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Back to Journal »Drug Design, Development and Treatment» Volume 15

Synthesis of a new coniferin amino acid derivative and its anti-aging effect on Caenorhabditis elegans by regulating DAF-16/FOXO

Authors: Wang Wen, Feng Xin, Du Ying, Liu C, Pang Xu, Jiang Ke, Wang Xu, Liu Yan

Published on October 5, 2021, Volume 2021: 15 pages, 4177-4193 pages

DOI https://doi.org/10.2147/DDDT.S330223

Single anonymous peer review

Editor approved for publication: Dr. Anastasios Lymperopoulos

Wenqi Wang,* Xin Feng,* Yu Du, Cen Liu, Xinxin Pang, Kunxiu Jiang, Xirui Wang, Yonggang Liu School of Chinese Medicine, Beijing University of Chinese Medicine, Beijing, 102488, People’s Republic of China *These authors have contributed equally. Corresponding author: Liu Yonggang Email [email protected] Purpose: Pine lignin is a dihydroflavonoid compound, which is widely present in many plant species. Although pinocembrin has good pharmacological activity, it has poor water solubility and low bioavailability. Therefore, we use a combination of different amino acids to modify the structure of rosin to solve this problem. In addition, the influence of their anti-aging activity has not been explored. Our purpose is to study the effect of rosin and its amino acid derivatives on the aging of Caenorhabditis elegans. Method: splicing coniferin with different amino acids to obtain the corresponding derivatives. Using the Caenorhabditis elegans model, the anti-aging effects of coniferin and its amino acid derivatives were preliminarily studied. Among all the compounds, the one with the best anti-aging effect, and then its anti-aging mechanism was studied. The ROS content of nematodes and the expression of sod-3p::GFP fusion protein were used to explain the protection of nematodes in emergency situations. The possible resistance of nematodes was determined by the DAF-16 nuclear localization experiment and the survival curve of the transgenic nematode model under stress conditions. Mechanism of aging. Results: Pb-3 has the best effect on improving the tolerance of heat stress and oxidative stress and reducing the accumulation of lipofuscin. In the measurement of C. elegans, pb-3 reduced the accumulation of intracellular ROS. The application of pb-3 to the transgenic mutant TJ356 induces the translocation of the transcription factor DAF-16 from the cytoplasm to the nucleus, and regulates the expression of SOD-3 (a downstream gene of daf-16), thereby regulating C. elegans. In addition, pb-3 did not extend the lifespan of daf-16, age-1, daf-2 and hsp16.2 mutants, indicating that these genetic pathways are involved in mediating the anti-aging effects of pb-3. Conclusion: The antioxidant and anti-aging properties of pb-3 may be involved in the transcription process of DAF-16/FOXO. Pinocembrin amino acid derivatives may be a new anti-aging treatment drug. Keywords: coniferin, anti-aging, oxidative stress, amino acid derivatives, Caenorhabditis elegans, DAF-16/FOXO transcription factor errata have been published

Aging is an inevitable biological process that occurs in every organism including humans. Aging is considered to be the main risk factor for the development of many human diseases, such as neurodegenerative diseases, cancer, diabetes or heart disease. 1 Therefore, anti-aging research has received more and more attention, which has important practical significance for human development. Drug research to prevent aging. ROS is one of the main factors leading to aging and age-related diseases. It is produced during aerobic respiration and various metabolic reactions. Importantly, overproduction of ROS can lead to oxidative stress. 2,3 In this case, medicinal plants provide a highly diverse natural product that can be used as a potential anti-aging agent. There are more than 300 compounds reported in the literature, including 185 natural compounds (such as resveratrol, astragaloside IV or rutin) and 55 natural product complexes or extracts. These compounds have significant resistance to oxidative stress. Oxidation activity. 4-8

Pinocembrin (PB; 5,7-dihydroxyflavanone, C15H12O4) is a natural flavonoid compound, which has been found in several plants, such as many genera of Piperaceae, including 14 genera and 1950 species. It is reported that these plants are rich in Contains pine lignin. 9 In fact, previous studies have shown that pine lignin has anti-inflammatory, anti-fungal, anti-oxidant, anti-cancer and anti-allergic activities. 9-15 In addition, pine lignin has a neuroprotective effect on cerebral ischemia injury by reducing reactive oxygen species (ROS), protects the blood-brain barrier, regulates mitochondrial function, and regulates cell apoptosis. 16-22

A number of studies have found that the biological activity of the compound is affected by amino acid groups. For example, using baicalein as the parent compound combined with amino acids to synthesize new neuroprotective agents, and synthesizing carboxamide derivatives through the reaction of amino acids and phthalic anhydride, showing better antibacterial activity. Compared with baicalein, 3-aminomethyl Glycine derivatives have better antibacterial activity. 23-25 ​​There are 24 small molecule drugs approved by the FDA in 2019, 13 of which contain amino acids, diamines or amino alcohols, and are generally considered to be derived from parent amino acids. 26 Therefore, modifying the structure of pinesin by introducing fragments containing amino acid residues may produce compounds with useful biological properties.

The free-living nematode C. elegans is an ideal model for genetic and anti-aging research because the complete sequence of the nematode is closely homologous to the human genome. The nematode body is small and transparent, easy to observe, and has a short lifespan (about three weeks), and the nematode culture is also easy to handle. 27 Since aging is characterized by progressive degenerative changes in tissues and functions, it is feasible to measure age-related changes in neuromuscular behavior (such as pharynx pumping) and biochemical substances (such as lipofuscin accumulation). The additional advantage of Cryptocaryon elegans. 28

In order to understand the effect of amino acid derivatives of rosin protein, we conducted experiments on the nematode model C. elegans. Pinocembrin is believed to have multiple effects on the prevention of many diseases, and we explored whether it can fight aging or extend the life of nematodes. Another purpose of this research is to determine whether coniferin derivatives have more effective anti-aging effects. In order to answer these questions, the following experiments were conducted to explore the effect of pine tree amino acid derivatives on nematode model nematodes.

Caenorhabditis elegans strains were obtained from the Caenorhabditis Genetics Center (CGC, University of Minnesota), including N2 (wild type), CF1553 (muIs84 [(pAD76) sod-3::GFP)]), DR26 daf-16 (m26) , TJ356 (zIs356[daf-16p::daf-16a/b::GFP rol-6]), VC475 hsp-16.2 (gk249), CB1370 daf-2 (e1370) III and TJ1052 age-1 (hx546)). Throughout the experiment, all strains were maintained on Nematode Growth Medium (NGM) plates inoculated with E. coli OP50.

Scheme 1 Synthesis of pinocembrin derivatives (Figure 1). Reagents and conditions: boc-amino acid, dichloromethane (DCM), 4-dimethylaminopyridine (DMAP), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) ); 25°C; 12 hours. Scheme 1 Synthesis of pinocembrin derivatives. Figure 1 The structure of pinocembrin derivatives 1-8.

Scheme 1 Synthesis of pinocembrin derivatives.

Figure 1 The structure of pinocembrin derivatives 1-8.

The synthetic route is shown in Figure 2 (take Boc-Gly-OH as an example), and all the designed derivatives are synthesized according to the following steps. The compound rosin (1 equivalent (equivalent)) was dissolved in anhydrous DCM (25 ml) and DMAP (0.5 equivalent), and then the protected amino acid (1.2 equivalent) and EDCI (1.5 equivalent) were added to the solution, and the mixture Stir at 25°C for 12 hours under nitrogen. After the reaction was completed (monitored by TLC), the solution was evaporated and washed with saturated sodium carbonate solution (20 mL). The aqueous layer was extracted with DCM (25 mL), and the combined organic extracts were washed with brine (20 mL), dried over sodium sulfate, filtered and evaporated. After concentration under reduced pressure, the wet mixture was eluted isocratically on a silica gel column with dichloromethane and methanol as eluents to purify the crude product. Figure 2 Synthetic route of Boc-Gly-OH.

Figure 2 Synthetic route of Boc-Gly-OH.

2.2.1 (S)-5-Hydroxy-4-oxo-2-phenylchroman-7-yl (tert-butoxycarbonyl) phenylalaninate (pb-1) yellow powder, yield 39.73%; 1H-NMR (400 MHz, DMSO- d6, Figure S1-1) δ: 11.94 (1H, s, OH-5), 7.63 (1H, d, J = 7.0 Hz, CONH), 7.53-7.55 (2H, m, H-2', 6') , 7.40–7.47 (3H, m, H-3', 4', 5'), 7.24–7.31 (5H, m, Ph-3''), 6.20 (1H, s, H-8), 6.15 (1H , s, H-6), 5.71–5.75 (1H, m, H-2), 4.34–4.39 (1H, m, H-2``), 3.39–3.47 (1H, m, H-3a), 3.01 –3.13 (2H, m, H-3''), 2.88–2.92 (1H, m, H-3b), 1.36 (9H, s, (CH3)3C); 13C-NMR (100 MHz, DMSO-d6, Figure S1-2) δ: 197.5 (C-4), 170.0 (C-1``), 162.1 (C-5), 162.0 (C-9), 157.8 (C-7), 155.4 (CONH), 138.1 (C-1'), 137.0 (C-1'''), 129.1 (C-5'), 128.6 (C-3'), 128.5 (C-5')''), 128.2 (C-3' ''), 126.5 (C-2', 4', 6', 2'''', 4''', 6'''), 105.9 (C-10), 102.3 (C-6), 101.2 ( C-8), 78.6 (C-2, (CH3)3C), 55.4 (C-2''), 42.2 (C-3), 36.0 (C-3''), 27.98 ((CH3)3C).

2.2.2 (S)-5-Hydroxy-4-oxo-2-phenylchroman-7-yl-N-(tert-butoxycarbonyl)-N-methylglycinate (pb-2) was obtained as a yellow powder with a yield of 35.22%; 1H-NMR (400 MHz, DMSO-d6, Figure S2-1) δ: 11.95 (1H, s, OH), 7.53–7.55 (2H, m, H-2', 6'), 7.40–7.47 (3H, m, H-3', 4', 5'), 6.34–6.39 (2H, m, H-6, 8), 5.72–5.76 (1H, m, H-2), 4.22–4.23 (2H, m, H-2''), 3.40–3.48 (1H, m, H-3a), 2.88–2.93 (4H, overlap, H-3b, CH3-N), 1.37--1.41 (9H, overlap, (CH3)3C) ; 13C-NMR (100 MHz, DMSO-d6, Figure S2-2) δ: 197.7 (C-4), 168.0 (C-1''), 162.2 (C-5), 157.5 (C-9), 155.3 (C-7), 154.6 (COO), 138.2 (C-1'), 128.6 (C-3', 5'), 126.7 (C-2', 4', 6'), 106.1 (C-10) , 102.4 (C-6), 101.3 (C-8), 79.3 (C-2), 78.8 ((CH3)3C), 50.6 (C-2``), 42.3 (C-3), 35.4 (CH3- N) ), 27.9 ((CH3)3C).

2.2.3 (S)-5-Hydroxy-4-oxo-2-phenylchroman-7-yl (tert-butoxycarbonyl)-alaninate (pb-3), yellow powder, yield 40.10%; 1H-NMR (400 MHz, DMSO-d6, Figure S3-1) δ: 11.95 (1H, s, OH), 7.55–7.58 (2H, m, H-2', 6'), 7.53 (1H, s, CONH), 7.43–7.46 ( 3H, m, H-3', 4', 5'), 6.32 (1H, s, H-8), 6.30 (1H, s, H-6), 5.75 (1H, s, H-2), 4.17 –4.23 (1H, m, H-2''), 3.39–3.47 (1H, m, H-3a), 2.88–2.93 (1H, m, H-3b), 1.36–1.39 (12H, overlap, (CH3 )3C，CH3-2''); 13C-NMR (100 MHz, DMSO-d6, Figure S3-2) δ: 197.5 (C-4), 171.0 (C-1''), 162.2 (C-5) , 162.0 (C-9), 158.0 (C-7), 155.3 (CONH), 138.1 (C-1'), 128.6 (C-3'), 128.5 (C-5'), 126.5 (C-2' , 4', 6'), 105.9 (C-10), 102.3 (C-6), 101.2 (C-8), 78.6 (C-2), 78.4 ((CH3)3C), 54.7 (C-2' '), 42.3 (C-3), 28.0 ((CH3)3C), 16.3 (CH3-2'').

2.2.4 (S)-5-Hydroxy-4-oxo-2-phenylchroman-7-yl (tert-butoxycarbonyl)-valinate (pb-4) is a yellow powder with a yield of 40.73%; 1H-NMR (400 MHz , DMSO-d6, Figure S4-1) δ: 12.00 (1H, s, OH), 7.55 (1H, s, CONH), 7.39-7.53 (5H, m, H-Ph), 6.31 (1H, s, H -8), 6.29 (1H, s, H-6), 5.69–5.72 (1H, m, H-2), 4.04–4.08 (1H, m, H-2``), 3.34 –3.42 (1H, m , H-3a), 2.88–2.93 (1H, m, H-3b), 2.17–2.24 (1H, m, H-3``), 1.42 (9H, s, (CH3)3C ), 0.99–1.01 ( 6H, overlap, (CH3)2–3''); 13C-NMR (100 MHz, DMSO-d6, Figure S4-2) δ: 197.4 (C-4), 169.9 (C-1''), 162.3 ( C-5), 162.0 (C-9), 157.8 (C-7), 155.9 (CONH), 138.1 (C-1'), 128.4 (C-3', 5'), 126.4 (C-2', 4', 6'), 105.9 (C-10), 102.3 (C-6), 101.2 (C-8), 78.7 (C-2), 78.4 ((CH3)3C), 59.7 (C-2'' ), 42.4 (C-3), 29.4 (C-3)''), 28.0 ((CH3)3C), 18.8 (C-4''), 18.4 (C-5'').

2.2.5 (S)-5-Hydroxy-4-oxo-2-phenylchroman-7-yl(tert-butoxycarbonyl)glycine (pb-5) yellow powder, yield 41.10%; 1H-NMR (400 MHz, DMSO-d6, Figure S5-1) δ: 11.94 (1H, s, OH), 7.55 (1H, brs, CONH), 7.53 (1H, brs, H-4'), 7.42-- 7.46 (4H, m , H-1', 2', 5', 6'), 6.35 (1H, s, H-8), 6.33 (1H, s, H-6), 5.72–5.75 (1H, m, H-2) , 3.95 (2H, d, J = 6 Hz), 3.43–3.47 (1H, m, H-3a), 2.88–2.93 (1H, m, H-3b), 1.40 (9H, s, (CH3)3C) ; 13C-NMR (100 MHz, DMSO-d6, Figure S5-2) δ: 197.5 (C-4), 168.4 (C-1''), 162.1 (C-5), 162.0 (C-9), 157.7 (C-7), 155.8 (CONH), 138.1 (C-1'), 128.6 (C-3'), 128.5 (C-5'), 126.5 (C-2', 4', 6'), 105.9 (C-10), 102.4 (C-6), 101.3 (C-8), 78.6 (C-2), 78.5 ((CH3)3C), 42.3 (C-3, 2``), 28.0 ((CH3 ) 3C).

2.2.6 (S)-5-Hydroxy-4-oxo-2-phenylchroman-7-yl (tert-butoxycarbonyl)-L-leucinate (pb-6) yellow powder, yield 41.09%; 1H-NMR (400 MHz , DMSO-d6, Figure S6-1) δ: 11.94 (1H, s, OH), 7.55 (1H, s, CONH), 7.53 (2H, brs, H-2', 6'), 7.40-7.46 (3H , m, H-3', 4', 5'), 6.31 (1H, s, H-8), 6.28 (1H, s, H-6), 5.72–5.75 (1H, m, H-2), 4.08–4.15 (1H, m, H-2``), 3.39–3.47 (1H, m, H-3a), 2.88–2.93 (1H, m, H-3b), 1.58–1.71 (2H, m, H -3''), 1.40 (10H, s, (CH3)3C, C-4''), 0.89-0.93 (6H, overlap, (CH3)2-4''); 13C-NMR (100 MHz, DMSO -d6, Figure S6-2) δ: 197.7 (C-4), 171.1 (C-1''), 162.2 (C-5), 162.1 (C-9), 158.0 (C-7), 155.7 (CONH ), 138.2 (C-1'), 128.7 (C-3'), 128.6 (C-5'), 126.7 (C-2', 4', 6'), 106.0 (C-10), 102.5 (C -6), 101.4 (C-8), 78.7 (C-2), 78.6 ((CH3)3C), 52.3 (C-2``), 42.3 (C-3, 3 ``), 28.1 ((CH3 )3C), 24.3 (C-4''), 22.8 (C-5''), 21.3 (C-6'').

2.2.7 (S)-5-Hydroxy-4-oxo-2-phenylchroman-7-yl (tert-butoxycarbonyl)-D-leucinate (pb-7) yellow powder, yield 42.80%; 1H-NMR (400 MHz , DMSO-d6, Figure S7-1) δ: 11.95 (1H, s, OH), 7.55 (1H, s, CONH), 7.49–7.53 (2H, m, H-2', 6'), 7.40–7.46 (3H, m, H-3', 4', 5'), 6.29 (1H, s, H-8), 6.26 (1H, s, H-6), 5.75 (1H, s, H-2), 4.03–4.07 (1H, m, H-2``), 3.40–3.47 (1H, m, H-3a), 2.88–2.92 (1H, m, H-3b), 1.88–1.89 (1H, m, H -3``a), 1.46--1.52 (1H, m, H-3''b), 1.40 (9H, s, (CH3)3C), 1.24--1.31 (1H, m, H -4''), 0.85--0.95 (6H, m, H-5``, 6''); 13C-NMR (100 MHz, DMSO-d6, Figure S7-2) δ: 197.5 (C-4), 169.9 (C-1' '), 162.2 (C-5), 162.1 (C-9), 157.7 (C-7), 155.8 (CONH), 138.1 (C-1'), 128.6 (C-3'), 128.5 (C-5 '), 126.5 (C-2', 4', 6'), 105.9 (C-10), 102.2 (C-6), 101.2 (C-8), 78.6 (C-2), 78.5 ((CH3) 3C), 58.6 (C-2''), 42.2 (C-3), 35.6 (C-3''), 28.0 ((CH3)3C), 25.0 (C-4''), 15.3 (C-5 ''), 10.9 (C-6'').

2.2.8 1-(tert-Butyl) 2-((S)-5-hydroxy-4-oxo-2-phenylchroman-7-yl) pyrrolidine-1,2-dicarboxylate (pb-8) yield of yellow powder 40.77%; 1H-NMR (400 MHz, DMSO-d6, Figure S8-1) δ: 11.95 (1H, s, OH), 7.53 (2H, brs, H-2', 6'), 7.41–7.44 ( 3H, m, H-3', 4', 5'), 6.36 (1H, s, H-8), 6.32 (1H, s, H-6), 5.75 (1H, brs, H-2), 4.37 –4.42 (1H), m, H-2``), 3.39–3.42 (2H, m, H-5''), 3.34–3.37 (1H, m, H-3a), 2.89–2.93 (1H, m , H-3b), 1.85–1.96 (4H, m, H-3”, 4”), 1.37–1.41 (9H, overlap, (CH3)3C); 13C-NMR (100 MHz, DMSO-d6, Figure S8-2) δ: 197.5 (C-4), 170.4 (C-1``), 163.4 (C-5), 162.6 (C-9), 157.6 (C-7), 155.8 (COO), 137.9 (C-1'), 128.5 (C-3', 5'), 126.5 (C-4'), 126.3 (C-2', 6'), 106.0 (C-10), 102.2 (C-6) , 101.1 (C-8), 79.1 (C-2), 78.7 ((CH3)3C), 58.6 (C-2''), 54.7 (C-5'' ), 30.1 (C-3''), 27.9 ((CH3)3C), 24.0 (C-4'').

The age-synchronized wild-type L4 larvae were transferred to NGM plates treated with or without 200 μM drug, and incubated at 20°C. Transfer them to a new NGM board every day. On the 5th day, incubate them at 37°C for 3 hours. Then, if you lightly touch it with a platinum wire pick once every 0.5 hours until all the nematodes die and none of these nematodes respond, it is recorded as dead. The experiment was repeated 3 times independently.

The age-synchronized wild-type L4 larvae were transferred to NGM plates treated with or without 200 μM drug, and incubated at 20°C. Transfer them to a new NGM board every day. On the 5th day, transfer them to a new plate containing hydrogen peroxide (4 μL of hydrogen peroxide solution per 1 mL of medium). Then, if the nematodes are lightly touched with a platinum wire pick every 0.5 hours until all the nematodes die and none of them respond, they are considered dead. The experiment was repeated 3 times independently.

Transfer age-synchronized wild-type L4 larvae to new plates treated with or without 200 μM drug and incubate at 20°C. On the second day, the nematodes in each experimental group were placed in the anesthetic placed in the center of the agarose pad on the microscope slide. A single image of a nematode was taken with a microscope and analyzed using NIS-Elements software to determine the length of each animal.

Transfer age-synchronized wild-type L4 larvae to new plates treated with or without 200 μM drug and incubate at 20°C. They are transferred to a new plate every day. On the 9th day, the nematodes of each experimental group were placed in the anesthetic in the center of the agarose pad on the microscope slide. The image of a single nematode was taken with a fluorescence microscope, and the fluorescence intensity of the nematode was analyzed and calculated using ImageJ software.

In order to study the phenotypic changes associated with aging, the pumping rate of the pharynx was also measured. Young people take an average of 250-300 puffs per minute, which decreases with age. The age-synchronized wild-type L4 larvae were transferred to NGM plates treated with or without 200 μM drug, and incubated at 20°C. They are transferred to a new plate every day. Calculate the pharynx blood draw rate for 20 seconds on the 3rd, 5th, and 7th days. Each group includes 10 worms.

The age-synchronized wild-type L4 larvae were transferred to NGM plates treated with amino acid derivatives of rosin protein and incubated at 20°C. Transfer them to a new NGM board every day. On the 9th day, the nematodes of each experimental group were placed in the anesthetic in the center of the agarose pad on the microscope slide. An image of a single nematode was taken with a fluorescence microscope and analyzed with ImageJ software to calculate the fluorescence intensity of the nematode.

The DCFH-DA fluorescent probe can penetrate the cell membrane. After entering the cell, DCFH-DA can be hydrolyzed by intracellular esterase to form DCF and cannot penetrate the cell membrane; therefore, the probe can be easily loaded into the cell. The reactive oxygen species in cells can oxidize non-fluorescent DCFH to produce fluorescent DCF. Reflect the level of reactive oxygen species in the cell by detecting the fluorescence of DCF. 29,30

The age-synchronized wild-type L4 larvae were transferred to NGM plates treated with or without 200 μM drug, and incubated at 20°C. Transfer them to a new NGM board every day. On the 5th day, they were transferred to a new NGM containing hydrogen peroxide and stimulated for 2 hours. Next, the nematodes were washed with M9 buffer and transferred to a 0.2 mL centrifuge tube. The nematodes were then treated with 20 μM DCFH-DA in the dark at 20°C for 2 hours. Wash the worm with M9 buffer to remove the dye, then place it in the anesthetic in the center of the agarose pad on the microscope slide. A fluorescence inverted microscope (excitation: 485 nm; emission: 530 nm) was used to take an image of a single nematode, and the ImageJ software was used for analysis to calculate the fluorescence intensity of the nematode.

A transgenic C. elegans CF1553 expressing the sod-3::GFP reporter gene was generated, and the GFP fluorescence intensity of each group of nematodes was observed by an inverted fluorescence inverted microscope to detect the expression of SOD-3 in the nematodes.

The age-synchronized wild-type L4 larvae were transferred to new NGM plates treated with or without 200 μM drug, and incubated at 20°C for 3 days. They were then transferred to a new NGM containing hydrogen peroxide and stimulated for 2 hours. A fluorescent inverted microscope was used to take an image of a single nematode, and the ImageJ software was used to analyze it to calculate the fluorescence intensity of the nematode.

The transgenic Caenorhabditis elegans TJ356 expressing the DAF-16::GFP reporter gene was used to detect the nuclear localization of DAF-16. DAF-16 protein is related to the formation, lifespan and stress resistance of nematode dauer stage, and is inhibited by the insulin signaling pathway. In order to study the effect of compound pb-3 on DAF-16, 200 μM drugs were used to treat the eggs of age-synchronized TJ356 nematodes. After three days, the nematodes developed into young adults. An inverted fluorescence microscope (excitation: 488 nm; emission: 500-530 nm) was used to take an image of a single nematode, and the fluorescence was analyzed to calculate nuclear localization.

The age-synchronized wild-type L4 larvae were transferred to NGM plates treated with or without 200 μM drug, and incubated at 20°C. Transfer them to a new NGM board every day. On the 5th day, transfer them to a new plate containing hydrogen peroxide (4 μL of hydrogen peroxide solution per 1 mL of medium). Then, if the nematodes are lightly touched with a platinum wire pick every 0.5 hours until all the nematodes die and none of them respond, they are considered dead. The experiment was repeated 3 times independently.

All graphs are generated using GraphPad Prism (GraphPad Software Inc.). Kaplan-Meier analysis was used to plot the data from the stress tolerance test, and the statistical significance was analyzed by log-rank test. The significant difference between the control group and the treatment group was analyzed by one-way analysis of variance (ANOVA). All P values ​​less than 0.05 are considered statistically significant. All values ​​expressed as percentages (%) are normalized to a control value set to 100%.

During the aging process, the organism's ability to respond to external stimuli gradually declines. Heat resistance is an important criterion for evaluating the aging process of organisms. 31 As shown in Figure 3, the protective effect of rosin protein derivatives was screened at a concentration of 200 μM. Compared with the control group, the survival curves of the pb-2-, pb-3-, pb-4- and pb-5- treatment groups showed a significant shift to the right (P <0.001). In addition, the median survival time of these four groups (all 3.5 hours) was higher than that of the control group (2.5 hours), while the median survival time of the coniferin group was 3 hours. Figure 3 The effect of pinocembrin amino acid derivatives on the lifespan of wild-type Caenorhabditis elegans under heat stress conditions.

Figure 3 The effect of pinocembrin amino acid derivatives on the lifespan of wild-type Caenorhabditis elegans under heat stress conditions.

In order to explore the protective effect against oxidative stress, we analyzed worms treated with derivatives of H2O2 oxidative stress. As shown in Figure 4, the protective effect of pinocembrin derivatives at a concentration of 200 mM was further screened. Pb-3 showed significant activity (P <0.001). The median survival time of the pb-3 group was 4 hours, and the median survival time of the pine lignin group was 3 hours, which was longer than the control group (2.5 hours). Figure 4 The effect of pinocembrin amino acid derivatives on the life span of wild-type C. elegans under oxidative stress.

Figure 4 The effect of pinocembrin amino acid derivatives on the life span of wild-type C. elegans under oxidative stress.

The change in body length reflects the growth rate and physiological state of Caenorhabditis elegans. In this study, body length was measured to check the growth rate of worms. Body length analysis showed that compared with the control worms, the average body length of the worms treated with 200 μM amino acid derivatives was not found (P> 0.05, Figure 5). Figure 5 The effect of pinocembrin amino acid derivatives on the body length of wild-type Caenorhabditis elegans.

Figure 5 The effect of pinocembrin amino acid derivatives on the body length of wild-type Caenorhabditis elegans.

Generally speaking, the content of lipofuscin in nematodes gradually increases with age. Lipofuscin is widely regarded as a biomarker of aging, cannot be excreted from the body through exocytosis, and will accumulate in cells over time. 32,33 Excessive lipofuscin precipitation can cause damage to the nematode's body and ultimately accelerate the senescence of the nematode. 34, 35 Under an inverted fluorescence microscope, blue autofluorescence of lipofuscin was observed in C. elegans (Figure 6A-J). Through the calculation of fluorescence density, we found that pb-2 and pb-3 can reduce the accumulation of lipofuscin (Figure 6K), which proves that these derivatives have the effect of delaying the senescence of nematodes. Figure 6 The effect of pinocembrin derivatives on the level of lipofuscin, the aging pigment of wild-type Caenorhabditis elegans. ((A) control group; (B) rosin group; (C) pb-1 group; (D) pb-2 group; (E) pb-3 group; (F) pb-4 group; (G) pb -5 groups; (H) pb-6 group; (I) pb-7 group; (J) pb-9 group) Representative pictures of lipofuscin accumulation in nematodes. (K) The relative fluorescence intensity of lipofuscin accumulation. Bars with different letters indicate significantly different values ​​(* p <0.05, ** p <0.01, *** p <0.001, and **** p <0.0001).

Figure 6 The effect of pinocembrin derivatives on the level of lipofuscin, the aging pigment of wild-type Caenorhabditis elegans. ((A) control group; (B) rosin group; (C) pb-1 group; (D) pb-2 group; (E) pb-3 group; (F) pb-4 group; (G) pb -5 groups; (H) pb-6 group; (I) pb-7 group; (J) pb-9 group) Representative pictures of lipofuscin accumulation in nematodes. (K) The relative fluorescence intensity of lipofuscin accumulation. Bars with different letters indicate significantly different values ​​(* p <0.05, ** p <0.01, *** p <0.001, and **** p <0.0001).

In conclusion, pb-3 is the most effective compound, and its protective effect was further screened at three concentrations. We measured the autofluorescence level of lipofuscin (Figure 7A-D), and the results showed that compared with the control group, pb-3-treated worms showed a significant decrease in the fluorescence intensity of intestinal lipofuscin (35% reduction; P <0.0001, Figure 7E). Figure 7 The effect of pb-3 on the level of the senescence-related pigment lipofuscin in wild-type Caenorhabditis elegans. ((A) control group; (B) 100 μM group; (C) 200 μM group; (D) 300 μM group) Representative images of lipofuscin accumulation in nematodes. (E) The relative fluorescence intensity of lipofuscin accumulation. Bars with different letters indicate significantly different values ​​(*p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001).

Figure 7 The effect of pb-3 on the level of the senescence-related pigment lipofuscin in wild-type Caenorhabditis elegans. ((A) control group; (B) 100 μM group; (C) 200 μM group; (D) 300 μM group) Representative images of lipofuscin accumulation in nematodes. (E) The relative fluorescence intensity of lipofuscin accumulation. Bars with different letters indicate significantly different values ​​(*p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001).

The pharynx suction rate is measured to determine the feeding behavior of the worm, because the healthy life span of an organism depends on its feeding behavior. 36 Slow feeding rate corresponds to the worm lifespan promotion effect induced by DR (diet restriction). elegans.37 In order to eliminate the possibility that the detected protective effects (for example, life extension during oxidative stress or antioxidant effects) may be caused by caloric restriction, the number of pharyngeal pumps of nematodes in 20 seconds was measured on 2 days. , Day 4 and Day 6. Experimental data showed that the frequency of pharynx suction of the control group of nematodes gradually decreased with age. This result may be due to the degeneration of the muscles and nervous system caused by aging. As shown in Figure 8, compared with the control group, the pharyngeal pump frequency of the experimental group increased slightly, but there was no significant difference (P>0.05), indicating that pb-3 had no effect on nematode intake. In addition, DR does not realize the mechanism of pb-3 to extend the lifespan of nematodes. Figure 8 The effect of pb-3 on the pharynx pumping rate of wild-type Caenorhabditis elegans.

Figure 8 The effect of pb-3 on the pharynx pumping rate of wild-type Caenorhabditis elegans.

The above results indicate that pb-3 can significantly improve the stress resistance of nematodes at the biological level. Therefore, the level of ROS was studied to determine the mechanism by which pb-3 protects oxidative damage. Fluorescence pictures of the control group, oxidatively stimulated nematodes under non-administration conditions and oxidatively stimulated nematodes under administration conditions are shown in Figure 9A-C. The content of ROS is directly related to oxidative stress. 38 As shown in Figure 9D, compared with H2O2 treatment, pb-3 treatment significantly down-regulated the level of ROS in nematodes (p <0.001). Figure 9 ((A) control group; (B) H2O2 group; (C) pb-3 group) fluorescence pictures of wild nematodes in each group. (D) The effect of pb-3 on ROS accumulation in wild-type Caenorhabditis elegans cells (***p <0.001).

Figure 9 ((A) control group; (B) H2O2 group; (C) pb-3 group) fluorescence pictures of wild nematodes in each group. (D) The effect of pb-3 on ROS accumulation in wild-type Caenorhabditis elegans cells (***p <0.001).

As shown in Figure 10A and B, after the nematode was exposed to pb-3 for 72 hours, green fluorescence only appeared on the head and tail of the nematode. Statistical analysis of the fluorescence level, as shown in Figure 10C, there is no significant difference in the total fluorescence yield between the experimental group and the control group (p> 0.05), indicating that pb-3 cannot promote sod-3 gene expression under normal living conditions . Figure 10 Fluorescence pictures of each group of CF1553C. Nematodes ((A) control group; (B) 200μM group). (C) The effect of pb-3 on the fluorescence expression of CF1553C. Nematodes (compared to the control group, p> 0.05).

Figure 10 Fluorescence pictures of each group of CF1553C. Nematodes ((A) control group; (B) 200μM group). (C) The effect of pb-3 on the fluorescence expression of CF1553C. Nematodes (compared to the control group, p> 0.05).

Therefore, we studied the effect of pb-3 on sod-3 gene expression under oxidative stress. After 2 hours of exposure to H2O2, the nematodes were almost full of green fluorescence when observed under a fluorescence microscope (Figure 11A and B). Compared with the control, pb-3 significantly increased the total amount of green fluorescent protein (p<0.001, Figure 11C), indicating that pb-3 can increase the expression of sod-3 gene and enhance the anti-oxidative stress under oxidative stress Capability. This finding further explains the increased survival rate and decreased ROS content under H2O2 oxidative stress in our previous study. The sod-3 gene is a downstream factor of the daf-16 gene, which is regulated by the daf-2/daf-16 gene and is related to longevity. 39 Figure 11 Fluorescence image showing sod-3::GFP in CF1553 Caenorhabditis elegans ((A) control group; (B) 200 μM group). (C) The sod-3::GFP intensity in the treated CF1553 was quantified and showed a significant difference from the control (***p <0.001).

Figure 11 Fluorescence image showing sod-3::GFP expression in CF1553 Caenorhabditis elegans ((A) control group; (B) 200 μM group). (C) The sod-3::GFP intensity in the treated CF1553 was quantified and showed a significant difference from the control (***p <0.001).

In order to verify the increased expression of daf-16 gene, we tested the TJ356 strain and the results showed the nuclear localization of daf-16 during stress. To confirm the involvement of the daf-16 gene, we used the TJ356 Caenorhabditis elegans strain, in which DAF-16 was labeled with GFP.

Under normal growth conditions, DAF-16 is mainly retained in the cytoplasm, and pb-3 does not promote the nuclear localization of DAF-16, but obviously promotes nuclear localization under the oxidative stress induced by H2O2, as shown in Figure 12A-D. Based on the count of fluorescent spots, pb-3 caused the transfer of DAF-16 from the cytoplasm to the nucleus (Figure 12E). The location of DAF-16 in the nucleus is crucial for activating the transcription of various genes that mediate stress resistance. Therefore, our research shows that pb-3 activates the DAF-16 transcription factor through the insulin/IGF-1 signaling pathway. Figure 12 DAF-16:: GFP position ((A) control group; (B) pb-3 group; (C) control group (H2O2); (D) pb-3 group (H2O2)). (E) Pb-3 induced a significant degree of DAF-16::GFP in mutant TJ356 worms (***p <0.001).

Figure 12 DAF-16:: GFP position ((A) control group; (B) pb-3 group; (C) control group (H2O2); (D) pb-3 group (H2O2)). (E) Pb-3 induced a significant degree of DAF-16::GFP in mutant TJ356 worms (***p <0.001).

In order to further confirm whether pb-3 mediated oxidative stress resistance requires daf-16, we studied the beneficial effects of pb-3 on daf-16 null mutants (DR26). The results show that pb-3 cannot improve the survival rate of these worms. Pb-3 protects Caenorhabditis elegans from stress conditions in a DAF-16 dependent manner (Figure 13A). Figure 13 The effect of pb-3 on the survival of the loss-of-function mutant strains daf-16 (A), hsp16.2 (B), daf-2 (C) and age-1 (D) mutants.

Figure 13 The effect of pb-3 on the survival of the loss-of-function mutant strains daf-16 (A), hsp16.2 (B), daf-2 (C) and age-1 (D) mutants.

DAF-16 plays an important role in regulating stress resistance through its downstream target genes such as sod-3 and hsp16.2.40. We evaluated the anti-aging effects of pb-3 under various culture conditions. Under heat stress, pb-3 can significantly increase the survival rate of N2 worms. This result may be due to the effect of pb-3 on the up-regulated expression of the heat shock protein hsp-16.2, which can improve stress resistance and serve as a pressure-sensitive reporter gene to predict the lifespan of Caenorhabditis elegans. 41,42 As shown in Figure 13B, pb-3 cannot improve the survival rate of worms. Pb-3 protects Caenorhabditis elegans from stress conditions in an hsp-16.2-dependent manner. Daf-2 can regulate the nuclear translocation of DAF-16.43,44 DAF-2 is a homolog of the mammalian IGF-1 receptor of C. elegans and is the only IGF-1/insulin signal present in C. elegans Receptor, and AGE-1 is a homologue of the catalytic subunit of mammalian PI3K. 45 It has been reported that mutations in age 1 and daf-2 can lead to longer lifespan. 46,47 Pb-3 treatment did not significantly increase the lifespan of daf-2 (e1370) or age-1 (hx546) mutants (Figure 13C and D). Therefore, the results indicate that the effect of pb-3 treatment depends on DAF-2, AGE-1 and DAF-16.

In this research, 8 new pine tree derivatives with different amino acids were designed and synthesized. All these derivatives are characterized by 1H-NMR and 13C-NMR. The Caenorhabditis elegans model system was used for the first time to prove their anti-aging effects. Among these derivatives, pb-3 was found to show the best effect, depending on the results of the survival curve under stress conditions and the low levels of lipofuscin in Caenorhabditis elegans.

This study shows that pb-3 can extend the lifespan and healthy lifespan of Caenorhabditis elegans, reduce the accumulation of lipofuscin, and improve stress resistance (including heat stress and oxidative stress). Compared with pinocembrin, pb-3 is obtained by introducing alanine and has better anti-aging activity. The study also showed that pb-3 can increase the SOD activity of nematodes and reduce the level of ROS, which is the reason for the improvement of stress resistance, especially the resistance to oxidative stress. The prolonged lifespan of Caenorhabditis elegans treated with pb-3 depends on the DAF-16 pathway. The translocation of DAF-16 to the nucleus can further trigger the transcriptional activation of genes, including sod-3 and hsp-16.2. In addition, genetic studies have shown that the IIS pathway may be involved in pb-3 mediated lifespan extension. We hypothesize that the mode of action of pb-3 is related to the inhibition of proteins in the insulin/IGF-1 signaling pathway and its inherent antioxidant properties.

In short, pb-3 exerts an anti-oxidative stress effect through the DAF-16/FOXO signaling pathway. It is worth discussing the difference between the anti-aging effects of pb-3 and other mature anti-aging compounds in the future. In addition, further research is needed to clarify the potential mechanism of pb-3 to extend the lifespan of nematodes, and more complex model organisms are needed for in vivo assays.

This research was supported by the National Key Research and Development Program (No.2020071620211) and the Beijing University of Traditional Chinese Medicine Science and Technology Development Key Program (No.2020-JYB-ZDGG-040).

The author declares that there is no conflict of interest.

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