2014-08-19 | 2017-02-22 | 天津市泉又今生物科技有限公司 | 生物强化细胞营养素在制备抗衰老功能食品中的应用 |
CN107875403A (zh) * | 2017-10-31 | 2018-04-06 | 华南理工大学 | 一种基于模式生物秀丽隐杆线虫的抗衰老评价方法与应用 |
CA3098481A1 (en) * | 2018-04-27 | 2019-10-31 | National Research Council Of Canada | High affinity monoclonal antibodies (mabs) against cell surface expressed human carbonic anhydrase ix (hca-ix), and uses thereof |
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WO2004031211A2 (en) * | 2002-10-03 | 2004-04-15 | Epimmune Inc. | Hla binding peptides and their uses |
CN104402975A (zh) * | 2014-12-11 | 2015-03-11 | 太原锦波生物医药科技有限公司 | 抗衰老短肽及其制备方法 |
CN109206480A (zh) * | 2017-07-03 | 2019-01-15 | 大连理工大学 | 一类促胶原分泌超短肽及其应用 |
CN109232714A (zh) * | 2017-07-03 | 2019-01-18 | 大连理工大学 | 一类用于促胶原分泌的超短肽及其应用 |
CN111704651A (zh) * | 2020-07-27 | 2020-09-25 | 南京安吉生物科技有限公司 | 具有抗衰老作用的多肽rv3和rv4及其应用 |
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