Jujubae Fructus extract prolongs lifespan and improves stress tolerance in Caenorhabditis elegans dependent on DAF-16/SOD-3

Jujubae Fructus extract prolongs lifespan and improves stress tolerance in Caenorhabditis elegans dependent on DAF-16/SOD-3 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Jujubae Fructus extract prolongs lifespan and improves stress tolerance in Caenorhabditis elegans dependent on DAF-16/SOD-3 zhi zhang, Jiajia Li, Feng Li, Tao Wang, Xiaoyan Luo, Bing Li, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3920171/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 14 Jun, 2024 Read the published version in Scientific Reports → Version 1 posted 8 You are reading this latest preprint version Abstract Jujubae Fructus , the fruit of Ziziphus jujuba Mill has been used as one of the medicine food homology species for thousands of years in China. Studies have shown that the active ingredients of Jujubae Fructus have a variety of biological effects, but its role in the aging process still lacks knowledge. Here, we investigated the effect of Jujubae Fructus extract (JE) on C. elegans lifespan and its potential mechanism. The lifespan of C. elegans treated with JE was signifificantly increased in a dose-dependent manner. In addition, JE treatment prolonged the reproductive period and increased normal activity during aging in C. elegans . Similarly, JE supplementation also enhanced the resistance to heat and oxidative stress in C. elegans . Furthermore, the mutant worms' lifespan assays demonstrated that JE requires daf-16 to prolong lifespan. DAF-16::GFP analysis of TJ356 showed that JE treatment translocates DAF-16::GFP to nucleus in transgenic worms. By analyzing the downstream of daf-16 , we identify that JE may regulate sod3 downstream of daf-16 . mutant worms' lifespan and transgenic reporter gene expression assays revealed that increasing SOD-3 expression was critical for extending longevity in C. elegans with JE therapy. Collectively, these data indicate that JE may have an important role in C. elegans longevity that is dependent on DAF-16 and SOD-3. Biological sciences/Cell biology/Senescence Health sciences/Molecular medicine Jujubae Fructus Caenorhabditis elegans lifespan stress tolerance daf-16 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Aging is the process of physiological and biochemical changes in the body and functional degradation with age ( 1 ). As the major risk factor for most of chronic diseases, it increases the incidence and mortality of obesity, diabetes, cancer, neurodegenerative diseases, and cardiovascular diseases ( 2 , 3 ). Therefore, delaying the aging process is of great significance to improve people's quality of life, preventing various diseases and even the sustainable development of society. Considering an explosive increase of senior citizens suffering from a number of chronic disorders, which could result in a tremendous social and economic burden, it is imperative to concentrate our collective efforts on developing novel remedies in order to decelerate senile changes and deter the inevitable physical alternations from advancing to the formidable ailments. Ziziphus Jujuba is a plant belonging to Ziziphus species in the buckthorn family Rhamnaceae. Historically, it has been widely cultivated in Europe, southern/eastern Asia, and Australia for over 4000 years, mainly because the fruit of Ziziphus Jujuba ( Jujubae Fructus ) has many active ingredients. The main biologically active components of Jujubae Fructus include cAMP, phenolics, flavonoids, triterpenic acids, and polysaccharides ( 4 ). In traditional Chinese medicine, Jujubae Fructus is often used to treat insomnia, spleen deficiency and palpitations, and used together with other medicinal materials to lower blood pressure, lower blood lipid and anti-anxiety. In the recent years, many studies have shown that the main biologically active components of Jujubae Fructus have a variety of biological effects ( 5 – 16 ). Through comparative analysis of the hypnotic and sedative effects of flavonoids, saponins, and polysaccharides extracted from Jujubae Fructus , saponins have been proved to have superior hypnotic and sedative effects than flavonoids, and may exert excellent sedative-hypnotic effects simultaneously through a variety of mechanisms ( 5 , 6 ). Jujubae Fructus can promote the learning and memory of the ovariectomized rats ( 7 ), and repair the memory and behavior disorders caused by NBM lesion in rats ( 8 ). It has been reported that Jujubae Fructus could protect the progression of CCl 4 -induced liver injury, which may be due to the fact that jujubae can bind to harmful free radicals and reduce their toxicity, as well as inhibit the inflammatory response of CCl 4 -induced liver injury ( 9 , 10 ). The active components of Jujubae Fructus , such as triterpenic acid, also have anticancer activity and can induce cell death by inhibiting cell proliferation and apoptosis ( 11 – 13 ). Jujubae Fructus also has an effect on glucose and lipid metabolism ( 14 , 15 ). The Jujubae Fructus polysaccharides can improve insulin resistance and dyslipidemia induced by fructose in mice ( 14 ). The infusion of Jujubae Fructus has beneficial effects on blood lipid and glycosylated hemoglobin in patients with type 2 diabetes mellitus ( 15 ). There are few studies on the effect of Jujubae Fructus on aging, Ghimire et al (2010) found that Jujubae Fructus feeding extended lifespan and health span of drosophila ( 16 ). About 60–80% of genes in C. elegans homologous to humans ( 17 ), the generation cycle of C. elegans is three days, the average life span is about 2–3 weeks, and it is easy to culture and observe. These advantages make C. elegans an ideal model for aging research ( 17 ). Numerous studies have focused on prolonging the lifespan of C. elegans, and the mechanism has been found to be related to daf-16. Polygonum multiflorum and Agrimonia procera extract have been shown to influence nematodes' lifespan via daf-16 ( 18 , 19 ). The longevity prolonging effect of blueberry on nematode and the enhancement of stress resistance were also confirmed to be mediated by daf-16 ( 20 ). The accumulation of ROS (reactive oxygen species) and oxidative stress injury in Glochidion zeylanicum treated nematodes were decreased, which was dependent on the DAF-16/FoxO and SKN-1/Nrf-2 signaling pathways ( 21 ). In addition, lyceum barbarum polysaccharides, Liangyi Gao, Holothuria leucospilota , and tiger milk mushroom were also found to improve the health status and increase lifespan of C. elegans via the daf-16 signaling pathway ( 22 – 25 ). Therefore, C. elegans is not only an ideal model to study the aging phenotype, but is also widely used to study the anti-aging mechanism. As a traditional Chinese medicine, Jujubae Fructus can be taken as a food and medicine, and have a variety of biological effects ( 5 – 16 ). There are limited studies on the effect of JE on lifespan, and the molecular mechanism of effects JE treatment based on C. elegans is still not clearly defined. Herein, to investigate the effect of JE treatment on C. elegans , we supplemented C. elegans with JE to investigate lifespan, vitality, fertility, and stress resistance of C. elegans , and further studied the mechanism of the effect of JE on C. elegans . MATERIALS AND METHODS Plant Extract Preparation The dried ripe fruit of Ziziphus jujube ( Jujubae Fructus ) was obtained from Bozhou Chinese medicinal materials market and identified as Zizyphus Jujuba cv. Jinsixiaozao by Professor Hongmei Gu (Zhoukou Normal University). The voucher specimen (ZF-180621) was deposited in the Institute of Translational Medicine, College of Life Science and Agronomy, Zhoukou Normal University under closed and dry conditions at 25 ± 5°C. Jujubae Fructus was cut up and extracted twice with boiling water for 2 h. The extract solution was filtrated immediately and the final filtrates were combined. The filtered extract was concentrated and dried under vacuum conditions by a rotary evaporator with 60°C water bath. The total yield of water extract from Jujubae Fructus (JE) was 51.7% (w/w). The chemical stability of JE aqueous solution was evaluated with an accelerate test by heating up to 60°C for 24 h, and comparative HPLC-PDAD-UV analysis was performed using an analytical Kromasil C18 column (5 µm, 100 Å, 4.6 mm × 250 mm; Akzo Nobel) on an Agilent 1100 HPLC system equipped with photo-diode array detector (G1316A). After filtered with 0.45 µm membrane, the JE sample solution in water (100 mg/ml) was injected (10 µL) into the column and eluted with a MeOH-water with 0.1% formic acid linear gradient (20%→100% MeOH in 20 min followed by 5 min with isocratic 100% MeOH) mobile phase (flow rate 1.0 mL/min flow rate). The acquired photodiode array data (PDAD) data were processed with Agilent OpenLAB software. UHPLC-Q-TOF-MS/MS Analysis of JE JE sample (10 mg) was added 1.0 ml of 80% methanol. The mixture was ultrasonicated for 15 min (200 W, 40 KHZ) at room temperature, and then centrifuged for 10 min at 12,000 rpm. Finally, the supernatants were filtered through a 0.22 µm membrane to obtain prepared samples for UHPLC-Q-TOF-MS analysis. Chromatographic separation was performed in a Nexera UHPLC LC-30A system (Shimadzu Corporation, Jappan) equipped with a SHIMADZU InerSustain C18 (100×2.1 mm, 2 µm) column maintained at 35°C with a flow rate of 1.0 mL/min. Gradient elution of analytes was carried out with acetonitrile (A) and 0.1% aqueous formic acid (B). The sample solution (5 µL) was injected after equilibration. An increasing linear gradient of solvent A (v/v) was then applied, as follows: 0–3 min, 5% A; 3–15 min, 5–20% A; 15–40 min, 20–100% A; 40–45 min, 100% A; 45–46 min, 100–5% A; 46–48 min, 5–5% A. The ESI-MSn experiments were carried out on a TripleTOF 5600 + Hybrid Quadrupole-TOF LC/MS/MS Mass Spectrometer (AB SCIEX™, United States) with spray voltages of 5.5 and 4.4 kV in positive and negative modes, respectively. Ion source gas and curtain gas were set at 50 and 25 arbitrary units, respectively. The source temperatures were 500 and 450°C in positive and negative modes, respectively. The analyzer scanned over mass ranges of m/z 100–1,200 Da and m/z 50–1,000 Da with accumulation time of 0.2s and 0.01s for TOF MS scan and product ion scan, respectively. Information-dependent acquisition (IDA) MS/MS experiments were performed with high sensitivity mode with Declustering Potential as ± 60V and Collision Energy as 35 ± 15eV. The analysis of UHPLC-MS data was performed using MS-DIAL 4.80 software (RIKEN Center for Sustainable Resource Science: Metabolome Informatics Research Team, Yokohama, Japan). We tentatively identified the compounds of JE by considering factors such as molecular weight, retention time, fragment information obtained from the MS/MS model, and further matching annotation in our prepared compounds, along with previous literature and the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database. C. elegans Strains and Maintenance Conditions The C. elegans strains used were N2: Bristol (wildtype), CF1038: daf-16 (mu86), CF1553: sod-3 (muIs84), and DA1116: eat-2 (ad1116), which were obtained from Shanghai Tech University. The PS3551: hsf-1 (sy441), CB1370: daf-2 (e1370), CL2070: hsp-16.2 (dvIs70), EU31: skn-1 (zu135), and TJ356: daf-16 (zIs356) were obtained from Tongji University of Life Sciences and Technology. All the strains were maintained using standard conditions at 20°C on NGM (nematode growth medium) plates. Worms were also allowed to grow in liquid S-medium with concentrated Escherichia coli OP50 (6mg/mL) as a food resource. Eggs were extracted from the nematodes before all experiments to synchronize the nematodes (worms at spawning stage were collected in M9 buffer solution (containing 0.5M NaOH and 0.8%NaClO), digested for 3–5 minutes and centrifuged to remove the supernatant at 1300g for 30s. The worms were washed twice with M9 buffer solution to retain the centrifuged precipitation ( 26 ). When the worms reached L1 stage, Live or dead (heat inactivated) E. coli OP50 was added to the NGM plates as food to feed the worms. OP50 was killed by heat shock temperature control 75°C during 2 hours. In L4 stage, different concentrations of JE were added to the culture, and 40 µM Fluoro-2'-deoxy-β-uridine (FUDR, Sigma-Aldrich) was also necessary to inhibit the growth of progen ( 26 ). Toxicity assay To perform the toxicity assay, the JE was dissolved in sterile water to prepare a stock solution of 100 mg/ml. The assay was performed using 100 µg/mL, 200 µg/mL, 1 mg/mL, 10 mg/mL, and 100 mg/mL concentrations. The control group was exposed to an equal volume of sterile water. On day 2 of adulthood, an age-synchronized population of worms was transferred to a 1 ml volume of S-medium supplemented with test doses of JE in a 12-well plate (Nest, China). A total of 90 worms were placed on triplicate wells with 30 worms per well for each group. The survival of worms was scored after every 2-hour interval and the data shown represents the survival rate of worms after 24 h ( 27 ). Lifespan assay N2, CF1038, DA1116, PS3551, CB1370, EU31, and CL2070 lifespan assays were carried out at 20°C. On day 2 of adulthood, an age-synchronized population of worms was transferred to NGM plates containing JE (20, 50, 100, 200 µg/mL) or an equal volume of sterile water (vehicle group). A total of 55–80 worms were placed on 3 to 4 replicate plates with 16 to 20 worms per 3.5 cm plate. Worms were transferred to new plates and scored every two days. Worms were scored as alive until there was no movement after repeated prodding. Lifespan assays were indicated in supplementary Table S1 , and the number of worms was pooled ( 28 ). ROS Assay Wild-type N2 worms from the adult stage were treated with 100 µg/mL JE or vehicle and were used for intracellular ROS determination. At day 2 and day 4 adult worms age, synchronized worms were collected in 500 µl of 0.1% PBST buffer and then homogenized and sonicated. The sample using Fluorescent probe H2DCF-DA (2′,7′-dichlorodihydroflfluoresceindiacetate) and BioTek microplate reader at emission FL fluorescence intensity at 485 nm and excitation at 530 nm to measure emission FL fluorescence intensity. The observations were recorded for 90 min at intervals of 20 min. The assay was performed in triplicate independently. Lipofuscin Assay Wild-type N2 worms raised from the adult stage were treated with 100 µg/mL JE or vehicle used for lipofuscin assay. On the 10th day of adulthood (n = 20) were randomly selected and mounted on 3% agarose pads and anesthetized with 2% sodium azide. Images were captured with fluorescence microscope (Olympus BX 61, Japan) using GFP filter (with excitation at 340–380 nm and emission at 435–485 nm) FL fluorescence microscope at 10×. The fluorescence levels were quantified using the ImageJ software. Pharyngeal Pumping Rate The pharyngeal pumping rate was quantified on the 5th, 7th, and 10th days of adulthood. Ten worms were treated with 100 µg/mL JE or vehicle randomly picked for measurement of pharyngeal pumping rate per 20 s. Movement assay To measure the frequency of body bending, on day 2 of adulthood, an age-synchronized population of worms was treated with 100 µg/mL JE or vehicle for 2 and 11 days. Worms were placed on NGM plates containing FUDR (40 µM), and the number of sinusoidal curves made during locomotion and swinging head in 1 min was scored. A total of 20–24 worms were detected in each group ( 29 ). Fertility measurement To determine fecundity, worms (N2/RNAi) were transferred to a medium without FUDR and treated with 100 µg/mL JE or vehicle treatment for 2 days. Hermaphrodites were serially transferred to fresh NGM plates at 24-hour intervals, until sterile. Two days after eggs were laid, the number of hatched progenies was counted. When the nematodes were no longer postpartum, their reproductive cycles were recorded. Each group included 13–14 worms ( 30 ). Heat stress resistance For the heat stress resistance assay, on day 2 of adulthood, an age-synchronized population of worms was treated with 100 µg/mL JE or vehicle for 2 days. Live or dead OP50 (killed by heat shock temperature control 75°C for 2 hours) was added to NGM plates. Worms were placed on NGM plates containing FUDR (40 µM) in 37°C conditions and then the number of worms was counted every hour until all the worms had died. Each group included 50 worms ( 31 ). Oxidative stress resistance On day 2 of adulthood, an age-synchronized population of worms was transferred to S-medium containing FUDR (40 µM) and with 100 µg/mL JE or vehicle treatment, respectively. After 2 days, 5 mM paraquat was added to S-medium, which induces lethal oxidative stress. The vitality of the worms was examined every 4 hours until all worms had died. Triplicate plates were used for each group. Each group included 150 worms ( 20 ). DAF-16 localization via fluorescence microscopy The TJ356 strain was used to examine the localization of DAF-16 in the living nematode. The aged-synchronized L4 larvae were transferred to NGM plates previously treated with 100 µg/mL JE or vehicle and incubated for 48 hours. DAF-16 localization was examined in 30 worms per treatment that were mounted in a drop of 20 mM levamisole hydrochloride. We scored each animal as having cytosolic localization, nuclear localization, or intermediate localization when there is a visible nuclear localization but one not as complete as nuclear. The number of worms with each level of nuclear translocation was counted. Fluorescence images were taken at constant exposure times (Olympus IX 73, Japan). Quantitative real-time PCR The contemporaneous 2-day-old worms were incubated in NGM containing the same concentration of JE for 7 days. After washing with M9 buffer, C. elegans were collected into 1.5 ml tubes and extraction RNA using the TransZol Up (Transgene) and stored at -80°C. Complementary DNA was prepared using HiScript III 1st Strand cDNA Synthesis Kit (+ gDNA wiper) (Vazyme) for real-time polymerase chain reaction (RT-PCR). Quantitative PCR (qPCR) was performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme). The mRNA expression levels of downstream genes of daf-16 in nematodes were monitored. β-actin was used as the housekeeping gene for normalization, and the experimental results were expressed as 2ΔΔCt values of PCR. The information of the primer sequence is included in supplementary Table S10. Statistical analysis Statistical analyses were carried out using Graphpad 7.0. A Kaplan–Meier lifespan analysis was carried out, and p values were calculated using the log-rank test. In all statistical analyses, p < 0.05 was accepted as statistically significant (* p < 0.05, ** p < 0.01, *** p < 0.001). RESULTS HPLC-PDAD-UV and UHPLC-Q-TOF-MS Analysis of JE In the accelerated test, the fingerprint chromatograms derived from HPLC-PDAD-UV analysis of JE aqueous solution showed no significant changes after 24 hours of heating (Fig. 1 ). Additionally, the UV absorption spectra of the main chromatographic peaks remained unchanged (data not shown). This result suggested that the JE is chemically stable in an aqueous solution during the lifespan assay under normal conditions. UHPLC-Q-TOF-MS analysis was conducted in both positive and negative modes, with Fig. 1 displaying the positive total ions chromatogram (TIC). A total of 22 phytochemicals were identified in JE, including three carbohydrates, five glycosides, two alkaloids, eleven triterpene acids, and cAMP (Table 1 ). Six of these compounds (cAMP, zizybeoside I and II, rutin, oleanonic acid, and ursolic acid) were identified by comparison to our previously prepared samples; the others were identified by comparison with literature ( 32 , 33 ). Table 1 Identified ingredients in JE. No. RT (min) Compound name Formular MS Error (ppm) MS/MS 1 1.01 Tartaric acid C 4 H 6 O 6 151.0241 [M + H] + 1.32 110.0111, 99.0442, 88.2338, 82.0182 2 1.16 Methose C 6 H 12 O 6 179.0544 [M-H]- 6.70 125.1130, 101.0300, 89.0345 3 1.21 Sucrose C 12 H 22 O 11 341.1055 [M-H] − 8.50 163.0637, 145.0529, 127.0410 4 1.79 cAMP C 10 H 12 N 5 O 6 P 330.0620 [M + H] + -5.15 232.0850, 136.0638 5 9.77 Zizybeoside I C 19 H 28 O 11 431.1527 [M-H] − 6.03 347.0983, 293.1010 6 10.44 Zizybeoside II C 25 H 38 O 16 617.2054 [M + Na] + 0.65 431.1798, 269.1303, 161.0566 7 10.89 Coclaurine C 17 H 19 NO 3 286.1449 [M + H] + -2.10 269.1149, 237.0914, 209.0965, 175.0787, 145.0646, 107.0539 8 11.48 Zizyvoside II C 31 H 50 O 18 709.2858 [M-H] − 8.60 547, 519.2244, 385.2117 9 11.60 Stepharine C 18 H 19 NO 3 298.1451 [M + H] + -2.68 269.1191, 192.1039, 161.0858 10 14.64 Rutin C 27 H 30 O 16 611.1597 [M + H] + 2.45 465.0975, 303.0503, 163.0670 11 16.11 Zizyvoside I C 25 H 40 O 12 533.2593 [M + H] + 0.94 435.1962, 393.1816, 277.2098 12 29.51 Ceanothic acid C 30 H 46 O 5 485.3259 [M-H] − 1.65 467.3810, 423.3476 13 30.51 Alphitolic acid C 30 H 48 O 4 473.3591 [M + H] + 8.45 455.3698, 390.9161 14 31.12 Maslinic acid C 30 H 48 O 4 495.3450 [M + Na] + 4.04 409.3433, 381.3086, 296.8615, 249.1672, 203.1779 15 31.38 2α-Hydroxyursolic acid C 30 H 48 O 4 473.3627 [M + H] + 0.85 437.3418, 409.3402, 391.3322, 285.2632, 223.1774, 205.1594, 187.1443 16 32.24 Zizyberanalic acid C 30 H 46 O 4 471.3475 [M + H] + -0.21 453.3368, 435.3225, 407.3355, 389.3332, 327.2283, 245.1501, 177.1640 17 33.20 3-O-cis-p-Coumaroylalphitolic acid C 39 H 54 O 6 619.3978 [M + H] + 3.39 437.3510, 391.3270, 259.1755, 202.5370, 173.1333, 135.1207 18 33.54 3-O-cis-p-Coumaroylmaslinic acid C 39 H 54 O 6 619.4019 [M + H] + -3.23 437.3447, 411.3292, 353.2572, 287.2157, 203.1819, 147.0457 19 34.07 3-O-trans-p-Coumaroylalphitolic acid C 39 H 54 O 6 619.4016 [M + H] + -2.74 437.3450, 409.3405, 391.3436, 363.2439, 201.1579, 177.1812 20 34.36 3-O-trans-p-Coumaroylmaslinic acid C 39 H 54 O 6 619.3944 [M + H] + 8.88 437.3506, 411.3207, 261.1825, 165.0583 21 36.94 Oleanonic acid C 30 H 48 O 3 455.3537 [M-H] − -2.64 437.3444, 409.3399, 259.1797, 177.1659 22 37.42 Ursonic acid C 30 H 48 O 3 455.3532 [M-H] − -1.54 437.3396, 409.3509, 261.1843, 208.1591, 163.1460 Effect of JE on the lifespan and stress resistance of C. elegans Before conducting lifespan experiments, we carried out an acute in vivo toxicity study. When the concentrations of JE were less than or equal to 1mg/mL, which were found to be non-toxic to C. elegans were chosen for further tests (Fig. 2 a). To address whether JE has a positive effect on the lifespan of C. elegans , N2 worms were treated with JE at treated with 20 ug/mL, 50 ug/mL, 100 ug/mL, and 200 ug/mL doses of standardized JE. The results showed that compared with the control group (equal volume sterile water was used as control), 50 ug/mL, 100 ug/mL, and 200 ug/mL JE treatment significantly increased the lifespan of the N2 worms in a dose-dependent manner, with the maximum lifespan increased from 19 days in control to 21, 24 and 23 days, respectively (Fig. 2 b, Table S1 ). The mean lifespan significantly increased to 114.1%, 123.3%, and 123.1% with the treatment of 50, 100, and 200 µg/mL (Table S1 ). The 100 and 200 µg/ml doses of JE were able to extend the mean lifespan and maximum lifespan maximally. In the follow-up experiments, 100 µg/mL of JE was used to cultivate worms to observe its effects on other physiological indicators of C. elegans . Meanwhile, JE treatment also increased the lifespan of worms exposed to 20°C or 37°C thermal shock when fed bacteria killed by heat (Figure S2). These data illustrated that JE-induced prolongation of lifespan occurs by a direct effect on C. elegans rather than indirectly through the bacteria. Aging is often accompanied by a decline in resistance to stress, we exposed C. elegans to heat and oxidative stress to observe the effects of JE on C. elegan's lifespan. Under the thermotolerance conditions, the mean lifespan of the JE treatment group significantly increased by 23.3% compared with the control group. The maximum lifespan of the control group was 12 hours, while that increased to 15 hours after JE treatment (Fig. 2 c, Table S2). The result of the oxidative stress showed JE treatment also could significantly increase the mean lifespan of C. elegan (Fig. 2 d, Table S3). JE decreases the pigment lipofuscin and intracellular ROS level in C. elegans As a marker of aging and oxidative damage, The rate of lipofuscin formation increases with age and it depends on the rate of oxidative damage ( 7 ). we detected lipofuscin levels of 100 µg/mL JE-treated or vehicle-treated wild-type C. elegans at day 5 and day 12 of adulthood. The result showed that, After receiving 5 or 12 days of JE treatment, the lipofuscin level in the intestine decreased by 65% and 20% respectively (Fig. 3 a-d). To investigate the effect of JE treatment on oxidative damage, intracellular ROS levels were evaluated in wild-type worms using H2DCF-DA, a widely known fluorescence probe for detecting intracellular ROS production. The results displayed that, with 2 or 4 days of JE treatment, the ROS accumulation of the wild-type worms had a significant decrease (Fig. 3 e-f). Effect of JE on pharyngeal pumping rate and fertility of C. elegans Worms with reduced pharyngeal pumping ingest fewer bacteria and exhibit numerous DR-like characteristics, such as decreased fecundity and prolonged lifespan ( 5 ). We tested whether JE had an effect on the pharyngeal pumping rate, and found that, compared to the control, 100 µg/mL JE treatment had no effect on pharyngeal pump rates on days 2 and 5, but significantly decreased pharyngeal pump rates in adults on days 7 and 10 (Fig. 4 a). This indicated that JE treatment just altered the feeding behavior of the older worms. Next, we explored whether the extension in lifespan was accompanied by any effect on the fertility of nematodes. The total offspring per worm in the control group was 225.8 ± 9.9. After administering 100 ug/ml JE, the total offspring decreased to 182.2 ± 10.9 (Fig. 4 b, c). To our surprise, the mean breeding days increased by 13.6% (3.5 days in control worms, 4.0 days in 100 ug/ml JE treatment worms, p < 0.05) (Fig. 4 b, d). Therefore, these results manifested that, with the lifespan extension, the JE treatment significantly decreased fertility and increased the breeding period of worms. Effect of JE on the mobility of C. elegans With the aging process, the worm's activity slows down and becomes insensitive to external stimuli ( 34 , 35 ). Stamper and Hosono previously reported that wild-type worms exhibit an age-dependent decline in movement ability, the decline of activity was rapid from days 7–10 ( 35 , 36 ). To investigate whether the increased lifespan was accompanied by the improvement of movement behavior, we conducted a movement behavior assay (body bending and head swing) at the age of 2 and 11 days of L4 stage With chronic JE treatment, the activity of body bending in adult worms at the age of 2 and 11 days showed a significant increase of 23.4% and 40.5% respectively (Fig. 5 a). Meanwhile, with chronic JE treatment, the head swing activity of 2 and 11 days worms significantly increased (24.6% in 2 days worms, p < 0.05 and 54.3% in 11 days worms, p < 0.001) (Fig. 5 b). Interestingly, the activity of body bending and head swing in vehicle group worms showed an age-dependent decline (Fig. 5 a, b). However, the activity of body bending and head swing in JE treatment group worms had no change with the aging process (Fig. 5 a, b). JE requires daf-16 to extend the lifespan of C. elegans In order to investigate the molecular mechanisms of JE on longevity extension and health improvement in C. elegans , we next dissected the longevity pathways required for the lifespan extension induced by JE by testing its effects in prototypical mutant worms for aging-related signaling pathways such as insulin/insulin-like growth factors-1 (IIS), caloric restriction, ROS and so on. The results showed that both eat-2 (caloric restriction), daf-2 (IIS), hsf-1 , skn-1 and hsp-16.2 mutant worms had an increased mean lifespan and maximum lifespan with JE treatment as compared with their respective mutant vehicle groups (Fig. 6 a-e, p < 0.05). In contrast, the effect of JE on lifespan was dependent on insulin/IGF1 signal pathway, as the improvement was entirely suppressed in daf-16 mutants, with the 100 ug/ml JE treatment, the mean lifespan and the maximum lifespan of daf-16 mutant worms had no change (Fig. 6 f, p = 0.92). All together, these results indicated that JE treatment requires daf-16 gene to extend the mean and maximum lifespan of C. elegans . JE cannot affect the mobility, fertility, and lifespan under stress resistance of mutant C. elegans ( daf-16 ) Next, we observed the effect of JE on physiological indexes of daf-16 mutant C. elegans . In the vehicle group, the worms bent their bodies 16.8 ± 2.0 times and swing heads 22.6 ± 1.5 times per minute, compared with 17.4 ± 1.9 and 22.5 ± 1.6 times per minute in the JE treatment group (Fig. 7 a-b). Similarly, JE treatment cannot significantly affect the number of eggs laid and breeding period of daf-16 mutant C. elegans . Under the treatment of JE, daf-16 mutant C. elegans breed 258 ± 19.7 eggs total, and the oviposition duration was 3.1 ± 0.2 days, which were not significantly different from the control group (Fig. 7 c-d). Then, the mutant worms were treated with JE for 48 hours and placed at 37°C and paraquat to observe their resistance to heat stress and oxidative stress. Our results showed that JE did not significantly increase the mean lifespan and maximum lifespan of daf-16 mutants under heat and oxidative stress conditions (Fig. 7 e-f). Effect of JE treatment on DAF-16 translocation The JE treatment did not change the lifespan, mobility, and fertility of daf-16 mutant worms, it indicated that the effects of JE treatment depend on the gene daf-16 in C. elegans . Daf-16 is a key factor in insulin/IGF1 signaling pathways transferred from the cytoplasm to the nucleus for multiple biological processes under stress. To determine whether JE is able to affect the cellular localization of DAF-16, we introduced the green fluorescent protein GFP-tagged daf-16 transgenic strain TJ365 to observe the location of DAF-16. The result showed that DAF-16::GFP of 9% transgenic worms localized in the nucleus with vehicle treatment, but heat stress and JE treatment translocates DAF-16::GFP to nucleus in transgenic worms (Fig. 8 a-b). JE requires sod-3 (the downstream of daf-16 ) to extend the lifespan of C. elegans To further explore the mechanism of daf-16 with JE treatment, the expression levels of genes downstream of daf-16 ( sod-3, mtl-1, gst-4, hsp-16.2 ctl-2 , old-1 ) were assessed. The expression level of genes downstream of daf-16 in the N2 worms without JE treatment was set to 1. JE treatment increased the relative expression of sod-3 by 2.1-fold, but had no influence on the expression of other daf-16 downstream genes. (Fig. 9 a). Meanwhile, we compared gene expression in daf-16 mutant worms with the vehicle and JE treatment. Compared with vehicle group, the JE-treated groups did not show significant differences in the expression of sod-3 (Fig. 9 b). Furthermore, The GFP-tagged sod-3 transgenic strain CF1553 (muls84) was introduced to investigate whether JE increased the protein level of sod-3 . Our results showed that 100 µg/mL JE treatment significantly induced the expression of SOD-3 (Fig. 9 c, d). In the present study, we found that JE treatment did not change the mean and maxium lifespan of sod-3 mutant worms (Fig. 9 e). In conclusion, our results indicated that JE treatment was able to induce high expression of gene sod-3 that might be depending on nuclear translocation of longevity-associated transcription factor DAF-16. DISCUSSION As a traditional Chinese medicine and food, Jujubae Fructus has been used in the treatment and late intervention of many diseases. However, there are few studies on its effect on aging. Here, we treated C. elegans with JE to observe the effects on longevity and health status, and further investigate the mechanisms involved. Our data showed that 50, 100 and 200 µg/mL of JE could significantly increase the lifespan of worms by 14.1%, 23.3% and 23.1%, respectively (Fig. 2 b). Increased longevity has been accompanied by improved health status. We also observed how JE affected the lifetime of worms under stress, and the findings revealed that worms treated with JE had greater resilience to heat stress and oxidative stress (Fig. 2 c, d). Many studies on Jujubae Fructus revealed various beneficial nutrients, including carbohydrate, mineral, vitamin, sugar and amino acid ( 37 ), as well as various kinds of secondary metabolites, such as nucleotides (71.98 mg/100g), flavonoids (48.62 mg/100g), triterpenic acids (343.43 mg/100g), polysaccharides (3.30 g/100g) ( 38 ). In this study, we identified 22 ingredients in the water extract of Jujubae Fructus by UPLC-MS analysis (Table 1 ). Among them, rutin treatment was reported to reduce polyglutamine (polyQ) protein aggregation in muscle and polyQ-mediated neuronal death in ASH sensory neurons, and extend lifespan in C. elegans . The possible mechanisms involved are antioxidant activity, activation of protein degradation (autophagy) and insulin/IGF1 signaling pathways ( 39 ). As a representative triterpenic acid, ursolic acid prolonged the life span of C. elegans , and significantly lowered reactive oxygen species (ROS), also could act through serotonin receptors to enhance stress resistance ( 40 ). Additionally, despite not identified, it is presumed that there were abundant polysaccharides in the JE prepared by decocting method, which were reported to be immunomodulating, antioxidative and hepatoprotective ( 4 , 41 ). These bioactive constituents may contribute to the beneficial effects of prolonging lifespan and improving stress tolerance in C. elegans by JE in this study. Gagnon et al found that telomere length in women who had given birth was significantly shorter than that of non-fertile women by analyzing the effects of fertility on the health status, and they indicated that reproduction may have accelerated the aging process ( 42 ). Some studies have shown that extension of lifespan is correlated with a decrease in fecundity ( 43 ). In addition, decreased worm fertility was associated with increased lifespan ( 20 ). Removal of reproductive precursor cells in normal and daf-2 mutant worms prolongs the lifespan of C. elegans ( 44 , 45 ). Blueberry extract treatment with extended lifespan of nematodes can simultaneously reduce the number of eggs laid and prolong the reeding period of C. elegans ( 20 ). Our results also showed that the C. elegans breeding period was prolonged, and the fecundity was decreased with the 100 ug/ml JE treatment (Fig. 4 ). Among long-lived humans, it was found that they had their last child at an older age, and the later the last child was born, the longer the females lived ( 46 , 47 ). In our hand, with the extension of lifespan, the 100 ug/ml JE treatment increased by 0.5 days in breeding period of C. elegans (Fig. 4 d). These data indicated that an extension in breeding period is correlated with a longer lifespan (Fig. 4 b,d). Dietary restriction is one of the ways to delay the senescence of nematodes. Eat-2 is a key gene in dietary restriction, it is a gene regulating pharyngeal suction rate, and its mutation will lead to feeding defects and reduce energy and nutrient intake of nematodes ( 48 ). In order to observe whether the longevity of C. elegans was prolonged by dietary restriction, we examined the effect of JE on the longevity of eat-2 mutants. The results showed that compared with the control group, JE significantly prolonged the mean and maximum lifespan of eat-2 mutants (Fig. 6 a, table S4). consistent with this, the result of pharyngeal pumping rate assay showed that JE treatment had no effect on pharyngeal pump rates on days 2 and 5 (Fig. 4 a). Our results suggested that the dietary restriction pathway is nonessential for the effect of JE on the longevity of worms. Interestingly, with JE treatment, the pharyngeal pumping rate of worms significantly decreased as aging progresses (Fig. 4 a). There are other signaling pathways involved in the aging process of nematodes ( 49 ). In the IIS signaling pathway, insulin or insulin-like growth factor-1(IGF-1) binding with daf-2 receptor induces the phosphorylation of pi3k/age-1, which activates the downstream akt-1 pathway to phosphorylate daf-16 and hsf-1 , preventing transcription factor transfer into the nucleus, thereby shortening the nematode lifespan ( 50 ). Daf-16 is homology with FOXO in mammals, and it is a key protein in nematode longevity ( 51 ). Herein, to further study the molecule mechanism of prolongated lifespan and improved health status of C. elegans with the JE supplementation, we investigated whether JE affected the longevity of nematodes deficient in eat-2 , daf-2 , hsf-1 , skn-1 , hsp-16.2 and daf-16 , respectively. However, compared with the control group, the life-prolonging effect of JE only disappeared in the mutant nematode daf-16 . Next, we observe the effects of JE on vitality, fertility, and stress resistance in mutant nematode daf-16 . Consistently, the effects of JE on these indices in wild-type nematodes were not found in daf-16 mutant nematodes (Fig. 7 a-f, Table S5-S9). These findings suggested that the activity of daf-16 gene is crucial for JE to prolong the lifespan and improve the health status of worms. Normally, DAF-16 is located in the cytoplasm, while under stress, DAF-16 is transferred from the cytoplasm to the nucleus, JE treatment also translocate DAF-16::GFP to nucleus in TJ356 worms compared to those untreated ones (Fig. 8 a,b). We also found that JE treatment increased the expression of sod-3 , downstream of daf-16 in N2 worms, but the effect was inhibited in mutant (daf-16) C. elegans. We also found that JE treatment doesn’t significantly increase the lifespan and the expression of sod-3 of mutant ( daf-16 ) worms as in wild type (Fig. 9 a,b). Our further study manifested that JE’s effect on prolonging the lifespan of worms disappeared with the silencing gene of sod-3 (Fig. 9 e). Sod-3 encodes a superoxide dismutase that can resist oxidative stress and prolong life. Trilobatin has been shown to effectively extend the lifespan of nematode worms by regulating the SKN1/SIRT3/DAF16 signaling pathway, as well as increasing the activity of antioxidant enzymes CAT and SOD-3 ( 52 ). Curcumin acetylsalicylate can also delay the aging of nematodes by activating the expression of daf16 and its downstream antioxidant genes sod-3 and gst-4 ( 53 ). Our findings indicated that JE treatment may induce nuclear translocation of longevity associated transcription factor DAF-16 depending on activation of downstream gene sod-3 to prolong lifespan of worms. Meanwhile, the specific small molecule in JE that prolongs the lifespan of C. elegans and its mechanism, as well as the important role of daf-16 in those effects, need further investigation. Jujubae Fructus has been poorly studied in terms of aging, and previous studies have reported a life-extending effect in drosophila ( 16 ). Our study makes up for the gap in the impact of Jujubae Fructus on lifespan of C. elegans and provides a certain basis for the relative studies on humans. CONCLUSION This study, for the first time, reports Jujubae Fructus extract supplementation promotes lifespan and improves the health status of the C. elegans model system. we identified 22 phytochemicals in JE, including three carbohydrates, five glycosides, two alkaloids, eleven triterpene acids and cAMP. Our data showed that JE treatment prolongs the worms' mean lifespan in both the standard laboratory and heat or oxidative stress conditions. The study also manifests that JE treatment enhances the vitality and breeding period. Meanwhile, the gene-specific mutant studies reveal that lifespan prolongation with JE supplementation was dependent on daf-16 and sod-3 (downstream of daf-16 ). The nuclear translocation of DAF-16 and the up expression of sod-3 , also supports that JE mediated lifespan extension is correlated with the insulin/IGF-1 signal pathway. Our study makes up for the gap in the impact of Jujubae Fructus on lifespan of C. elegans , the present study may provide a novel avenue against aging and aging related disorders. Abbreviations FUDR Fluoro-2'-deoxy-β-uridine JE Jujubae Fructus extract H2DCF-DA 2′,7′-dichlorodihydroflfluoresceindiacetate IDA Information-dependent acquisition NGM nematode growth medium PDAD photodiode array data ROS reactive oxygen species TCMSP Traditional Chinese Medicine Systems Pharmacology TIC total ions chromatogram. Declarations FUNDING This work is supported by the Starting Research Fund from the Xin Xiang Medical University (XYBSKYZZ202135), Science and technology innovation talent Program of Henan Province (19HASTIT015), Henan Science and Technology Research Plan Project of China (222102110412). 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Supplementary Files supportinginformation.docx Cite Share Download PDF Status: Published Journal Publication published 14 Jun, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 26 Mar, 2024 Reviews received at journal 08 Mar, 2024 Reviewers agreed at journal 29 Feb, 2024 Reviewers invited by journal 29 Feb, 2024 Editor assigned by journal 29 Feb, 2024 Editor invited by journal 14 Feb, 2024 Submission checks completed at journal 13 Feb, 2024 First submitted to journal 02 Feb, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3920171","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":272839316,"identity":"d385b455-ea23-4c33-a35f-c58177d6c3cb","order_by":0,"name":"zhi zhang","email":"","orcid":"","institution":"Xinxiang Medical University","correspondingAuthor":false,"prefix":"","firstName":"zhi","middleName":"","lastName":"zhang","suffix":""},{"id":272839317,"identity":"ed3aecf0-5f9e-46ad-bf39-f96e4175e3f0","order_by":1,"name":"Jiajia Li","email":"","orcid":"","institution":"Zhoukou Normal University","correspondingAuthor":false,"prefix":"","firstName":"Jiajia","middleName":"","lastName":"Li","suffix":""},{"id":272839318,"identity":"7dd2e3b8-b36c-40a4-a699-0452d484452d","order_by":2,"name":"Feng Li","email":"","orcid":"","institution":"Xinxiang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Feng","middleName":"","lastName":"Li","suffix":""},{"id":272839319,"identity":"00485e04-784b-4aa6-95b3-4f6d233041e2","order_by":3,"name":"Tao Wang","email":"","orcid":"","institution":"Zhoukou Normal University","correspondingAuthor":false,"prefix":"","firstName":"Tao","middleName":"","lastName":"Wang","suffix":""},{"id":272839320,"identity":"c8f05376-3775-4f72-8ca0-b526f5d38272","order_by":4,"name":"Xiaoyan Luo","email":"","orcid":"","institution":"Xinxiang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoyan","middleName":"","lastName":"Luo","suffix":""},{"id":272839321,"identity":"6a62f4bf-07eb-4b49-8663-64399e28397e","order_by":5,"name":"Bing Li","email":"","orcid":"","institution":"Zhoukou Normal University","correspondingAuthor":false,"prefix":"","firstName":"Bing","middleName":"","lastName":"Li","suffix":""},{"id":272839322,"identity":"1c2fb2b2-b726-48b2-923c-40f9260fe486","order_by":6,"name":"Yilin You","email":"","orcid":"","institution":"China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yilin","middleName":"","lastName":"You","suffix":""},{"id":272839323,"identity":"1a628a9f-7538-4f74-bba0-76855860ee63","order_by":7,"name":"Changjing Wu","email":"","orcid":"","institution":"Zhoukou Normal University","correspondingAuthor":false,"prefix":"","firstName":"Changjing","middleName":"","lastName":"Wu","suffix":""},{"id":272839324,"identity":"b022088c-8706-44e4-bd15-d01355e768be","order_by":8,"name":"Xiaomeng Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAt0lEQVRIiWNgGAWjYBACPghlI8fPzHzwAVFa2CBUmrFkO1uyASlaDiduOM9jJkCcFonsNKkbQC2bDzOYMTDU2EQT1sJzdrNxDkO68bbDDGkPGI6l5TYQ1MLeu/FxDoO1LFDLcQPGhsNEaGHm3XA4h4GZcXMzY5sEcVogtjgrbmBmZiNSC8QvacYSh9mYDRKI8Qu/RO426RxQVPaf//jgQ40NYS1gwPgPykggSvkoGAWjYBSMAoIAAENJN6JGNeiOAAAAAElFTkSuQmCC","orcid":"","institution":"Xinxiang Medical University","correspondingAuthor":true,"prefix":"","firstName":"Xiaomeng","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2024-02-02 08:33:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3920171/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3920171/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-64045-0","type":"published","date":"2024-06-14T14:56:39+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":51198565,"identity":"ba1e2f04-0538-41cd-b43e-6c4f7bfc3a06","added_by":"auto","created_at":"2024-02-15 19:41:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":63327,"visible":true,"origin":"","legend":"\u003cp\u003eThe chemical stability test for JE aqueous solution by HPLC-PDAD-UV analysis\u003c/p\u003e","description":"","filename":"figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3920171/v1/765c39ac7a4c549fb326e1b2.png"},{"id":51198452,"identity":"3e5f3c0c-d94e-4dac-8411-4b5960c5c99b","added_by":"auto","created_at":"2024-02-15 19:33:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":58912,"visible":true,"origin":"","legend":"\u003cp\u003eLifespan and stress resistance of \u003cem\u003eC. elegans \u003c/em\u003eexposed to JE. (a) Acute toxicity of JE on C. elegans feeding in S-medium. (n = 90). (b ) Lifespan of C. elegans treated with 20 μg/mL, 50 μg /mL, 100 μg/ml and 200 ug/mL, or the corresponding concentration of vehicle. (n ≥ 146). (c) Lifespan of C. elegans exposed to 37 °C thermal shock treated with 100 μg/mL JE or vehicle. (n ≥ 120), **P ≤ 0.01. (d) Lifespan of C. elegans exposed to 50 μM paraquat treated with 100 ug/mL JE or vehicle. (n ≥ 139). **P ≤ 0.05*, P ≤ 0.01 , ***P ≤ 0.001 (vehicle vs treated ). The experiment was carried out several times, and a typical trial is shown.\u003c/p\u003e","description":"","filename":"figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3920171/v1/acbbdacc63f400531e3dbdb5.png"},{"id":51198566,"identity":"ae601187-1443-4537-be19-4497fcb3a3e1","added_by":"auto","created_at":"2024-02-15 19:41:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":155048,"visible":true,"origin":"","legend":"\u003cp\u003eJE treatment decreases intestinal autofluorescence (lipofuscin) and ROS levels in adult wild-type worms. (a) day 5 adult wild-type worms. (b) Mean RFU value. (c) day 12 adult wild-type worms. (d) Mean RFU value . (e) Assessment of ROS level with 36 hours JE or vehicle treatment. (f) Assessment of ROS level with 84 hours JE or vehicle treatment\u003c/p\u003e","description":"","filename":"figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3920171/v1/82802b72489ed04dbcde3cc1.png"},{"id":51198454,"identity":"e8abca67-effe-43d3-86b0-4e532b794e28","added_by":"auto","created_at":"2024-02-15 19:33:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":33307,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of JE on the fertility of \u003cem\u003eC. elegans\u003c/em\u003e. (a) Mean pumping rates (pumps per 20 s) are shown for each time point (n = 10). (b-c) the number of eggs laid of \u003cem\u003eC. elegans \u003c/em\u003etreated with 100 μg/mL JE or. (d) breeding period of \u003cem\u003eC. elegans \u003c/em\u003etreated with 100 μg/mL JE or vehicle(n = 16). Values are mean ± SE. *P ≤ 0.05, **P ≤ 0.01 ***P ≤ 0.001.\u003c/p\u003e","description":"","filename":"figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3920171/v1/59aa9668b990411ae70a3468.png"},{"id":51198459,"identity":"40c6e44c-23bd-4080-82f4-eaadd2b836e6","added_by":"auto","created_at":"2024-02-15 19:33:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":37153,"visible":true,"origin":"","legend":"\u003cp\u003eJE increases the motility of \u003cem\u003eC. elegans\u003c/em\u003e. (a) The counts of body bending (n = 20). (b) The counts of head swing (n = 20). Values are mean ± SE. *P ≤ 0.05, ***P ≤ 0.001.\u003c/p\u003e","description":"","filename":"figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3920171/v1/9f2f5500771d52cd99b3b5b2.png"},{"id":51198456,"identity":"e45229e3-ee03-4975-9634-9cc331845602","added_by":"auto","created_at":"2024-02-15 19:33:34","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":86403,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of JE on Lifespan of mutant \u003cem\u003eC. elegans\u003c/em\u003e. \u0026nbsp;(a) The lifespan of \u003cem\u003eeat-2\u003c/em\u003e mutant treated with 100 μg/mL JE or vehicle (n ≥ 122). (b) The lifespan of \u003cem\u003ehsp-16.2\u003c/em\u003e treated with 100 ug/mL JE or vehicle (n ≥ 119). (c) The lifespan of \u003cem\u003ehsf-1\u003c/em\u003emutant treated with 100 μg/mL JE or vehicle (n =120). (d) The lifespan of \u003cem\u003eskn-1\u003c/em\u003emutant treated with 100 μg/mL JE or vehicle (n =120). (e) The lifespan of \u003cem\u003edaf-2\u003c/em\u003e mutant treated with 100 ug/mL JE or vehicle (n ≥ 121). (f) The lifespan of \u003cem\u003eeat-16\u003c/em\u003e mutant treated with 100 μg/mL JE or vehicle(n ≥ 116). *P ≤ 0.05, **P ≤ 0.01.\u003c/p\u003e","description":"","filename":"figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-3920171/v1/30778c929d2d9a1a4bc82b05.png"},{"id":51198458,"identity":"3d1ef76b-ae69-4cda-924d-b18ca82248c8","added_by":"auto","created_at":"2024-02-15 19:33:34","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":62097,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of JE on the motility, fertility and resistance to stress of daf-16 mutant \u003cem\u003eC. elegans\u003c/em\u003e. (a) body bending (n ≥ 20). (b) head swing (n ≥ 20). (c) number of eggs laid (n = 13). (d) breeding period (n =20) . (e) Exposed to 37 °C thermal shock (n ≥ 55). (f) Exposed to 50 μM paraquat (n ≥ 127). Values are mean ± SE. *P ≤ 0.05.\u003c/p\u003e","description":"","filename":"figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-3920171/v1/e4e797e37a2a095de97917b7.png"},{"id":51198460,"identity":"d749ee82-8417-46ad-9205-a15a5e1c1b83","added_by":"auto","created_at":"2024-02-15 19:33:34","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":113839,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of JE on daf16 of \u003cem\u003eC. elegans\u003c/em\u003e. (a) Representative images of GFP in TJ356 (DAF16-GFP) worm treated with vehicle, heat shock (37 \u003csup\u003e◦ \u003c/sup\u003eC, 20 min) and 100\u0026nbsp; μg/ml JE. (b) DAF-16 translocation assay (n=30).\u003c/p\u003e","description":"","filename":"figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-3920171/v1/046aa664589defac2a052130.png"},{"id":51198457,"identity":"42081b8e-1b29-425e-890f-735a1280ffb2","added_by":"auto","created_at":"2024-02-15 19:33:34","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":99393,"visible":true,"origin":"","legend":"\u003cp\u003eGenes affected by JE treatment in \u003cem\u003eC. elegans \u003c/em\u003ewild-type（a) The relative expression level of the downstream gene of \u003cem\u003edaf-16\u003c/em\u003e in N2 strain after JE treatment. (b) The relative expression level of the downstream gene of \u003cem\u003edaf-16\u003c/em\u003e in GR1307 strain (\u003cem\u003edaf-16\u003c/em\u003emutant) after JE treatment. (c) Representative images of GFP in CF1553 (SOD3-GFP) worm treated with vehicle and 100 μg/ml JE. (d) The expression of SOD-3 assay (n=30). (e) The lifespan of \u003cem\u003esod-3\u003c/em\u003e mutant treated with 100 μg/mL JE or vehicle (n = 129)Values are mean ± SE. **P ≤ 0.01, ***P ≤ 0.001.\u003c/p\u003e","description":"","filename":"figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-3920171/v1/88fd0f427b58dd1ff1812f44.png"},{"id":58822493,"identity":"0d69daca-8f66-40be-8ca0-5934e99eb9bd","added_by":"auto","created_at":"2024-06-21 16:45:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1545880,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3920171/v1/5b567432-bef7-47f8-ad4a-f0d49135a1f1.pdf"},{"id":51198461,"identity":"cf897fe0-5239-48a3-8d95-7ee62c1c91f4","added_by":"auto","created_at":"2024-02-15 19:33:34","extension":"docx","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":297117,"visible":true,"origin":"","legend":"","description":"","filename":"supportinginformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-3920171/v1/26062236087b3782f9fd3fdd.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Jujubae Fructus extract prolongs lifespan and improves stress tolerance in Caenorhabditis elegans dependent on DAF-16/SOD-3","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAging is the process of physiological and biochemical changes in the body and functional degradation with age (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). As the major risk factor for most of chronic diseases, it increases the incidence and mortality of obesity, diabetes, cancer, neurodegenerative diseases, and cardiovascular diseases (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Therefore, delaying the aging process is of great significance to improve people's quality of life, preventing various diseases and even the sustainable development of society. Considering an explosive increase of senior citizens suffering from a number of chronic disorders, which could result in a tremendous social and economic burden, it is imperative to concentrate our collective efforts on developing novel remedies in order to decelerate senile changes and deter the inevitable physical alternations from advancing to the formidable ailments.\u003c/p\u003e \u003cp\u003e \u003cem\u003eZiziphus Jujuba\u003c/em\u003e is a plant belonging to Ziziphus species in the buckthorn family Rhamnaceae. Historically, it has been widely cultivated in Europe, southern/eastern Asia, and Australia for over 4000 years, mainly because the fruit of \u003cem\u003eZiziphus Jujuba\u003c/em\u003e (\u003cem\u003eJujubae Fructus\u003c/em\u003e) has many active ingredients. The main biologically active components of \u003cem\u003eJujubae Fructus\u003c/em\u003e include cAMP, phenolics, flavonoids, triterpenic acids, and polysaccharides (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). In traditional Chinese medicine, \u003cem\u003eJujubae Fructus\u003c/em\u003e is often used to treat insomnia, spleen deficiency and palpitations, and used together with other medicinal materials to lower blood pressure, lower blood lipid and anti-anxiety. In the recent years, many studies have shown that the main biologically active components of \u003cem\u003eJujubae Fructus\u003c/em\u003e have a variety of biological effects (\u003cspan additionalcitationids=\"CR6 CR7 CR8 CR9 CR10 CR11 CR12 CR13 CR14 CR15\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Through comparative analysis of the hypnotic and sedative effects of flavonoids, saponins, and polysaccharides extracted from \u003cem\u003eJujubae Fructus\u003c/em\u003e, saponins have been proved to have superior hypnotic and sedative effects than flavonoids, and may exert excellent sedative-hypnotic effects simultaneously through a variety of mechanisms (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). \u003cem\u003eJujubae Fructus\u003c/em\u003e can promote the learning and memory of the ovariectomized rats (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e), and repair the memory and behavior disorders caused by NBM lesion in rats (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). It has been reported that \u003cem\u003eJujubae Fructus\u003c/em\u003e could protect the progression of CCl\u003csub\u003e4\u003c/sub\u003e-induced liver injury, which may be due to the fact that jujubae can bind to harmful free radicals and reduce their toxicity, as well as inhibit the inflammatory response of CCl\u003csub\u003e4\u003c/sub\u003e-induced liver injury (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). The active components of \u003cem\u003eJujubae Fructus\u003c/em\u003e, such as triterpenic acid, also have anticancer activity and can induce cell death by inhibiting cell proliferation and apoptosis (\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). \u003cem\u003eJujubae Fructus\u003c/em\u003e also has an effect on glucose and lipid metabolism (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). The \u003cem\u003eJujubae Fructus\u003c/em\u003e polysaccharides can improve insulin resistance and dyslipidemia induced by fructose in mice (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). The infusion of \u003cem\u003eJujubae Fructus\u003c/em\u003e has beneficial effects on blood lipid and glycosylated hemoglobin in patients with type 2 diabetes mellitus (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). There are few studies on the effect of \u003cem\u003eJujubae Fructus\u003c/em\u003e on aging, Ghimire et al (2010) found that \u003cem\u003eJujubae Fructus\u003c/em\u003e feeding extended lifespan and health span of \u003cem\u003edrosophila\u003c/em\u003e (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAbout 60\u0026ndash;80% of genes in \u003cem\u003eC. elegans\u003c/em\u003e homologous to humans (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e), the generation cycle of \u003cem\u003eC. elegans\u003c/em\u003e is three days, the average life span is about 2\u0026ndash;3 weeks, and it is easy to culture and observe. These advantages make \u003cem\u003eC. elegans\u003c/em\u003e an ideal model for aging research (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Numerous studies have focused on prolonging the lifespan of C. elegans, and the mechanism has been found to be related to daf-16. \u003cem\u003ePolygonum multiflorum\u003c/em\u003e and \u003cem\u003eAgrimonia procera\u003c/em\u003e extract have been shown to influence nematodes' lifespan via \u003cem\u003edaf-16\u003c/em\u003e (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). The longevity prolonging effect of blueberry on nematode and the enhancement of stress resistance were also confirmed to be mediated by \u003cem\u003edaf-16\u003c/em\u003e (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). The accumulation of ROS (reactive oxygen species) and oxidative stress injury in \u003cem\u003eGlochidion zeylanicum\u003c/em\u003e treated nematodes were decreased, which was dependent on the DAF-16/FoxO and SKN-1/Nrf-2 signaling pathways (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). In addition, \u003cem\u003elyceum barbarum\u003c/em\u003e polysaccharides, Liangyi Gao, \u003cem\u003eHolothuria leucospilota\u003c/em\u003e, and tiger milk mushroom were also found to improve the health status and increase lifespan of \u003cem\u003eC. elegans\u003c/em\u003e via the \u003cem\u003edaf-16\u003c/em\u003e signaling pathway (\u003cspan additionalcitationids=\"CR23 CR24\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Therefore, \u003cem\u003eC. elegans\u003c/em\u003e is not only an ideal model to study the aging phenotype, but is also widely used to study the anti-aging mechanism. As a traditional Chinese medicine, \u003cem\u003eJujubae Fructus\u003c/em\u003e can be taken as a food and medicine, and have a variety of biological effects (\u003cspan additionalcitationids=\"CR6 CR7 CR8 CR9 CR10 CR11 CR12 CR13 CR14 CR15\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). There are limited studies on the effect of JE on lifespan, and the molecular mechanism of effects JE treatment based on \u003cem\u003eC. elegans\u003c/em\u003e is still not clearly defined. Herein, to investigate the effect of JE treatment on \u003cem\u003eC. elegans\u003c/em\u003e, we supplemented \u003cem\u003eC. elegans\u003c/em\u003e with JE to investigate lifespan, vitality, fertility, and stress resistance of \u003cem\u003eC. elegans\u003c/em\u003e, and further studied the mechanism of the effect of JE on \u003cem\u003eC. elegans\u003c/em\u003e.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant Extract Preparation\u003c/h2\u003e \u003cp\u003eThe dried ripe fruit of \u003cem\u003eZiziphus jujube\u003c/em\u003e (\u003cem\u003eJujubae Fructus\u003c/em\u003e) was obtained from Bozhou Chinese medicinal materials market and identified as \u003cem\u003eZizyphus Jujuba\u003c/em\u003e cv. Jinsixiaozao by Professor Hongmei Gu (Zhoukou Normal University). The voucher specimen (ZF-180621) was deposited in the Institute of Translational Medicine, College of Life Science and Agronomy, Zhoukou Normal University under closed and dry conditions at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u0026deg;C. \u003cem\u003eJujubae Fructus\u003c/em\u003e was cut up and extracted twice with boiling water for 2 h. The extract solution was filtrated immediately and the final filtrates were combined. The filtered extract was concentrated and dried under vacuum conditions by a rotary evaporator with 60\u0026deg;C water bath. The total yield of water extract from \u003cem\u003eJujubae Fructus\u003c/em\u003e (JE) was 51.7% (w/w). The chemical stability of JE aqueous solution was evaluated with an accelerate test by heating up to 60\u0026deg;C for 24 h, and comparative HPLC-PDAD-UV analysis was performed using an analytical Kromasil C18 column (5 \u0026micro;m, 100 \u0026Aring;, 4.6 mm \u0026times; 250 mm; Akzo Nobel) on an Agilent 1100 HPLC system equipped with photo-diode array detector (G1316A). After filtered with 0.45 \u0026micro;m membrane, the JE sample solution in water (100 mg/ml) was injected (10 \u0026micro;L) into the column and eluted with a MeOH-water with 0.1% formic acid linear gradient (20%\u0026rarr;100% MeOH in 20 min followed by 5 min with isocratic 100% MeOH) mobile phase (flow rate 1.0 mL/min flow rate). The acquired photodiode array data (PDAD) data were processed with Agilent OpenLAB software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eUHPLC-Q-TOF-MS/MS Analysis of JE\u003c/h2\u003e \u003cp\u003eJE sample (10 mg) was added 1.0 ml of 80% methanol. The mixture was ultrasonicated for 15 min (200 W, 40 KHZ) at room temperature, and then centrifuged for 10 min at 12,000 rpm. Finally, the supernatants were filtered through a 0.22 \u0026micro;m membrane to obtain prepared samples for UHPLC-Q-TOF-MS analysis. Chromatographic separation was performed in a Nexera UHPLC LC-30A system (Shimadzu Corporation, Jappan) equipped with a SHIMADZU InerSustain C18 (100\u0026times;2.1 mm, 2 \u0026micro;m) column maintained at 35\u0026deg;C with a flow rate of 1.0 mL/min. Gradient elution of analytes was carried out with acetonitrile (A) and 0.1% aqueous formic acid (B). The sample solution (5 \u0026micro;L) was injected after equilibration. An increasing linear gradient of solvent A (v/v) was then applied, as follows: 0\u0026ndash;3 min, 5% A; 3\u0026ndash;15 min, 5\u0026ndash;20% A; 15\u0026ndash;40 min, 20\u0026ndash;100% A; 40\u0026ndash;45 min, 100% A; 45\u0026ndash;46 min, 100\u0026ndash;5% A; 46\u0026ndash;48 min, 5\u0026ndash;5% A.\u003c/p\u003e \u003cp\u003eThe ESI-MSn experiments were carried out on a TripleTOF 5600\u003csup\u003e+\u003c/sup\u003e Hybrid Quadrupole-TOF LC/MS/MS Mass Spectrometer (AB SCIEX\u0026trade;, United States) with spray voltages of 5.5 and 4.4 kV in positive and negative modes, respectively. Ion source gas and curtain gas were set at 50 and 25 arbitrary units, respectively. The source temperatures were 500 and 450\u0026deg;C in positive and negative modes, respectively. The analyzer scanned over mass ranges of m/z 100\u0026ndash;1,200 Da and m/z 50\u0026ndash;1,000 Da with accumulation time of 0.2s and 0.01s for TOF MS scan and product ion scan, respectively. Information-dependent acquisition (IDA) MS/MS experiments were performed with high sensitivity mode with Declustering Potential as \u0026plusmn;\u0026thinsp;60V and Collision Energy as 35\u0026thinsp;\u0026plusmn;\u0026thinsp;15eV. The analysis of UHPLC-MS data was performed using MS-DIAL 4.80 software (RIKEN Center for Sustainable Resource Science: Metabolome Informatics Research Team, Yokohama, Japan). We tentatively identified the compounds of JE by considering factors such as molecular weight, retention time, fragment information obtained from the MS/MS model, and further matching annotation in our prepared compounds, along with previous literature and the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database.\u003c/p\u003e \u003cp\u003e \u003cb\u003eC. elegans\u003c/b\u003e \u003cb\u003eStrains and Maintenance Conditions\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eC. elegans\u003c/em\u003e strains used were N2: Bristol (wildtype), CF1038: \u003cem\u003edaf-16\u003c/em\u003e (mu86), CF1553: \u003cem\u003esod-3\u003c/em\u003e (muIs84), and DA1116: \u003cem\u003eeat-2\u003c/em\u003e (ad1116), which were obtained from Shanghai Tech University. The PS3551: \u003cem\u003ehsf-1\u003c/em\u003e (sy441), CB1370: \u003cem\u003edaf-2\u003c/em\u003e (e1370), CL2070: \u003cem\u003ehsp-16.2\u003c/em\u003e (dvIs70), EU31: \u003cem\u003eskn-1\u003c/em\u003e (zu135), and TJ356: \u003cem\u003edaf-16\u003c/em\u003e (zIs356) were obtained from Tongji University of Life Sciences and Technology. All the strains were maintained using standard conditions at 20\u0026deg;C on NGM (nematode growth medium) plates. Worms were also allowed to grow in liquid S-medium with concentrated \u003cem\u003eEscherichia coli\u003c/em\u003e OP50 (6mg/mL) as a food resource. Eggs were extracted from the nematodes before all experiments to synchronize the nematodes (worms at spawning stage were collected in M9 buffer solution (containing 0.5M NaOH and 0.8%NaClO), digested for 3\u0026ndash;5 minutes and centrifuged to remove the supernatant at 1300g for 30s. The worms were washed twice with M9 buffer solution to retain the centrifuged precipitation (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). When the worms reached L1 stage, Live or dead (heat inactivated) E. coli OP50 was added to the NGM plates as food to feed the worms. OP50 was killed by heat shock temperature control 75\u0026deg;C during 2 hours. In L4 stage, different concentrations of JE were added to the culture, and 40 \u0026micro;M Fluoro-2'-deoxy-β-uridine (FUDR, Sigma-Aldrich) was also necessary to inhibit the growth of progen (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eToxicity assay\u003c/h2\u003e \u003cp\u003eTo perform the toxicity assay, the JE was dissolved in sterile water to prepare a stock solution of 100 mg/ml. The assay was performed using 100 \u0026micro;g/mL, 200 \u0026micro;g/mL, 1 mg/mL, 10 mg/mL, and 100 mg/mL concentrations. The control group was exposed to an equal volume of sterile water. On day 2 of adulthood, an age-synchronized population of worms was transferred to a 1 ml volume of S-medium supplemented with test doses of JE in a 12-well plate (Nest, China). A total of 90 worms were placed on triplicate wells with 30 worms per well for each group. The survival of worms was scored after every 2-hour interval and the data shown represents the survival rate of worms after 24 h (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eLifespan assay\u003c/h2\u003e \u003cp\u003eN2, CF1038, DA1116, PS3551, CB1370, EU31, and CL2070 lifespan assays were carried out at 20\u0026deg;C. On day 2 of adulthood, an age-synchronized population of worms was transferred to NGM plates containing JE (20, 50, 100, 200 \u0026micro;g/mL) or an equal volume of sterile water (vehicle group). A total of 55\u0026ndash;80 worms were placed on 3 to 4 replicate plates with 16 to 20 worms per 3.5 cm plate. Worms were transferred to new plates and scored every two days. Worms were scored as alive until there was no movement after repeated prodding. Lifespan assays were indicated in supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, and the number of worms was pooled (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eROS Assay\u003c/h2\u003e \u003cp\u003eWild-type N2 worms from the adult stage were treated with 100 \u0026micro;g/mL JE or vehicle and were used for intracellular ROS determination. At day 2 and day 4 adult worms age, synchronized worms were collected in 500 \u0026micro;l of 0.1% PBST buffer and then homogenized and sonicated. The sample using Fluorescent probe H2DCF-DA (2\u0026prime;,7\u0026prime;-dichlorodihydroflfluoresceindiacetate) and BioTek microplate reader at emission FL fluorescence intensity at 485 nm and excitation at 530 nm to measure emission FL fluorescence intensity. The observations were recorded for 90 min at intervals of 20 min. The assay was performed in triplicate independently.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eLipofuscin Assay\u003c/h2\u003e \u003cp\u003eWild-type N2 worms raised from the adult stage were treated with 100 \u0026micro;g/mL JE or vehicle used for lipofuscin assay. On the 10th day of adulthood (n\u0026thinsp;=\u0026thinsp;20) were randomly selected and mounted on 3% agarose pads and anesthetized with 2% sodium azide. Images were captured with fluorescence microscope (Olympus BX 61, Japan) using GFP filter (with excitation at 340\u0026ndash;380 nm and emission at 435\u0026ndash;485 nm) FL fluorescence microscope at 10\u0026times;. The fluorescence levels were quantified using the ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003ePharyngeal Pumping Rate\u003c/h2\u003e \u003cp\u003eThe pharyngeal pumping rate was quantified on the 5th, 7th, and 10th days of adulthood. Ten worms were treated with 100 \u0026micro;g/mL JE or vehicle randomly picked for measurement of pharyngeal pumping rate per 20 s.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eMovement assay\u003c/h2\u003e \u003cp\u003eTo measure the frequency of body bending, on day 2 of adulthood, an age-synchronized population of worms was treated with 100 \u0026micro;g/mL JE or vehicle for 2 and 11 days. Worms were placed on NGM plates containing FUDR (40 \u0026micro;M), and the number of sinusoidal curves made during locomotion and swinging head in 1 min was scored. A total of 20\u0026ndash;24 worms were detected in each group (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eFertility measurement\u003c/h2\u003e \u003cp\u003eTo determine fecundity, worms (N2/RNAi) were transferred to a medium without FUDR and treated with 100 \u0026micro;g/mL JE or vehicle treatment for 2 days. Hermaphrodites were serially transferred to fresh NGM plates at 24-hour intervals, until sterile. Two days after eggs were laid, the number of hatched progenies was counted. When the nematodes were no longer postpartum, their reproductive cycles were recorded. Each group included 13\u0026ndash;14 worms (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eHeat stress resistance\u003c/h2\u003e \u003cp\u003eFor the heat stress resistance assay, on day 2 of adulthood, an age-synchronized population of worms was treated with 100 \u0026micro;g/mL JE or vehicle for 2 days. Live or dead OP50 (killed by heat shock temperature control 75\u0026deg;C for 2 hours) was added to NGM plates. Worms were placed on NGM plates containing FUDR (40 \u0026micro;M) in 37\u0026deg;C conditions and then the number of worms was counted every hour until all the worms had died. Each group included 50 worms (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eOxidative stress resistance\u003c/h2\u003e \u003cp\u003eOn day 2 of adulthood, an age-synchronized population of worms was transferred to S-medium containing FUDR (40 \u0026micro;M) and with 100 \u0026micro;g/mL JE or vehicle treatment, respectively. After 2 days, 5 mM paraquat was added to S-medium, which induces lethal oxidative stress. The vitality of the worms was examined every 4 hours until all worms had died. Triplicate plates were used for each group. Each group included 150 worms (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eDAF-16 localization via fluorescence microscopy\u003c/h2\u003e \u003cp\u003eThe TJ356 strain was used to examine the localization of DAF-16 in the living nematode. The aged-synchronized L4 larvae were transferred to NGM plates previously treated with 100 \u0026micro;g/mL JE or vehicle and incubated for 48 hours. DAF-16 localization was examined in 30 worms per treatment that were mounted in a drop of 20 mM levamisole hydrochloride. We scored each animal as having cytosolic localization, nuclear localization, or intermediate localization when there is a visible nuclear localization but one not as complete as nuclear. The number of worms with each level of nuclear translocation was counted. Fluorescence images were taken at constant exposure times (Olympus IX 73, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative real-time PCR\u003c/h2\u003e \u003cp\u003eThe contemporaneous 2-day-old worms were incubated in NGM containing the same concentration of JE for 7 days. After washing with M9 buffer, \u003cem\u003eC. elegans\u003c/em\u003e were collected into 1.5 ml tubes and extraction RNA using the TransZol Up (Transgene) and stored at -80\u0026deg;C. Complementary DNA was prepared using HiScript III 1st Strand cDNA Synthesis Kit (+\u0026thinsp;gDNA wiper) (Vazyme) for real-time polymerase chain reaction (RT-PCR). Quantitative PCR (qPCR) was performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme). The mRNA expression levels of downstream genes of \u003cem\u003edaf-16\u003c/em\u003e in nematodes were monitored. β-actin was used as the housekeeping gene for normalization, and the experimental results were expressed as 2ΔΔCt values of PCR. The information of the primer sequence is included in supplementary Table S10.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were carried out using Graphpad 7.0. A Kaplan\u0026ndash;Meier lifespan analysis was carried out, and p values were calculated using the log-rank test. In all statistical analyses, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was accepted as statistically significant (* p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, *** p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003eHPLC-PDAD-UV and UHPLC-Q-TOF-MS Analysis of JE\u003c/p\u003e\n\u003cp\u003eIn the accelerated test, the fingerprint chromatograms derived from HPLC-PDAD-UV analysis of JE aqueous solution showed no significant changes after 24 hours of heating (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Additionally, the UV absorption spectra of the main chromatographic peaks remained unchanged (data not shown). This result suggested that the JE is chemically stable in an aqueous solution during the lifespan assay under normal conditions. UHPLC-Q-TOF-MS analysis was conducted in both positive and negative modes, with Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e displaying the positive total ions chromatogram (TIC). A total of 22 phytochemicals were identified in JE, including three carbohydrates, five glycosides, two alkaloids, eleven triterpene acids, and cAMP (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Six of these compounds (cAMP, zizybeoside I and II, rutin, oleanonic acid, and ursolic acid) were identified by comparison to our previously prepared samples; the others were identified by comparison with literature (\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eIdentified ingredients in JE.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"7\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNo.\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRT (min)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCompound name\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFormular\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMS\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eError (ppm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMS/MS\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTartaric acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e4\u003c/sub\u003eH\u003csub\u003e6\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e151.0241 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e110.0111, 99.0442, 88.2338, 82.0182\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMethose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e179.0544 [M-H]-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e125.1130, 101.0300, 89.0345\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSucrose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e12\u003c/sub\u003eH\u003csub\u003e22\u003c/sub\u003eO\u003csub\u003e11\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e341.1055 [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e163.0637, 145.0529, 127.0410\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ecAMP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e10\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003eP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e330.0620 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-5.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e232.0850, 136.0638\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eZizybeoside I\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e28\u003c/sub\u003eO\u003csub\u003e11\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e431.1527 [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e347.0983, 293.1010\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eZizybeoside II\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e25\u003c/sub\u003eH\u003csub\u003e38\u003c/sub\u003eO\u003csub\u003e16\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e617.2054 [M\u0026thinsp;+\u0026thinsp;Na]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e431.1798, 269.1303, 161.0566\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCoclaurine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e17\u003c/sub\u003eH\u003csub\u003e19\u003c/sub\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e286.1449 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e269.1149, 237.0914, 209.0965, 175.0787, 145.0646, 107.0539\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e11.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eZizyvoside II\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e31\u003c/sub\u003eH\u003csub\u003e50\u003c/sub\u003eO\u003csub\u003e18\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e709.2858 [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e547, 519.2244, 385.2117\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e11.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eStepharine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e19\u003c/sub\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e298.1451 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e269.1191, 192.1039, 161.0858\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e14.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRutin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e27\u003c/sub\u003eH\u003csub\u003e30\u003c/sub\u003eO\u003csub\u003e16\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e611.1597 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e465.0975, 303.0503, 163.0670\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eZizyvoside I\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e25\u003c/sub\u003eH\u003csub\u003e40\u003c/sub\u003eO\u003csub\u003e12\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e533.2593 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e435.1962, 393.1816, 277.2098\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e29.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCeanothic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e30\u003c/sub\u003eH\u003csub\u003e46\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e485.3259 [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e467.3810, 423.3476\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e30.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAlphitolic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e30\u003c/sub\u003eH\u003csub\u003e48\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e473.3591 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e455.3698, 390.9161\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e31.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMaslinic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e30\u003c/sub\u003eH\u003csub\u003e48\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e495.3450 [M\u0026thinsp;+\u0026thinsp;Na]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e409.3433, 381.3086, 296.8615, 249.1672, 203.1779\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e31.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u0026alpha;-Hydroxyursolic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e30\u003c/sub\u003eH\u003csub\u003e48\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e473.3627 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e437.3418, 409.3402, 391.3322, 285.2632, 223.1774, 205.1594, 187.1443\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eZizyberanalic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e30\u003c/sub\u003eH\u003csub\u003e46\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e471.3475 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e453.3368, 435.3225, 407.3355, 389.3332, 327.2283, 245.1501, 177.1640\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e33.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3-O-cis-p-Coumaroylalphitolic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e39\u003c/sub\u003eH\u003csub\u003e54\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e619.3978 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e437.3510, 391.3270, 259.1755, 202.5370, 173.1333, 135.1207\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e33.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3-O-cis-p-Coumaroylmaslinic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e39\u003c/sub\u003eH\u003csub\u003e54\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e619.4019 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-3.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e437.3447, 411.3292, 353.2572, 287.2157, 203.1819, 147.0457\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e34.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3-O-trans-p-Coumaroylalphitolic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e39\u003c/sub\u003eH\u003csub\u003e54\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e619.4016 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e437.3450, 409.3405, 391.3436, 363.2439, 201.1579, 177.1812\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e34.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3-O-trans-p-Coumaroylmaslinic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e39\u003c/sub\u003eH\u003csub\u003e54\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e619.3944 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e437.3506, 411.3207, 261.1825, 165.0583\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e36.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOleanonic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e30\u003c/sub\u003eH\u003csub\u003e48\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e455.3537 [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e437.3444, 409.3399, 259.1797, 177.1659\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e37.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUrsonic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e30\u003c/sub\u003eH\u003csub\u003e48\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e455.3532 [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e437.3396, 409.3509, 261.1843, 208.1591, 163.1460\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eEffect of JE on the lifespan and stress resistance of \u003cem\u003eC. elegans\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eBefore conducting lifespan experiments, we carried out an acute in vivo toxicity study. When the concentrations of JE were less than or equal to 1mg/mL, which were found to be non-toxic to \u003cem\u003eC. elegans\u003c/em\u003e were chosen for further tests (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea). To address whether JE has a positive effect on the lifespan of \u003cem\u003eC. elegans\u003c/em\u003e, N2 worms were treated with JE at treated with 20 ug/mL, 50 ug/mL, 100 ug/mL, and 200 ug/mL doses of standardized JE. The results showed that compared with the control group (equal volume sterile water was used as control), 50 ug/mL, 100 ug/mL, and 200 ug/mL JE treatment significantly increased the lifespan of the N2 worms in a dose-dependent manner, with the maximum lifespan increased from 19 days in control to 21, 24 and 23 days, respectively (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb, Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e). The mean lifespan significantly increased to 114.1%, 123.3%, and 123.1% with the treatment of 50, 100, and 200 \u0026micro;g/mL (Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e). The 100 and 200 \u0026micro;g/ml doses of JE were able to extend the mean lifespan and maximum lifespan maximally. In the follow-up experiments, 100 \u0026micro;g/mL of JE was used to cultivate worms to observe its effects on other physiological indicators of \u003cem\u003eC. elegans\u003c/em\u003e. Meanwhile, JE treatment also increased the lifespan of worms exposed to 20\u0026deg;C or 37\u0026deg;C thermal shock when fed bacteria killed by heat (Figure S2). These data illustrated that JE-induced prolongation of lifespan occurs by a direct effect on C. elegans rather than indirectly through the bacteria.\u003c/p\u003e\n\u003cp\u003eAging is often accompanied by a decline in resistance to stress, we exposed \u003cem\u003eC. elegans\u003c/em\u003e to heat and oxidative stress to observe the effects of JE on \u003cem\u003eC. elegan\u0026apos;s\u003c/em\u003e lifespan. Under the thermotolerance conditions, the mean lifespan of the JE treatment group significantly increased by 23.3% compared with the control group. The maximum lifespan of the control group was 12 hours, while that increased to 15 hours after JE treatment (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec, Table S2). The result of the oxidative stress showed JE treatment also could significantly increase the mean lifespan of \u003cem\u003eC. elegan\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ed, Table S3).\u003c/p\u003e\n\u003cp\u003eJE decreases the pigment lipofuscin and intracellular ROS level in C. elegans\u003c/p\u003e\n\u003cp\u003eAs a marker of aging and oxidative damage, The rate of lipofuscin formation increases with age and it depends on the rate of oxidative damage (\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e). we detected lipofuscin levels of 100 \u0026micro;g/mL JE-treated or vehicle-treated wild-type C. elegans at day 5 and day 12 of adulthood. The result showed that, After receiving 5 or 12 days of JE treatment, the lipofuscin level in the intestine decreased by 65% and 20% respectively (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea-d). To investigate the effect of JE treatment on oxidative damage, intracellular ROS levels were evaluated in wild-type worms using H2DCF-DA, a widely known fluorescence probe for detecting intracellular ROS production. The results displayed that, with 2 or 4 days of JE treatment, the ROS accumulation of the wild-type worms had a significant decrease (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ee-f).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of JE on pharyngeal pumping rate and fertility of\u003c/strong\u003e \u003cstrong\u003eC. elegans\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWorms with reduced pharyngeal pumping ingest fewer bacteria and exhibit numerous DR-like characteristics, such as decreased fecundity and prolonged lifespan (\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e). We tested whether JE had an effect on the pharyngeal pumping rate, and found that, compared to the control, 100 \u0026micro;g/mL JE treatment had no effect on pharyngeal pump rates on days 2 and 5, but significantly decreased pharyngeal pump rates in adults on days 7 and 10 (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea). This indicated that JE treatment just altered the feeding behavior of the older worms. Next, we explored whether the extension in lifespan was accompanied by any effect on the fertility of nematodes. The total offspring per worm in the control group was 225.8\u0026thinsp;\u0026plusmn;\u0026thinsp;9.9. After administering 100 ug/ml JE, the total offspring decreased to 182.2\u0026thinsp;\u0026plusmn;\u0026thinsp;10.9 (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb, c). To our surprise, the mean breeding days increased by 13.6% (3.5 days in control worms, 4.0 days in 100 ug/ml JE treatment worms, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb, d). Therefore, these results manifested that, with the lifespan extension, the JE treatment significantly decreased fertility and increased the breeding period of worms.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of JE on the mobility of\u003c/strong\u003e \u003cstrong\u003eC. elegans\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWith the aging process, the worm\u0026apos;s activity slows down and becomes insensitive to external stimuli (\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e). Stamper and Hosono previously reported that wild-type worms exhibit an age-dependent decline in movement ability, the decline of activity was rapid from days 7\u0026ndash;10 (\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e). To investigate whether the increased lifespan was accompanied by the improvement of movement behavior, we conducted a movement behavior assay (body bending and head swing) at the age of 2 and 11 days of L4 stage With chronic JE treatment, the activity of body bending in adult worms at the age of 2 and 11 days showed a significant increase of 23.4% and 40.5% respectively (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea). Meanwhile, with chronic JE treatment, the head swing activity of 2 and 11 days worms significantly increased (24.6% in 2 days worms, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and 54.3% in 11 days worms, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eb). Interestingly, the activity of body bending and head swing in vehicle group worms showed an age-dependent decline (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea, b). However, the activity of body bending and head swing in JE treatment group worms had no change with the aging process (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea, b).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJE requires\u003c/strong\u003e \u003cstrong\u003edaf-16\u003c/strong\u003e \u003cstrong\u003eto extend the lifespan of\u003c/strong\u003e \u003cstrong\u003eC. elegans\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to investigate the molecular mechanisms of JE on longevity extension and health improvement in \u003cem\u003eC. elegans\u003c/em\u003e, we next dissected the longevity pathways required for the lifespan extension induced by JE by testing its effects in prototypical mutant worms for aging-related signaling pathways such as insulin/insulin-like growth factors-1 (IIS), caloric restriction, ROS and so on. The results showed that both \u003cem\u003eeat-2\u003c/em\u003e (caloric restriction), \u003cem\u003edaf-2\u003c/em\u003e (IIS), \u003cem\u003ehsf-1\u003c/em\u003e, \u003cem\u003eskn-1\u003c/em\u003e and \u003cem\u003ehsp-16.2\u003c/em\u003e mutant worms had an increased mean lifespan and maximum lifespan with JE treatment as compared with their respective mutant vehicle groups (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea-e, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In contrast, the effect of JE on lifespan was dependent on insulin/IGF1 signal pathway, as the improvement was entirely suppressed in \u003cem\u003edaf-16\u003c/em\u003e mutants, with the 100 ug/ml JE treatment, the mean lifespan and the maximum lifespan of \u003cem\u003edaf-16\u003c/em\u003e mutant worms had no change (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ef, p\u0026thinsp;=\u0026thinsp;0.92). All together, these results indicated that JE treatment requires \u003cem\u003edaf-16\u003c/em\u003e gene to extend the mean and maximum lifespan of \u003cem\u003eC. elegans\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJE cannot affect the mobility, fertility, and lifespan under stress resistance of mutant\u003c/strong\u003e \u003cstrong\u003eC. elegans\u003c/strong\u003e \u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003edaf-16\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, we observed the effect of JE on physiological indexes of \u003cem\u003edaf-16\u003c/em\u003e mutant \u003cem\u003eC. elegans\u003c/em\u003e. In the vehicle group, the worms bent their bodies 16.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0 times and swing heads 22.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5 times per minute, compared with 17.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9 and 22.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6 times per minute in the JE treatment group (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ea-b). Similarly, JE treatment cannot significantly affect the number of eggs laid and breeding period of \u003cem\u003edaf-16\u003c/em\u003e mutant \u003cem\u003eC. elegans\u003c/em\u003e. Under the treatment of JE, \u003cem\u003edaf-16\u003c/em\u003e mutant \u003cem\u003eC. elegans\u003c/em\u003e breed 258\u0026thinsp;\u0026plusmn;\u0026thinsp;19.7 eggs total, and the oviposition duration was 3.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 days, which were not significantly different from the control group (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ec-d). Then, the mutant worms were treated with JE for 48 hours and placed at 37\u0026deg;C and paraquat to observe their resistance to heat stress and oxidative stress. Our results showed that JE did not significantly increase the mean lifespan and maximum lifespan of \u003cem\u003edaf-16\u003c/em\u003e mutants under heat and oxidative stress conditions (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ee-f).\u003c/p\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003eEffect of JE treatment on DAF-16 translocation\u003c/h2\u003e\n \u003cp\u003eThe JE treatment did not change the lifespan, mobility, and fertility of \u003cem\u003edaf-16\u003c/em\u003e mutant worms, it indicated that the effects of JE treatment depend on the gene \u003cem\u003edaf-16\u003c/em\u003e in \u003cem\u003eC. elegans\u003c/em\u003e. \u003cem\u003eDaf-16\u003c/em\u003e is a key factor in insulin/IGF1 signaling pathways transferred from the cytoplasm to the nucleus for multiple biological processes under stress. To determine whether JE is able to affect the cellular localization of DAF-16, we introduced the green fluorescent protein GFP-tagged \u003cem\u003edaf-16\u003c/em\u003e transgenic strain TJ365 to observe the location of DAF-16. The result showed that DAF-16::GFP of 9% transgenic worms localized in the nucleus with vehicle treatment, but heat stress and JE treatment translocates DAF-16::GFP to nucleus in transgenic worms (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003ea-b).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eJE requires\u003c/strong\u003e \u003cstrong\u003esod-3\u003c/strong\u003e \u003cstrong\u003e(the downstream of\u003c/strong\u003e \u003cstrong\u003edaf-16\u003c/strong\u003e\u003cstrong\u003e) to extend the lifespan of\u003c/strong\u003e \u003cstrong\u003eC. elegans\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eTo further explore the mechanism of \u003cem\u003edaf-16\u003c/em\u003e with JE treatment, the expression levels of genes downstream of \u003cem\u003edaf-16\u003c/em\u003e (\u003cem\u003esod-3, mtl-1, gst-4, hsp-16.2 ctl-2\u003c/em\u003e, \u003cem\u003eold-1\u003c/em\u003e) were assessed. The expression level of genes downstream of \u003cem\u003edaf-16\u003c/em\u003e in the N2 worms without JE treatment was set to 1. JE treatment increased the relative expression of sod-3 by 2.1-fold, but had no influence on the expression of other \u003cem\u003edaf-16\u003c/em\u003e downstream genes. (Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003ea). Meanwhile, we compared gene expression in \u003cem\u003edaf-16\u003c/em\u003e mutant worms with the vehicle and JE treatment. Compared with vehicle group, the JE-treated groups did not show significant differences in the expression of \u003cem\u003esod-3\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eb). Furthermore, The GFP-tagged \u003cem\u003esod-3\u003c/em\u003e transgenic strain CF1553 (muls84) was introduced to investigate whether JE increased the protein level of \u003cem\u003esod-3\u003c/em\u003e. Our results showed that 100 \u0026micro;g/mL JE treatment significantly induced the expression of SOD-3 (Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003ec, d). In the present study, we found that JE treatment did not change the mean and maxium lifespan of \u003cem\u003esod-3\u003c/em\u003e mutant worms (Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003ee). In conclusion, our results indicated that JE treatment was able to induce high expression of gene \u003cem\u003esod-3\u003c/em\u003e that might be depending on nuclear translocation of longevity-associated transcription factor DAF-16.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eAs a traditional Chinese medicine and food, \u003cem\u003eJujubae Fructus\u003c/em\u003e has been used in the treatment and late intervention of many diseases. However, there are few studies on its effect on aging. Here, we treated \u003cem\u003eC. elegans\u003c/em\u003e with JE to observe the effects on longevity and health status, and further investigate the mechanisms involved. Our data showed that 50, 100 and 200 \u0026micro;g/mL of JE could significantly increase the lifespan of worms by 14.1%, 23.3% and 23.1%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Increased longevity has been accompanied by improved health status. We also observed how JE affected the lifetime of worms under stress, and the findings revealed that worms treated with JE had greater resilience to heat stress and oxidative stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, d). Many studies on \u003cem\u003eJujubae Fructus\u003c/em\u003e revealed various beneficial nutrients, including carbohydrate, mineral, vitamin, sugar and amino acid (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e), as well as various kinds of secondary metabolites, such as nucleotides (71.98 mg/100g), flavonoids (48.62 mg/100g), triterpenic acids (343.43 mg/100g), polysaccharides (3.30 g/100g) (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). In this study, we identified 22 ingredients in the water extract of \u003cem\u003eJujubae Fructus\u003c/em\u003e by UPLC-MS analysis (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Among them, rutin treatment was reported to reduce polyglutamine (polyQ) protein aggregation in muscle and polyQ-mediated neuronal death in ASH sensory neurons, and extend lifespan in \u003cem\u003eC. elegans\u003c/em\u003e. The possible mechanisms involved are antioxidant activity, activation of protein degradation (autophagy) and insulin/IGF1 signaling pathways (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). As a representative triterpenic acid, ursolic acid prolonged the life span of \u003cem\u003eC. elegans\u003c/em\u003e, and significantly lowered reactive oxygen species (ROS), also could act through serotonin receptors to enhance stress resistance (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Additionally, despite not identified, it is presumed that there were abundant polysaccharides in the JE prepared by decocting method, which were reported to be immunomodulating, antioxidative and hepatoprotective (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). These bioactive constituents may contribute to the beneficial effects of prolonging lifespan and improving stress tolerance in \u003cem\u003eC. elegans\u003c/em\u003e by JE in this study.\u003c/p\u003e \u003cp\u003eGagnon et al found that telomere length in women who had given birth was significantly shorter than that of non-fertile women by analyzing the effects of fertility on the health status, and they indicated that reproduction may have accelerated the aging process (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). Some studies have shown that extension of lifespan is correlated with a decrease in fecundity (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). In addition, decreased worm fertility was associated with increased lifespan (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Removal of reproductive precursor cells in normal and \u003cem\u003edaf-2\u003c/em\u003e mutant worms prolongs the lifespan of \u003cem\u003eC. elegans\u003c/em\u003e (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). Blueberry extract treatment with extended lifespan of nematodes can simultaneously reduce the number of eggs laid and prolong the reeding period of \u003cem\u003eC. elegans\u003c/em\u003e (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Our results also showed that the \u003cem\u003eC. elegans\u003c/em\u003e breeding period was prolonged, and the fecundity was decreased with the 100 ug/ml JE treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Among long-lived humans, it was found that they had their last child at an older age, and the later the last child was born, the longer the females lived (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). In our hand, with the extension of lifespan, the 100 ug/ml JE treatment increased by 0.5 days in breeding period of \u003cem\u003eC. elegans\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). These data indicated that an extension in breeding period is correlated with a longer lifespan (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb,d).\u003c/p\u003e \u003cp\u003eDietary restriction is one of the ways to delay the senescence of nematodes. \u003cem\u003eEat-2\u003c/em\u003e is a key gene in dietary restriction, it is a gene regulating pharyngeal suction rate, and its mutation will lead to feeding defects and reduce energy and nutrient intake of nematodes (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). In order to observe whether the longevity of \u003cem\u003eC. elegans\u003c/em\u003e was prolonged by dietary restriction, we examined the effect of JE on the longevity of \u003cem\u003eeat-2\u003c/em\u003e mutants. The results showed that compared with the control group, JE significantly prolonged the mean and maximum lifespan of \u003cem\u003eeat-2\u003c/em\u003e mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, table S4). consistent with this, the result of pharyngeal pumping rate assay showed that JE treatment had no effect on pharyngeal pump rates on days 2 and 5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Our results suggested that the dietary restriction pathway is nonessential for the effect of JE on the longevity of worms. Interestingly, with JE treatment, the pharyngeal pumping rate of worms significantly decreased as aging progresses (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eThere are other signaling pathways involved in the aging process of nematodes (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). In the IIS signaling pathway, insulin or insulin-like growth factor-1(IGF-1) binding with \u003cem\u003edaf-2\u003c/em\u003e receptor induces the phosphorylation of pi3k/age-1, which activates the downstream \u003cem\u003eakt-1\u003c/em\u003e pathway to phosphorylate \u003cem\u003edaf-16\u003c/em\u003e and \u003cem\u003ehsf-1\u003c/em\u003e, preventing transcription factor transfer into the nucleus, thereby shortening the nematode lifespan (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). \u003cem\u003eDaf-16\u003c/em\u003e is homology with FOXO in mammals, and it is a key protein in nematode longevity (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). Herein, to further study the molecule mechanism of prolongated lifespan and improved health status of \u003cem\u003eC. elegans\u003c/em\u003e with the JE supplementation, we investigated whether JE affected the longevity of nematodes deficient in \u003cem\u003eeat-2\u003c/em\u003e, \u003cem\u003edaf-2\u003c/em\u003e, \u003cem\u003ehsf-1\u003c/em\u003e, \u003cem\u003eskn-1\u003c/em\u003e, \u003cem\u003ehsp-16.2\u003c/em\u003e and \u003cem\u003edaf-16\u003c/em\u003e, respectively. However, compared with the control group, the life-prolonging effect of JE only disappeared in the mutant nematode \u003cem\u003edaf-16\u003c/em\u003e. Next, we observe the effects of JE on vitality, fertility, and stress resistance in mutant nematode \u003cem\u003edaf-16\u003c/em\u003e. Consistently, the effects of JE on these indices in wild-type nematodes were not found in \u003cem\u003edaf-16\u003c/em\u003e mutant nematodes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea-f, Table S5-S9). These findings suggested that the activity of \u003cem\u003edaf-16\u003c/em\u003e gene is crucial for JE to prolong the lifespan and improve the health status of worms. Normally, DAF-16 is located in the cytoplasm, while under stress, DAF-16 is transferred from the cytoplasm to the nucleus, JE treatment also translocate DAF-16::GFP to nucleus in TJ356 worms compared to those untreated ones (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea,b). We also found that JE treatment increased the expression of \u003cem\u003esod-3\u003c/em\u003e, downstream of \u003cem\u003edaf-16\u003c/em\u003e in N2 worms, but the effect was inhibited in mutant (daf-16) C. elegans. We also found that JE treatment doesn\u0026rsquo;t significantly increase the lifespan and the expression of \u003cem\u003esod-3\u003c/em\u003e of mutant (\u003cem\u003edaf-16\u003c/em\u003e) worms as in wild type (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea,b). Our further study manifested that JE\u0026rsquo;s effect on prolonging the lifespan of worms disappeared with the silencing gene of \u003cem\u003esod-3\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ee). \u003cem\u003eSod-3\u003c/em\u003e encodes a superoxide dismutase that can resist oxidative stress and prolong life. Trilobatin has been shown to effectively extend the lifespan of nematode worms by regulating the SKN1/SIRT3/DAF16 signaling pathway, as well as increasing the activity of antioxidant enzymes CAT and SOD-3 (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). Curcumin acetylsalicylate can also delay the aging of nematodes by activating the expression of daf16 and its downstream antioxidant genes \u003cem\u003esod-3\u003c/em\u003e and \u003cem\u003egst-4\u003c/em\u003e (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e). Our findings indicated that JE treatment may induce nuclear translocation of longevity associated transcription factor DAF-16 depending on activation of downstream gene \u003cem\u003esod-3\u003c/em\u003e to prolong lifespan of worms. Meanwhile, the specific small molecule in JE that prolongs the lifespan of \u003cem\u003eC. elegans\u003c/em\u003e and its mechanism, as well as the important role of \u003cem\u003edaf-16\u003c/em\u003e in those effects, need further investigation. \u003cem\u003eJujubae Fructus\u003c/em\u003e has been poorly studied in terms of aging, and previous studies have reported a life-extending effect in drosophila (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Our study makes up for the gap in the impact of \u003cem\u003eJujubae Fructus\u003c/em\u003e on lifespan of C. elegans and provides a certain basis for the relative studies on humans.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThis study, for the first time, reports \u003cem\u003eJujubae Fructus\u003c/em\u003e extract supplementation promotes lifespan and improves the health status of the \u003cem\u003eC. elegans\u003c/em\u003e model system. we identified 22 phytochemicals in JE, including three carbohydrates, five glycosides, two alkaloids, eleven triterpene acids and cAMP. Our data showed that JE treatment prolongs the worms' mean lifespan in both the standard laboratory and heat or oxidative stress conditions. The study also manifests that JE treatment enhances the vitality and breeding period. Meanwhile, the gene-specific mutant studies reveal that lifespan prolongation with JE supplementation was dependent on \u003cem\u003edaf-16\u003c/em\u003e and \u003cem\u003esod-3\u003c/em\u003e (downstream of \u003cem\u003edaf-16\u003c/em\u003e). The nuclear translocation of DAF-16 and the up expression of \u003cem\u003esod-3\u003c/em\u003e, also supports that JE mediated lifespan extension is correlated with the insulin/IGF-1 signal pathway. Our study makes up for the gap in the impact of \u003cem\u003eJujubae Fructus\u003c/em\u003e on lifespan of \u003cem\u003eC. elegans\u003c/em\u003e, the present study may provide a novel avenue against aging and aging related disorders.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFUDR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFluoro-2'-deoxy-β-uridine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eJE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eJujubae Fructus extract\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eH2DCF-DA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e2\u0026prime;,7\u0026prime;-dichlorodihydroflfluoresceindiacetate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIDA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInformation-dependent acquisition\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNGM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enematode growth medium\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePDAD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ephotodiode array data\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eROS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ereactive oxygen species\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTCMSP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTraditional Chinese Medicine Systems Pharmacology\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTIC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etotal ions chromatogram.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFUNDING\u003c/h2\u003e \u003cp\u003eThis work is supported by the Starting Research Fund from the Xin Xiang Medical University (XYBSKYZZ202135), Science and technology innovation talent Program of Henan Province (19HASTIT015), Henan Science and Technology Research Plan Project of China (222102110412).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eLiu Xiaomeng, Wu Changjing and Zhang Zhi: Data curation, Writing- Original draft preparationLi Jiajia, Li Feng and Wang Tao: Methodology, Software Luo Xiaoyan and Li Bin: Visualization, InvestigationWu C and Li Bin: Extraction and UHPLC-MS analysis.Wu C and You Yilin: Writing- Reviewing and Editing\u003c/p\u003e\n\u003cp\u003eStatement\u003c/p\u003e\n\u003cp\u003eThis study was in accordance with relevant institutional, national, and international guidelines and legislation.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eC. 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Molecules 26, (2021).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Jujubae Fructus, Caenorhabditis elegans, lifespan, stress tolerance, daf-16","lastPublishedDoi":"10.21203/rs.3.rs-3920171/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3920171/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eJujubae Fructus\u003c/em\u003e, the fruit of \u003cem\u003eZiziphus jujuba\u003c/em\u003e Mill has been used as one of the medicine food homology species for thousands of years in China. Studies have shown that the active ingredients of \u003cem\u003eJujubae Fructus\u003c/em\u003e have a variety of biological effects, but its role in the aging process still lacks knowledge. Here, we investigated the effect of \u003cem\u003eJujubae Fructus\u003c/em\u003e extract (JE) on \u003cem\u003eC. elegans\u003c/em\u003e lifespan and its potential mechanism. The lifespan of \u003cem\u003eC. elegans\u003c/em\u003e treated with JE was signifificantly increased in a dose-dependent manner. In addition, JE treatment prolonged the reproductive period and increased normal activity during aging in \u003cem\u003eC. elegans\u003c/em\u003e. Similarly, JE supplementation also enhanced the resistance to heat and oxidative stress in \u003cem\u003eC. elegans\u003c/em\u003e. Furthermore, the mutant worms' lifespan assays demonstrated that JE requires \u003cem\u003edaf-16\u003c/em\u003e to prolong lifespan. DAF-16::GFP analysis of TJ356 showed that JE treatment translocates DAF-16::GFP to nucleus in transgenic worms. By analyzing the downstream of \u003cem\u003edaf-16\u003c/em\u003e, we identify that JE may regulate \u003cem\u003esod3\u003c/em\u003e downstream of \u003cem\u003edaf-16\u003c/em\u003e. mutant worms' lifespan and transgenic reporter gene expression assays revealed that increasing SOD-3 expression was critical for extending longevity in \u003cem\u003eC. elegans\u003c/em\u003e with JE therapy. Collectively, these data indicate that JE may have an important role in \u003cem\u003eC. elegans\u003c/em\u003e longevity that is dependent on DAF-16 and SOD-3.\u003c/p\u003e","manuscriptTitle":"Jujubae Fructus extract prolongs lifespan and improves stress tolerance in Caenorhabditis elegans dependent on DAF-16/SOD-3","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-15 19:33:29","doi":"10.21203/rs.3.rs-3920171/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-03-26T07:32:25+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-03-08T09:37:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"1c7be686-c1bc-4f48-8995-a9c6c29950be","date":"2024-03-01T04:08:52+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-02-29T20:29:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-29T12:24:45+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-02-14T12:25:34+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-02-14T04:58:24+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-02-02T08:18:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3d4ea2f9-ef32-44be-acfe-cdf39c2431a2","owner":[],"postedDate":"February 15th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":28759123,"name":"Biological sciences/Cell biology/Senescence"},{"id":28759124,"name":"Health sciences/Molecular medicine"}],"tags":[],"updatedAt":"2024-06-21T14:56:39+00:00","versionOfRecord":{"articleIdentity":"rs-3920171","link":"https://doi.org/10.1038/s41598-024-64045-0","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-06-14 14:56:39","publishedOnDateReadable":"June 14th, 2024"},"versionCreatedAt":"2024-02-15 19:33:29","video":"","vorDoi":"10.1038/s41598-024-64045-0","vorDoiUrl":"https://doi.org/10.1038/s41598-024-64045-0","workflowStages":[]},"version":"v1","identity":"rs-3920171","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3920171","identity":"rs-3920171","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}