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
|
C4H6O6
|
151.0241 [M + H]+
|
1.32
|
110.0111, 99.0442, 88.2338, 82.0182
|
2
|
1.16
|
Methose
|
C6H12O6
|
179.0544 [M-H]-
|
6.70
|
125.1130, 101.0300, 89.0345
|
3
|
1.21
|
Sucrose
|
C12H22O11
|
341.1055 [M-H]−
|
8.50
|
163.0637, 145.0529, 127.0410
|
4
|
1.79
|
cAMP
|
C10H12N5O6P
|
330.0620 [M + H]+
|
-5.15
|
232.0850, 136.0638
|
5
|
9.77
|
Zizybeoside I
|
C19H28O11
|
431.1527 [M-H]−
|
6.03
|
347.0983, 293.1010
|
6
|
10.44
|
Zizybeoside II
|
C25H38O16
|
617.2054 [M + Na]+
|
0.65
|
431.1798, 269.1303, 161.0566
|
7
|
10.89
|
Coclaurine
|
C17H19NO3
|
286.1449 [M + H]+
|
-2.10
|
269.1149, 237.0914, 209.0965, 175.0787, 145.0646, 107.0539
|
8
|
11.48
|
Zizyvoside II
|
C31H50O18
|
709.2858 [M-H]−
|
8.60
|
547, 519.2244, 385.2117
|
9
|
11.60
|
Stepharine
|
C18H19NO3
|
298.1451 [M + H]+
|
-2.68
|
269.1191, 192.1039, 161.0858
|
10
|
14.64
|
Rutin
|
C27H30O16
|
611.1597 [M + H]+
|
2.45
|
465.0975, 303.0503, 163.0670
|
11
|
16.11
|
Zizyvoside I
|
C25H40O12
|
533.2593 [M + H]+
|
0.94
|
435.1962, 393.1816, 277.2098
|
12
|
29.51
|
Ceanothic acid
|
C30H46O5
|
485.3259 [M-H]−
|
1.65
|
467.3810, 423.3476
|
13
|
30.51
|
Alphitolic acid
|
C30H48O4
|
473.3591 [M + H]+
|
8.45
|
455.3698, 390.9161
|
14
|
31.12
|
Maslinic acid
|
C30H48O4
|
495.3450 [M + Na]+
|
4.04
|
409.3433, 381.3086, 296.8615, 249.1672, 203.1779
|
15
|
31.38
|
2α-Hydroxyursolic acid
|
C30H48O4
|
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
|
C30H46O4
|
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
|
C39H54O6
|
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
|
C39H54O6
|
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
|
C39H54O6
|
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
|
C39H54O6
|
619.3944 [M + H]+
|
8.88
|
437.3506, 411.3207, 261.1825, 165.0583
|
21
|
36.94
|
Oleanonic acid
|
C30H48O3
|
455.3537 [M-H]−
|
-2.64
|
437.3444, 409.3399, 259.1797, 177.1659
|
22
|
37.42
|
Ursonic acid
|
C30H48O3
|
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. 2a). 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. 2b, 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. 2c, Table S2). The result of the oxidative stress showed JE treatment also could significantly increase the mean lifespan of C. elegan (Fig. 2d, 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. 3a-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. 3e-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. 4a). 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. 4b, 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. 4b, 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. 5a). 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. 5b). Interestingly, the activity of body bending and head swing in vehicle group worms showed an age-dependent decline (Fig. 5a, b). However, the activity of body bending and head swing in JE treatment group worms had no change with the aging process (Fig. 5a, 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. 6a-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. 6f, 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. 7a-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. 7c-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. 7e-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. 8a-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. 9a). 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. 9b). 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. 9c, d). In the present study, we found that JE treatment did not change the mean and maxium lifespan of sod-3 mutant worms (Fig. 9e). 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.