Abstract
As a significant global issue, aging is prompting people’s interest in
the potential anti-aging properties of Anoectochilus roxburghii (A.
roxburghii), a plant traditionally utilized in various Asian countries
for its purported benefits in treating diabetes and combating aging.
However, the specific anti-aging components and mechanisms of A.
roxburghii remain unclear. This study aims to investigate the
anti-aging effects and mechanisms of A. roxburghii extract E (ARE).
Caenorhabditis elegans (C. elegans) were exposed to media containing
different concentrations of ARE whose superior in vitro radical
scavenging capacity was thus identified. Lifespan assays, stress
resistance tests, and RT-qPCR analyses were conducted to evaluate
anti-aging efficacy, reactive oxygen species (ROS) levels, antioxidant
enzyme activity, and daf-16, sod-3, and gst-4 levels. Additionally,
transcriptomic and metabolomic analyses were performed to elucidate the
potential anti-aging mechanisms of ARE. Fluorescence protein assays and
gene knockout experiments were employed to validate the impacts of ARE
on anti-aging mechanisms. Our results revealed that ARE not only
prolonged the lifespan of C. elegans but also mitigated ROS and
lipofuscin accumulation, and boosted resistance to UV and heat stress.
Furthermore, ARE modulated the expression of pivotal anti-aging genes
including daf-16, sod-3, and gst-4, facilitating the nuclear
translocation of DAF-16. Significantly, ARE failed to extend the
lifespan of daf-16-deficient C. elegans (CF1038), indicating its
dependency on the daf-16/FoxO signaling pathway. These results
underscored the effectiveness of ARE as a natural agent for enhancing
longevity and stress resilience to C. elegans, potentially to human.
Keywords: Anoectochilus roxburghii, Caenorhabditis elegans, daf-16/FoxO
pathway, anti-aging, lifespan, stress resilience
1. Introduction
Aging is a complex biological process characterized by the gradual
decline in the physiological functions of tissues and organs, as well
as a loss of repair capabilities, which increases the risk to various
diseases [[36]1,[37]2,[38]3]. To delay aging, several strategies have
been proposed, including calorie restriction, regular exercise, and
dietary supplementation [[39]4,[40]5]. Recently, there has been a
marked increase in interest in identifying natural compounds that can
delay aging and extend lifespan. Among the range of natural products,
medicinal plants have shown considerable promise due to their bioactive
compounds which offer potential health benefits
[[41]6,[42]7,[43]8,[44]9].
Caenorhabditis elegans (C. elegans) has emerged as a valuable model
organism in aging research due to its short lifespan, well-defined
genetics, and highly conserved aging pathways similar to those in
mammals [[45]10,[46]11]. The daf-16/FoxO signaling pathway in C.
elegans plays a pivotal role in regulating lifespan and stress
resistance, making it an exemplary system for exploring the molecular
mechanisms of potential anti-aging compounds [[47]12,[48]13,[49]14].
Previous research has demonstrated that natural compounds can modulate
the daf-16/FoxO pathway, effectively enhancing the lifespan and stress
resilience of C. elegans [[50]15,[51]16,[52]17].
Anoectochilus roxburghii (A. roxburghii), a rare orchid species, is
widely found in tropical and subtropical regions of Asia, particularly
in mountainous areas of southern China [[53]18]. As a traditional
medicinal herb, it is highly valued in Asian countries, especially in
China and Japan, for its numerous therapeutic properties. Notably, A.
roxburghii has significant anti-inflammatory effects and is
traditionally used to treat inflammation, detoxify the body, and
alleviate throat infections and bronchitis [[54]19]. Additionally, it
is believed to strengthen the immune system, enhance the body’s ability
to combat diseases, and protect the liver, aiding in the treatment of
hepatitis and other liver conditions [[55]20,[56]21,[57]22].
Recent advances in science and technology have led to a deeper
understanding of the bioactive components of A. roxburghii and their
potential health benefits. Research has indicated that it is rich in
antioxidants, such as flavonoids and polysaccharides, which can
scavenge free radicals and reduce oxidative stress, thereby potentially
slowing the aging process [[58]23]. Additionally, A. roxburghii extract
has also been found to lower blood sugar levels through various
mechanisms, including enhancing insulin sensitivity and improving
glucose metabolism [[59]24]. However, the specific effects of A.
roxburghii extract on the lifespan, stress resistance, and underlying
molecular mechanisms in C. elegans have not been extensively explored.
This study employed lifespan assays, stress resistance tests,
transcriptomics, and metabolomics to examine the influence of A.
roxburghii extract on the lifespan, stress resistance, gene expression,
and to elucidate the potential mechanisms underpinning the observed
phenotypic changes in C. elegans. The results would provide a
theoretical foundation for the development of anti-aging health
products based on A. roxburghii.
2. Materials and Methods
2.1. Reagents and Materials
E. coli OP50 and C. elegans (N2, TJ356, CF1553, CL2166, CF1038) were
acquired from the Caenorhabditis Genetics Center (CGC, Minneapolis, MN,
USA). A. roxburghii was provided from the Jinhua Academy of
Agricultural Sciences (Jinhua, Zhejiang, China). Plant specimens were
preserved at the Southern Agriculture and Forestry Resources Laboratory
of Xingzhi College, Zhejiang Normal University. The following reagents
were utilized in the experiments: agar, KH[2]PO[4], Na[2]HPO[4],
NaH[2]PO[4], NaCl, NaClO, CaCl[2], NaOH, MgSO[4], cholesterol, yeast
extract, tryptone, and peptone were purchased from Sangon Biotech
(Shanghai, China). TB Green^® Premix Ex Taq™ and PrimeScript™ RT
reagent Kit with gDNA Eraser were supplied by Takara (Beijing, China).
Commercial kits for measuring SOD, MDA, CAT, and GSH-px were provided
by the Nanjing Institute of Biological Engineering (Nanjing, China).
Reactive Oxygen Species Assay Kit and Detergent Compatible Bradford
Protein Assay Kit were obtained from Beyotime (Shanghai, China). A BCA
assay kit was purshased from Nanjing Institute of Biological
Engineering (Nanjing, China).
2.2. Preparation and Identification of AREs
The A. roxburghii were divided into five categories based on different
polarities: A, B, C, D, and E. The detailed methods for extraction and
screening are provided in [60]Appendix A. The A. roxburghii fraction E,
named ARE, had exhibited superior antioxidant properties compared to
other fractions during in vitro antioxidant experiments. Subsequently,
the LC-MS was used to identify the components of ARE and the condition
as follows.
After freeze-drying, 8 mg ARE were weighed into a 1.5 mL EP tube, two
small steel balls were added, and 400 μL methanol-water (v:v = 4:1,
including mixed internal standard, 4 μg/mL) was added. After
pre-cooling for 2 min in the −40 °C refrigerator, it was put it into
the grinder for grinding (60 Hz, 2 min). It then underwent ultrasonic
extraction in an ice water bath for 10 min and was left overnight at
−40 °C. Then the sample was centrifuged for 10 min (12,000 rpm, 4 °C),
extracted 150 μL supernatant with a syringe, filtered with 0.22 μm
organic phase pinhole filter, transfered to LC sample vial, and stored
at −80 °C until LC-MS analysis.
Metabolites were separated and detected using a liquid
chromatography-mass spectrometry system composed of a Waters (Milford,
MA, USA) ACQUITY UPLC I-Class plus and Thermo (Waltham, MA, USA) QE
high-resolution mass spectrometer. The chromatographic separation was
performed on an ACQUITY UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 μm)
with the following gradient program: column temperature at 45 °C;
mobile phase A (water containing 0.1% formic acid) and mobile phase B
(acetonitrile); flow rate at 0.35 mL/min with a linear gradient setting
as follows: 5% B (0–2 min), 5–30% B (2–4 min), 50% B (4–8 min), 50–80%
B (8–10 min), 80–100% B (10–14 min), 100–100% B (14–15 min), 100–5% B
(15–15.1 min), 5% B (15.1–16 min).
The mass spectra were acquired ranging from 70 to 1050 m/z with a
primary resolution of 70,000 and a secondary resolution of 17,500. The
sheath gas flow rate was 35, and the auxiliary gas flow rate was 8. The
spray voltages were set at 3.8 kV (positive ion) and 3 kV (negative
ion). The capillary temperature was maintained at 320 °C, and the
auxiliary gas heater temperature was at 350 °C. Progenesis QI v3.0
software (Nonlinear Dynamics, Newcastle, UK) was used for baseline
filtering, peak recognition, integration, retention time correction,
peak alignment, and normalization of the original data.
2.3. Culture and Treatment of C. elegans
The C. elegans were cultured at 20 °C in nematode growth media (NGM)
with E. coli OP50 serving as the food source and treated with sodium
hypochlorite to synchronize the populations. The protocols for
cultivation and maintenance followed those outlined in the WormBook
[[61]25]. The dosed plates were prepared by adding 10 μg/mL and 100
μg/mL ARE into the NGM and then coated with OP50 on the surface and
incubated at 37 °C for 12 h.
Subsequently, synchronized C. elegans (L1 stage) were cultured at 20 °C
for 3 days in NGM with OP50, water, 10 μg/mL, and 100 μg/mL ARE to
evaluate the toxicity of ARE. The body length, locomotion, and pumping
rates of the C. elegans were measured using an Mshot ML31 biomicroscope
with an MShot Image Analysis System (Guangzhou Micro-shot Technology
Co., Guangzhou, China). Statistical analysis was performed using a
Student’s t-test. The data are presented as mean ± SD.
Furthermore, to evaluate the preference of C. elegans for OP50 mixed
with ARE, 50 μL of OP50 alone and 50 μL of OP50 mixed with 100 μg/mL
ARE were seeded on opposite sides of an NGM plate. Then, 200 worms were
placed in the center of the plate. After 1 h, the number of worms on
each bacterial lawn was counted. This experiment was repeated three
times.
2.4. Lifespan Analysis
Synchronized L4 stage C. elegans were picked and placed on standard NGM
plates containing different concentrations of ARE, with ample OP50
provided as food. The NGM plates also contained 100 μM
5-fluoro-2-deoxyuridine (FUDR). The C. elegans were observed every 24 h
until all had died.
After culturing synchronized L4 stage C. elegans for 2 days, they were
subjected to UV irradiation and heat treatment at 37 °C to induce
stress responses, which differ slightly from conventional lifespan
assays. The UV treatment group was monitored every 24 h, whereas the
heat treatment group was observed every hour until all had died. The
data were statistically analyzed using the log-rank test.
2.5. Lipofuscin Level Assay
L4 stage larvae were incubated with 0 μg/mL, 10 μg/mL, or 100 μg/mL
ARE. In 7-day-old worms, the autofluorescence of intestinal lipofuscin
was measured through the use of a fluorescence microscopy (Guangzhou
Micro-shot Technology Co., Guangzhou, China). The fluorescence
intensity of 20 worms was quantified using the Image J software
(version 15.4f) to determine lipofuscin levels.
2.6. Stress Tolerance Tests
The L4 stage C. elegans were treated with water, 10 μg/mL or 100 μg/mL
ARE for 48 h, and then randomly divided into the control and treated
groups with 200 worms per group. Afterward, the C. elegans were exposed
to 100 mj of energy under a UV for 5 min to evaluate the UV protection
by ARE.
In addition, the C. elegans were incubated at 37 °C, and their death
number was recorded hourly to evaluate the resistance to heat stress of
ARE.
In each experiment, death was determined by a lack of response to touch
with a platinum wire. Unexpected deaths, such as those from loss or
wall climbing, were excluded. Statistical analysis of the data was
performed using the log-rank test.
2.7. Determination of ROS and Antioxidant Enzymes
Synchronized L4 stage C. elegans were treated in the NGM containing
water, 10 μg/mL and 100 μg/mL ARE for 48 h, respectively. Subsequently,
M9 buffer was used to wash C. elegans 3 times. Then, the active oxygen
probe (DCFH-DA) was added at a ratio of 1:1000 and incubated at 20 °C
for 25 min. After incubation, C. elegans were washed three times with
M9 buffer and then placed on 2% agarose pads for photographing. ROS
levels were measured using a microplate reader (Spark03030923, Tecan,
Mannedorf, Switzerland) at an excitation wavelength of 488 nm and an
emission wavelength of 525 nm.
Synchronized L4 stage C. elegans were transferred onto NGM containing 0
μg/mL, 10 μg/mL, and 100 μg/mL ARE, respectively. A total of 500 C.
elegans were selected and washed with M9 buffer to remove residual
OP50. The supernatant was then collected by grinding, crushing, and
centrifugation (3000 rpm, 10 min) in an ice bath. The levels of SOD,
CAT, and GSH-px were measured following the instructions of the
commercial kits. Subsequently, protein concentrations were determined
using a BCA assay kit. Statistical analysis was performed using the
Student’s t-test. The data are presented as mean ± SD.
2.8. RNA-seq
The L4 stage worms were cultured on NGM plates containing 100 μg/mL ARE
at 20°C for 48 h. Subsequently, they were washed with M9 buffer to
remove OP50, then flash-frozen in liquid nitrogen and stored at −80 °C.
Afterwards, they were sent to OE Biotech (Shanghai, China) for
subsequent library construction analysis. Alignment to the C. elegans
reference genome was performed using HISAT2 software (version 2.1.0) to
calculate gene expression levels (FPKM). DESeq2 software (version
1.22.2) was used to conduct differential expression gene (DEG)
analysis, with DEGs defined as genes having a p-value <0.05 and a fold
change >2 or <0.5.
2.9. Metabonomics
The L4 stage worms were cultured on NGM plates containing 100 μg/mL ARE
at 20 °C for 48 h. Subsequently, they were washed off with M9 buffer to
remove OP50, then flash-frozen in liquid nitrogen and stored at −80 °C.
Afterwards, they were sent to OE Biotech (Shanghai, China) for
subsequent differential metabolite analysis. Metabolites were
identified based on multiple dimensions including retention time (RT),
accurate mass, secondary fragmentation, and isotope distribution using
The Human Metabolome Database (HMDB), Lipidmaps (v2.3), METLIN
database, and the LuMet-Animal3.0 local database. Statistical
significance (p-value) was computed using a single-factor analysis
(t-test). Metabolites with VIP > 1 and a p-value < 0.05 were considered
differentially expressed metabolites (DEMs).
2.10. Real-Time Quantitative PCR
The worms were treated with 100 μg/mL ARE for 2 days, and total RNA was
extracted from the worms using the RNAiso Plus kit (Takara, Dalian,
China) following the manufacturer’s instructions. Possible genomic DNA
contamination in the extracted RNA was removed using the PrimeScript™
RT reagent kit with gDNA Eraser kit (Takara, Dalian, China), and then
the RNA was reverse transcribed into cDNA using the same PrimeScript™
RT reagent kit with gDNA Eraser. Subsequently, real-time quantitative
PCR was performed on the cDNA obtained from C. elegans treated with 100
μg/mL ARE, using the TB Green^® Premix Ex Taq™ kit (Takara, Dalian,
China). The relative gene expression was calculated using the 2^−ΔΔCt
method, with Actin-1 selected as the housekeeping gene. Primer
sequences are provided in the [62]Appendix A.
2.11. Nuclear Localization of DAF-16
To determine DAF-16::GFP nuclear localization, synchronized L4 larvae
of DAF-16::GFP-expressing TJ356 worms were randomly divided into
control and ARE groups, which cultured on NGM plates containing 100
μg/mL ARE at 20 °C for 48 h. Then, the worms (n = 20) were transferred
to a 2% agarose pad on a glass slide and anesthetized by 50 μg/mL
levamisole. Fluorescence microscopy was used to capture and determine
the location of DAF-16::GFP (cytoplasmic, intermediate between
cytoplasm and nucleus, and nuclear). Representative images and number
of DAF-16::GFP nuclear localization of C. elegans were obtained and
counted. Statistical analysis was performed using the Student’s t-test.
The data are presented as mean ± SD.
2.12. Quantitation of GST-4::GFP and SOD-3::GFP Expression
The synchronized L4 stage of CF1553 and CL2166 worms were utilized to
quantified the protein of SOD-3 and GST-4 expression, respectively. The
CF1553 and CL2166 worms were incubated on NGM plates containing 100
μg/mL ARE at 20 °C for 2 days. Subsequently, the worms were
anesthetized with 50 μg/mL levamisole hydrochloride and imaged using a
fluorescence microscope (n = 20).
3. Results
3.1. Screening and Identification of Active Substances from A. roxburghii
DPPH and ABTS assays were performed to evaluate the antioxidant
capacity of A. roxburghii A, B, C, D, and ARE. The results demonstrated
that 2 mg/mL ARE exhibited stronger scavenging abilities than 2 mg/mL
A. roxburghii A, B, C, and D, reaching 83.55 ± 1.9% for DPPH and 64.11
± 0.41% for ABTS ([63]Figure 1a,b). The IC50 values of ARE for DPPH and
ABTS were determined to be 0.244 mg/mL and 0.7681 mg/mL, respectively.
The IC50 values for A. roxburghii A, B, C, and D can be found in
[64]Table A1. Therefore, ARE was selected and served as the primary
active substance of A. roxburghii. Furthermore, UHPLC-MS/MS was
employed to analyze the composition of ARE. The positive and negative
total ion chromatograms of ARE are displayed in [65]Figure 1c,d. After
a detailed comparison of the mass-to-charge ratios (m/z) and secondary
fragment information, a total of 65 compounds were identified in ARE
([66]Table 1).
Figure 1.
[67]Figure 1
[68]Open in a new tab
Screening and identification of active substances from A. roxburghii.
(a) DPPH scavenging capacity of different 2 mg/mL extracts. (b) ABTS
scavenging capacity of different 2 mg/mL extracts. (c) Positive ion
chromatogram of ARE. (d) Negative ion chromatogram of ARE.
Table 1.
ARE composition.
Components RT (Min) Precursor m/z Reference m/z Error (ppm) Adduct
Formula
(5E)-4-methoxy-5-[methoxy-[(2R,3S)-3-phenyloxiran-2-yl]methylidene]fura
n-2-one 1.158633 273.10920 273.10904 0.586 [M−H]^− C[15]H[14]O[5]
3-Methylxanthine 1.24045 165.04170 165.04179 0.545 [M−H]^−
C[6]H[6]N[4]O[2]
Arabinose 1.24045 149.04580 149.04555 1.677 [M−H]^− C[5]H[10]O[5]
Carbetamide 1.344417 237.12330 237.12337 −0.295 [M+H]^+
C[12]H[16]N[2]O[3]
trans-5-O-Caffeoylquinic acid 1.362117 353.10940 353.10901 1.104
[M−H]^− C[16]H[18]O[9]
N-Benzyloxycarbonylglycine 4.972417 208.06190 208.06154 1.730 [M−H]^−
C[10]H[11]NO[4]
N-Isovalerylglycine 6.510334 158.08210 158.08226 −1.012 [M−H]^−
C[7]H[13]NO[3]
3Alpha-Hydroxy-3-Deoxyangolensic acid methyl ester 6.676167 471.24510
471.24536 −0.552 [M−H]^− C[27]H[36]O[7]
Okaramine J_120151 6.780283 525.28610 525.28601 0.171 [M+H]^+
C[32]H[36]N[4]O[3]
(2S,3R,4S,5S,6R)-2-[[(1S,4aR,7aS)-7-(hydroxymethyl)-1,4a,5,7a-tetrahydr
ocyclopenta[c]pyran-1-yl]oxy]-6-(hydroxymethyl)oxane-3,4,5-triol
6.883483 329.12400 329.12418 −0.547 [M−H]^− C[15]H[22]O[8]
Cytisine 6.903934 229.06770 229.06781 −0.480 [M+H]^+ C[11]H[14]N[2]O
4-acetyloxy-8-(3-oxo-2-pent-2-enylcyclopenten-1-yl) octanoic acid
7.344133 349.11430 349.11429 0.029 [M−H]^− C[20]H[30]O[5]
4-[2-[(1R,4aS,5R,6R,8aS)-6-hydroxy-5-(hydroxymethyl)-5,8a-dimethyl-2-me
thylidene-3,4,4a,6,7,8-hexahydro-1H-naphthalen-1-yl]-1-hydroxyethyl]-2H
-furan-5-one 7.344133 349.11460 349.11481 −0.602 [M−H]^− C[20]H[30]O[5]
MBOA 7.428117 164.03480 164.03461 1.158 [M−H]^− C[8]H[7]NO[3]
5,6-dihydropenicillic acid 7.428117 170.98500 170.98529 −1.696 [M−H]^−
C[8]H[12]O[4]
1,4-Cyclohexanedicarboxylic acid 7.470117 171.06640 171.06628 0.701
[M−H]^− C[8]H[12]O[4]
Communesin-B 7.484583 509.29140 509.29108 0.628 [M+H]^+
C[32]H[36]N[4]O[2]
Meglutol 7.880767 161.04580 161.04555 1.552 [M−H]^− C[6]H[10]O[5]
2-[5-[2-[2-[5-(2-hydroxypropyl)oxolan-2-yl]propanoyloxy]propyl]oxolan-2
-yl]propanoic acid 8.028216 409.21950 409.21967 −0.415 [M+H]^+
C[20]H[34]O[7]
Wikstromol 8.238367 307.15160 307.15158 0.065 [M+H]^+ C[15]H[24]O[5]
Melatonin 8.245916 231.12240 231.12207 1.428 [M−H]^− C[13]H[16]N[2]O[2]
Isopropylmalic acid 8.733883 175.05960 175.05991 −1.771 [M−H]^−
C[7]H[12]O[5]
Caffeine 9.06815 217.07030 217.07001 1.336 [M+H]^+ C[8]H[10]N[4]O[2]
15,17-dihydroxy-9-methyl-4,10-dioxatricyclo
[11.4.0.0,]heptadeca-1(17),13,15-triene-2,11-dione 9.096033 305.10300
305.10306 −0.197 [M−H]^− C[16]H[18]O[6]
(3,4,5-trihydroxy-6-methyloxan-2-yl) 2-(methylamino)benzoate 9.4975
296.11350 296.11395 −1.520 [M−H]^− C[14]H[19]NO[6]
MITOMYCIN C 9.4975 333.12020 333.12045 −0.750 [M−H]^−
C[15]H[18]N[4]O[5]
Beclomethasone dipropionate 9.5138 521.23270 521.23297 −0.518 [M+H]^+
C[28]H[37]CO[7]
Indole-3-acetyl-L-glutamic acid 9.577333 303.09860 303.09863 −0.099
[M−H]^− C H[16]N[2]O[5]
3′,5′-Dimethoxy-4′-hydroxyacetophenone 9.617333 195.06640 195.06628
0.615 [M−H]^− C[10]H[12]O[4]
3-hydroxy-2-octylpentanedioic acid 9.698167 259.15520 259.15509 0.424
[M−H]^− C[13]H[24]O[5]
methyl 2-benzamido-3-phenylpropanoate 9.698167 282.11370 282.11356
0.496 [M−H]^− C[17]H[17]NO[3]
FERULATE 9.82215 193.05020 193.05000 1.036 [M−H]^− C[10]H[10]O[4]
[4-[3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyphenyl]methyl
3-acetyloxy-2-hydroxy-2-[(4-hydroxyphenyl)methyl]butanoate 9.846617
575.15250 575.15253 −0.052 [M+H]^+ C[26]H[32]O[12]
Shikimic acid 10.37175 197.04200 197.04204 −0.203 [M+H]^+ C[7]H[10]O[5]
Alverine citrate 10.41242 282.22160 282.22198 −1.346 [M+H]^+
C[26]H[35]NO[7]
[3-(4-hydroxy-3-methoxybenzoyl)-2,3-dimethyloxiran-2-yl]-(4-hydroxy-3-m
ethoxyphenyl)methanone 10.42645 371.07480 371.07471 0.243 [M−H]^−
C[20]H[20]O[7]
C17_Sphingosine 10.65907 286.27370 286.27399 −1.013 [M+H]^+
C[17]H[35]NO[2]
3-[(2S,3R,4S,5S,6R)-4,5-dihydroxy-6-(hydroxymethyl)-3-[(2S,3R,4S,5R)-3,
4,5-trihydroxyoxan-2-yl]oxyoxan-2-yl]oxy-5,7-dihydroxy-2-(4-hydroxyphen
yl)chromen-4-one 11.07492 579.15290 579.15253 0.639 [M−H]^−
C[26]H[28]O[15]
Styrene 11.29803 105.06960 105.06988 −2.665 [M+H]^+ C[8]H[8]
Mollugin 11.49802 307.10000 307.10001 −0.033 [M+H]^+ C[17]H[16]O[4]
Pesticide3_Bifenazate_C17H20N2O3_1-Methylethyl
2-(4-methoxybiphenyl-3-yl)hydrazinecarboxylate 11.53785 301.15370
301.15399 −0.963 [M+H]^+ C[17]H[20]N[2]O[3]
8-acetamido-2-methyl-7-oxononanoic acid 11.59707 242.13950 242.13977
−1.115 [M−H]^− C[12]H[21]NO[4]
[(3aS,4S,5S,6E,10Z,11aR)-6-formyl-5-hydroxy-10-(hydroxymethyl)-3-methyl
idene-2-oxo-3a,4,5,8,9,11a-hexahydrocyclodeca[b]furan-4-yl]
2-methylprop-2-enoate 11.65785 401.09960 401.09970 −0.249 [M+H]^+
C[19]H[22]O[7]
Licoagroside B (Not validated) 11.75722 431.11870 431.11880 −0.232
[M−H]^− C[18]H[24]O[12]
Scytophycin B 12.05967 842.50730 842.50702 0.332 [M+H]^+
C[45]H[73]NO[12]
[5-hydroxy-3-(hydroxymethyl)-2-oxo-6-propan-2-ylcyclohex-3-en-1-yl]
3-methylpentanoate 12.39685 297.17090 297.17075 0.505 [M−H]^−
C[16]H[26]O[5]
Chrysanthemic acid, ethyl ester 12.756 195.13920 195.13905 0.769
[M−H]^− C[12]H[20]O[2]
(2R)-2-[(2R,5S)-5-[(2S)-2-hydroxybutyl]oxolan-2-yl]propanoic acid
12.83583 215.12860 215.12888 −1.302 [M−H]^− C[11]H[20]O[4]
DGMG 18:3 13.1938 721.36410 721.36407 0.042 [M−H]^− C[33]H[56]O[14]
MEGxp0_001388 15.65933 955.29820 955.29828 −0.084 [M−H]^−
C[41]H[32]O[27]
2-[4a-methyl-8-methylidene-4-(3-methylpentanoyloxy)-1,2,3,4,5,6,7,8a-oc
tahydronaphthalen-2-yl]prop-2-enoic acid 15.94167 347.22250 347.22278
−0.806 [M−H]^− C[21]H[32]O[4]
2-(8-hydroxyoctyl)-6-methoxybenzoic acid 15.98182 279.15920 279.15900
0.716 [M−H]^− C[16]H[24]O[4]
Phosphatidylinositol 16 15.98182 857.51860 857.51855 0.058 [M−H]^−
C[45]H[79]O[13]P
Carylophyllene oxide 16.02265 205.15980 205.15977 0.146 [M−H]^−
C[14]H[22]O
Roccellic acid 16.06382 299.22230 299.22278 −1.604 [M−H]^−
C[17]H[32]O[4]
Lauric acid 16.30847 199.17040 199.17035 0.251 [M−H]^− C[12]H[24]O[2]
4-({5-[5-hydroxy-3-({[(2Z)-2-methylbut-2-enoyl]oxy}methyl)pentyl]-8a-(h
ydroxymethyl)-5,6-dimethyl-3,4,4a,5,6,7,8,8a-octahydronaphthalen-1-yl}m
ethoxy)-4-oxobutanoic acid 16.67412 521.31180 521.31195 −0.288 [M−H]^−
C[29]H[46]O[8]
Ketoisovaleric acid 18.08483 117.05470 117.05462 0.683 [M+H]^+
C[5]H[8]O[3]
Isopalmitic Acid 18.32648 257.24750 257.24750 0.000 [M+H]^+
C[16]H[32]O[2]
FA 18:1+3O 18.38903 329.23240 329.23251 −0.334 [M−H]^− C[18]H[34]O[5]
Grossamide or its isomer (not validated) 18.97012 625.25680 625.25671
0.144 [M+H]^+ C[36]H[36]N[2]O[8]
Heptadecanoic acid 19.17193 271.26040 271.26001 1.438 [M+H]^+
C[17]H[34]O[2]
(E)-5-[(1S,4aR,8aR)-2-formyl-5,5,8a-trimethyl-1,4,4a,6,7,8-hexahydronap
hthalen-1-yl]-3-(acetyloxymethyl)pent-2-enoic acid 19.32013 375.27600
375.27643 −1.146 [M−H]^− C[22]H[32]O[5]
6-Hydroxycaproic acid 19.77323 133.08550 133.08592 −3.156 [M+H]^+
C[6]H[12]O[3]
Avocadene 4-acetate 20.71465 327.25420 327.25406 0.428 [M−H]^−
C[19]H[36]O[4]
[69]Open in a new tab
3.2. ARE Did Not Affect Food Selection, Proliferation, and Development of C.
elegans
It was demonstrated that 100 μg/mL of ARE did not significantly affect
OP50 proliferation ([70]Figure 2a) and food choice of C. elegans
([71]Figure 2b). Additionally, the average body length, egg laying, and
movement were recorded to evaluate the impact of ARE on the growth and
development of C. elegans. As shown in [72]Figure 2c–e, the body length
of C. elegans in the control, 10 of μg/mL ARE, and 100 μg/mL of ARE
group was 1053.2 ± 85 μm, 1026.6 ± 52.7 μm, and 1041.3 ± 48.1 μm,
respectively. The number of eggs laid by C. elegans in the control, 10
μg/mL of ARE, and 100 μg/mL of ARE group was 321.3 ± 56.1, 348.1 ±
28.6, and 311.7 ± 33.8, respectively. The average locomotion frequency
of C. elegans was 12.6 ± 3.2 times, 12.6 ± 3.1 times, and 13.3 ± 4
times per 60 s in the control, 10 μg/mL of ARE, and 100 μg/mL of ARE
group, respectively. These results reveal that ARE has no significant
effect on the proliferation and development of C. elegans.
Figure 2.
[73]Figure 2
[74]Open in a new tab
Effects of ARE on OP50 and C. elegans. (a) Effect of ARE on the
proliferation of OP50. (b) Preference of C. elegans for food containing
ARE (n = 3). (c) The effect of ARE on the body length of C. elegans.
(d) The effects of ARE on C. elegans reproduction (e) The effects of
ARE on C. elegans locomotion. Statistical analysis was conducted using
Student’s t-test. Data are shown as mean ± SD. ns—not significant.
3.3. ARE Reduced the Accumulation of Lipofuscin and Prolonged the Life Span
of C. elegans
As aging progresses, lipofuscin gradually accumulates in the intestines
of C. elegans. As shown in [75]Figure 3a,b, compared with the control
group, the relative level of lipofuscin in C. elegans decreased to 66.6
± 2.7% (p < 0.01) and 55.3 ± 2.8% (p < 0.01) after treatment with 10
μg/mL and 100 μg/mL ARE. The result of the survival experiment was
displayed in [76]Figure 3c. In the control group, the median lifespan
of C. elegans was 17 days, with an average lifespan of 17.38 days and a
maximum lifespan of 22 days. In the 10 μg/mL ARE group, the median
lifespan was 19 days, with an average lifespan of 18.38 days and a
maximum lifespan of 23 days, resulting in a 5.7% increase in average
lifespan compared to the control group. In the 100 μg/mL ARE group, the
median lifespan increased to 20 days, with an average lifespan of 20.21
days and a maximum lifespan extending to 27 days, representing a 16.3%
increase in average lifespan compared to the control group.
Figure 3.
[77]Figure 3
[78]Open in a new tab
Effects of ARE on C. elegans. (a) The effect of ARE on the accumulation
of lipofuscin in C. elegans (n = 20). Scale bar = 50 μm. (b) Effect of
ARE on the lifespan of C. elegans (n = 200). (c) Effect of ARE on the
lifespan of C. elegans under UV stress (n = 200). (d) Effect of ARE on
the lifespan of C. elegans under 37 °C heat stress (n = 200).
Statistical analysis was conducted using Student’s t-test. Data from
lifespan analysis were analyzed using Kaplan–Meier analysis and a
log-rank test. Data are shown as mean ± SD. **** p < 0.0001.
3.4. ARE Enhanced the Stress Resistance of C. elegans
After 0.1 J of UV irradiation, the average lifespan of C. elegans in
the control group was 4.43 days, with a maximum survival time of 8
days. In the 10 μg/mL ARE group, the average lifespan was 4.51 days,
with a maximum survival time of 8 days, showing no significant
difference between the two groups. However, in the 100 μg/mL ARE group,
the average lifespan increased to 5.08 days, representing a 14.7%
increase (p < 0.01), with the maximum survival time extending to 9 days
([79]Figure 3c).
At 37 °C, the average survival time of the control group was 7.82 h,
with a maximum survival time of 15 h. In the 10 μg/mL ARE group, the
average lifespan was 8.28 h, representing a 5.8% increase (p < 0.05),
with a maximum survival time extending to 16 h. In the 100 μg/mL ARE
group, the average lifespan was 9.04 h, representing a 15.6% increase
(p < 0.01), with the maximum survival time extending to 17 h. These
results indicate a significant improvement in the stress resistance of
C. elegans treated with 100 μg/mL ARE ([80]Figure 3d).
3.5. ARE Increased Antioxidant Enzyme Activity and Decreased ROS Accumulation
in C. elegans
ROS accumulation in C. elegans was detected after treatment with ARE
for 28 h. The results demonstrated that, compared to the control group,
10 μg/mL and 100 μg/mL of ARE significantly decreased the level of ROS
in C. elegans (p < 0.01) ([81]Figure 4a).
Figure 4.
[82]Figure 4
[83]Open in a new tab
ROS content and antioxidant enzyme activity. (a) Effect of different
concentrations of ARE on ROS accumulation in C. elegans. (b) Effect of
different concentrations of ARE on SOD enzyme activity in C. elegans.
(c) Effect of different concentrations of ARE on MDA content in C.
elegans. (d) Effect of different concentrations of ARE on CAT enzyme
activity in C. elegans. (e) Effect of different concentrations of ARE
on GSH-px enzyme activity in C. elegans. Statistical analysis was
conducted using Student’s t-test. Data are shown as mean ± SD. * p <
0.05, ** p < 0.01, **** p < 0.0001, ns—not significant.
The results of antioxidant enzyme activity showed that compared to the
control group, 10 μg/mL ARE significantly increased SOD activity while
having no significant effect on MDA content, CAT, and GSH-px activity
(p > 0.05). Interestingly, 100 μg/mL ARE significantly decreased MDA
and increased SOD, CAT, and GSH-px activity (p < 0.05). Specifically,
as shown in [84]Figure 4b–e, compared to the control group, 100 μg/mL
ARE decreased MDA to 84.4 ± 6.4%, but increased SOD, CAT, and GSH-px
activities to 242.7 ± 3.2%, 152.6 ± 15.7%, and 138.5 ± 2.7%,
respectively (p < 0.01).
3.6. RNA-Seq and Enrichment Analysis
RNA-seq analysis was further performed to explore the potential
differentially expressed genes (DEGs) and mechanisms of ARE in
prolonging the lifespan of C. elegans. The OPLS-DA results showed that
the control group and 100 μg/mL ARE group were well clustered
([85]Figure 5a). Subsequently, compared to the control group, 581
differentially expressed genes (DEGs) were identified (p < 0.05,
|log2FC| > 1), with 250 upregulated genes and 331 downregulated genes
([86]Figure 5b,c). The results of GO enrichment analysis revealed that
the biological process (BP) entries were predominantly linked to fatty
acid beta-oxidation, xenobiotic metabolic processes, organic acid
metabolic processes, and exogenous drug catabolic processes.
Additionally, molecular function (MF) entries were identified and
primarily associated with activities such as the structural composition
of the cuticle, aromatase activity, heme binding, iron ion binding,
UDP-glycosyltransferase activity, glucuronosyltransferase activity, and
carboxylic ester hydrolase activity. The cellular components (CCs)
entries were mainly linked to collagen trimers, organelle membranes,
intracellular membrane-bounded organelles, peroxisomes, extracellular
regions, and endoplasmic reticulum membranes. The BP, CC, and MF were
displayed based on p value ranking ([87]Figure 5d).
Figure 5.
[88]Figure 5
[89]Open in a new tab
RNA seq and enrichment analysis E—100 μg/mL ARE; C—Control. (a)
Orthogonal partial least squares discriminant analysis (OPLS-DA). (b)
Volcano plot of significant DEGs (FC > 1.5, p < 0.05). (c) DEGs orange
represents upregulation, while blue represents downregulation (FC >
1.5, p < 0.05). (d) DEGs expression GO analysis. (e) Differential gene
expression KEGG analysis. The size of the circles corresponds to the
number of DEGs and are color-coded according to −log10 (p value). The
x-axis shows the enrichment factor value.
Furthermore, KEGG pathway enrichment analysis further highlighted the
alteration of 29 related signaling pathways, significantly enriching
pathways involved in peroxisome, FoxO signaling pathway, TGF-beta
signaling pathway, and Wnt signaling pathway. The top 20 signaling
pathways are displayed in [90]Figure 5e. These results indicate that
the expression of antioxidant enzymes in the worms was activated, and
ARE might extend the lifespan of C. elegans through the FoxO signaling
pathway.
3.7. Metabolomics Analysis
Metabolomics analysis was further performed to explore the differential
metabolites and mechanism of ARE in prolonging the lifespan of C.
elegans. The OPLS-DA results showed that the control group and 100
μg/mL ARE group were well clustered ([91]Figure 6a). In this study, 384
differential metabolites (263 upregulated and 121 downregulated) were
identified based on the criteria of p < 0.05 and VIP > 1.0 ([92]Figure
6b,c). Subsequently, the results of KEGG pathway enrichment analysis
showed that the top 20 pathways mainly involved ABC transporters,
arginine biosynthesis, alanine, aspartate and glutamate metabolism,
aminoacyl-tRNA biosynthesis, the mTOR signaling pathway, glyoxylate and
dicarboxylate metabolism, and the FoxO signaling pathway ([93]Figure
6d). Coincidentally, the FoxO signaling pathway has also been
identified in metabolomics after treatment with ARE in C. elegans.
Figure 6.
[94]Figure 6
[95]Open in a new tab
Identification of differential metabolites and enrichment analysis of
metabolic pathways. E—100 μg/mL ARE. C—control. (a) Orthogonal partial
least squares discriminant analysis (OPLS-DA). (b) Volcano plot of
differential metabolites. (c) Heatmap of differential metabolites. (d)
KEGG pathway analysis of differential metabolites. The size of the
circles corresponds to the number of DEMs and are color-coded according
to p value.
3.8. Integration of Transcriptomics and Metabolomics Networks in C. elegans
The random forest method was used to screen the important biomarkers.
The top 30 biomarkers were displayed in [96]Figure 7a. Furthermore, as
shown in [97]Figure 7b, the top 20 genes and the top 20 metabolites
were obtained and used to build correlation heatmaps. The association
network and the number of association nodes were constructed and
counted by integrating the absolute value of the correlation
coefficient into the top 100 genes and metabolites ([98]Figure 7c,d).
As depicted in [99]Figure 7e, we further constructed a differential
network by integrating significantly altered genes and metabolites,
subsequently mapping these entities to their corresponding pathways. As
a result, 19 commonly affected pathways were identified ([100]Figure
7f), including the following: arginine biosynthesis, biosynthesis of
unsaturated fatty acids, FoxO signaling pathway, and mTOR signaling
pathway.
Figure 7.
[101]Figure 7
[102]Open in a new tab
Metabonomic and transcriptome integration analysis. (a) Top 30
biomarkers. (b) Heatmap of differential genes and metabolites. (c)
Network plot of differential genes and metabolites. (d) Top 30
associated network nodes. (e) Venn diagram of differential genes
pathway and metabolites pathway. (f) Common differential pathways. * p
< 0.05, ** p < 0.01, *** p < 0.001.
3.9. ARE Upregulated the Expression Levels of the daf-16, sod-3, and gst-4
Real-time quantitative PCR was used to verify the expression of key
genes in the FoxO signaling pathway, including akt-1, akt-2, daf-16,
sod-3, and gst-4 genes, identified through multi-omics analysis. As
shown in [103]Figure 8a, compared to the control group, ARE
significantly upregulated the expression levels of daf-16, sod-3, and
gst-4 genes in C. elegans, while it did not significantly alter the
expression levels of akt-1 and akt-2 genes. Specifically, the
expression levels of daf-16, sod-3, and gst-4 genes increased to 290.8
± 13.6%, 216.2 ± 17.4%, and 139.4 ± 20.7%, respectively, after
treatment with ARE compared to the control group.
Figure 8.
[104]Figure 8
[105]Open in a new tab
Gene expression and knockout verification. (a) Real-time PCR. (b)
Fluorescent image of DAF-16. (c) Cellular localization of DAF-16 (n =
20). (d) Fluorescent image of SOD-3. (e) SOD-3 expression analysis (n =
20). (f) Fluorescent image of GST-4. (g) GST-4 expression analysis (n =
20). (h) Effect of ARE on lifespan of daf-16-deficient C. elegans
(CF1038). *** p < 0.001, **** p < 0.0001. ns—not significant.
Subsequently, nematode TJ356 was used to evaluate the effects of ARE on
the subcellular localization of daf-16. As shown in [106]Figure 8b,c,
the number of nuclear-localized daf-16 significantly increased in worms
after treatment with ARE. Additionally, transgenic nematodes CF1553 and
CL2166 were used to evaluate the translation of gst-4 and sod-3 genes
downstream of daf-16 in C. elegans, respectively. The results of the
CL2166 and CF1553 transgenic worms showed that sod-3::GFP expression
increased to 250.1 ± 50.9% (p < 0.01) and GST-4::GFP expression
increased to 133 ± 19.7% (p < 0.01) ([107]Figure 8d–g).
3.10. ARE Prolongs the Lifespan of C. elegans through the daf-16/FoxO Pathway
To further clarify the role of ARE in promoting the expression of
downstream genes sod-3 and gst-4 by upregulating daf-16, we used
daf-16-deficient nematodes (CF1038) to evaluate the effect of ARE on
the lifespan of C. elegans. The results showed that the lifespan of
daf-16-deficient nematodes treated with ARE was not significantly
different from that of the control group, confirming that ARE could
prolong the lifespan of nematodes by promoting the expression of daf-16
([108]Figure 8h).
4. Discussion
In this study, we focused on A. roxburghii, an Asian plant with
traditional medicinal and dietary uses, to explore its anti-aging
properties in C. elegans. This research is the first to demonstrate
that A. roxburghii can extend the lifespan of C. elegans. Using mutant
nematode strains, transcriptomic, metabolomic, and other methods, we
found that the activation of DAF-16/FoxO transcriptional nuclear
activity, mediated by A. roxburghii, is crucial for the observed
lifespan extension in C. elegans.
Previous studies have indicated that A. roxburghii possesses strong in
vitro antioxidant activity [[109]18,[110]21], and our research has
confirmed this finding. To further investigate the potential in vivo
antioxidant activity of A. roxburghii, we used C. elegans as a model
organism. The results showed that A. roxburghii extended the lifespan
of C. elegans by 16.3%, reduced ROS and lipofuscin accumulation, and
had no adverse effects on the growth, development, or reproduction of
the nematodes. In summary, these results support the effectiveness of
A. roxburghii as an in vivo antioxidant and demonstrate its
bioavailability. These findings are consistent with several other
studies that have clearly demonstrated the antioxidant properties of A.
roxburghii [[111]21,[112]26].
To elucidate the potential mechanisms underlying the anti-aging effects
on C. elegans, differential gene and metabolite analysis revealed
significant enrichment of the FoxO signaling pathway. The FoxO
signaling pathway plays a crucial role in regulating various cellular
processes such as the cell cycle, proliferation, apoptosis, and
antioxidant stress response [[113]27,[114]28]. In C. elegans, this
pathway includes daf-2/IGFR, age-1/IP3K, and daf-16/FoxO [[115]29].
Studies have shown that the increased nuclear translocation of daf-16
is one of the main mechanisms for extending the lifespan of C. elegans.
The nuclear translocation of daf-16 stimulates the expression of
downstream antioxidant enzymes, thereby reducing the accumulation of
oxidative damage [[116]30,[117]31,[118]32]. Treatment with ARE
significantly increased the nuclear translocation of daf-16 in C.
elegans and upregulated the expression of downstream antioxidant
enzymes SOD-3 and GST-4. When the daf-16 was knocked out, the lifespan
extension effect of ARE was abolished. Additionally, we found that ARE
had no effect on the reproduction of C. elegans, confirming our
hypothesis that reproductive signaling is not involved in ARE-induced
lifespan extension. Therefore, we propose that ARE activates the FoxO
signaling pathway, promotes the nuclear translocation of daf-16/FoxO
transcription factors, and subsequently stimulates and regulates the
expression of these two downstream proteins, SOD-3 and GST-4.
However, this study has some limitations. Firstly, it only demonstrated
that ARE can activate daf-16 to extend the lifespan of C. elegans and
its primary and secondary metabolites have been identified. The
specific relationship between these metabolites and daf-16 remains
unclear, including which substances play a role in extending lifespan
and how they influence daf-16 expression to exert their effects.
Additionally, further research is needed to investigate the potential
of ARE in improving age-related diseases.
In conclusion, ARE demonstrates anti-aging effects on C. elegans and
holds potential for developing anti-aging products, providing
significant benefits for middle-aged and elderly populations.
Appendix A. Anoectochilus roxburghii Extract Extends the Lifespan of
Caenorhabditis elegans through Activating the daf-16/FoxO Pathway
Appendix A.1. Extraction Method for A. roxburghii
Fresh A. roxburghii was pre-cooled at −80 °C for 2 h, followed by
freeze-drying. After freeze-drying, the A. roxburghii was ground into a
fine powder for later use. Five grams of the dried powder were weighed
and subjected to Soxhlet extraction with 100 mL of petroleum ether at a
1:20 solid–liquid ratio for 18 h, yielding residue 1 and a petroleum
ether extract. The petroleum ether extract, designated as Extract A,
was concentrated using rotary evaporation and dried at 60 °C. The
resulting mass of Extract A after drying was 0.058 g, indicating an
extraction yield of approximately 1.16%. This extract predominantly
comprises non-polar or weakly polar compounds, including fats, waxes,
specific steroids, and non-polar aliphatic compounds.
Residue 1 was dried and subjected to ultrasonic extraction using 100 mL
of 60% ethanol at 60 °C and 300 W for 1 h. This process was repeated
five times, with the filtrates combined and concentrated by rotary
evaporation, yielding residue 2. The ethanol extract was divided into
supernatant and precipitate fractions by adjusting the pH to 2.0 using
dilute hydrochloric acid and allowing it to stand in the refrigerator
for 12 h, followed by centrifugation. After concentrating the
supernatant through rotary evaporation at 60 °C, it was dried to obtain
Extract B, weighing 1.13 g with a yield of 22.6%. Similarly, the
precipitate was dried at the same temperature to yield Extract C,
weighing 0.504 g with a yield of 10.08%. Extracts B and C primarily
contain highly polar compounds, such as certain organic acids, phenolic
compounds, and water-soluble flavonoids.
Residue 2 was dried at a low temperature and then subjected to Soxhlet
extraction using 100% ethanol at a solid–liquid ratio of 1:20 for 1 h.
Subsequently, the filtered drug residue 3 was extracted with boiling
water (1:5 w/v) for 1 h. It was then mixed with four volumes of
anhydrous ethanol, left to precipitate at 4 °C for 12 h, and the
resulting precipitate was treated with a chloroform–butanol solution in
a 1:4 ratio (v/v) for three washes before drying. This product was
labeled as Extract D, yielding 0.012 g with an extraction rate of
0.24%. Meanwhile, the supernatant was concentrated by rotary
evaporation at 60 °C, resulting in Extract E with a mass of 0.072 g and
an extraction rate of 1.44%. Extracts D and E primarily contain
moderately polar compounds, such as polysaccharides, proteins, certain
alkaloids, and high-molecular-weight flavonoids.
Figure A1.
[119]Figure A1
[120]Open in a new tab
Extraction method for A. roxburghii flowchart.
Appendix A.2. DPPH Radical Scavenging Assay of Extracts
First, dissolve Extract A in petroleum ether. Dissolve Extracts B, C,
D, and E in ethanol. Dissolve Extract F in deionized water to prepare
different concentrations of the extract.
Next, prepare a 0.1 mM DPPH solution in anhydrous ethanol. Mix 3 mL of
the DPPH solution with 1 mL of each sample solution, vortex thoroughly,
and incubate the mixture at room temperature in the dark for 30 min to
allow the free radicals in the liquid to be fully scavenged. Then,
measure the absorbance of the samples at 517 nm, using a water and
ethanol mixture as a blank. Use glutathione as the positive control
group. The scavenging activity is calculated using the following
formula:
[MATH: Scavenging Rate (%)=1−A1−A3A2×100% :MATH]
* A1 is the absorbance of 3 mL sample + 1 mL DPPH.
* A2 is the absorbance of 3 mL sample solvent + 1 mL DPPH.
* A3 is the absorbance of 3 mL sample + 1 mL ethanol.
Appendix A.3. ABTS Radical Scavenging Assay
Prepare the ABTS stock solution by mixing equal volumes of 7 mmol/L
ABTS aqueous solution and 4.9 mmol/L potassium persulfate solution.
Store the mixture in the dark. Dilute the ABTS stock solution to create
a series of gradient dilutions. In a microplate, add 180 μL of each
diluted ABTS solution and 20 μL of deionized water. Incubate the
mixture at 37 °C in the dark for 5 min, then measure the absorbance at
405 nm. Select the dilution with an absorbance value around 0.7 as the
ABTS working solution for subsequent experiments (prepare fresh as
needed).
Add 20 μL of different concentrations of the extract sample solution to
a microplate well, followed by 180 μL of ABTS working solution. Gently
vortex to mix and incubate at 37 °C in the dark for 5 min. Measure the
absorbance at 405 nm (A1). Use the sample solvent instead of the
extract solution for the blank control (A2), and use distilled water
instead of the ABTS working solution for the zero-adjustment control
(A3). Use reduced GSH at the same concentration as the positive control
group. The scavenging activity is calculated using the following
formula:
[MATH: Scavenging Rate (%)=1−A1−A3A2×100% :MATH]
Table A1.
Different Anoectochilus roxburghii extracts IC50.
Anoectochilus roxburghii Extract DPPH (IC50) ABTS (IC50)
A 0.2068 mg/mL 4.872 mg/mL
B 0.2692 mg/mL 5.942 mg/mL
C 0.0488 mg/mL 2.107 mg/mL
D 0.1471 mg/mL 9.961 mg/mL
E 0.0319 mg/mL 0.8797 mg/mL
[121]Open in a new tab
Figure A2.
[122]Figure A2
[123]Open in a new tab
We found that the fluorescence of lipofuscin in C. elegans began to
weaken when the concentration of ARE reached 10 μg/mL, and there was no
significant change in the decrease in lipofuscin when the concentration
reached more than 100 μg/mL compared with 150 μg/mL. Therefore, we
chose the two concentrations of 10 μg/mL and 100 μg/mL. Scale bar = 50
μm.
Author Contributions
P.X. and J.W. (Junyi Wang): investigation, formal analysis, and
writing—original draft preparation. S.L. and W.W.: investigation and
formal analysis. J.W. (Jianfeng Wang) and Y.S.: conceptualization, data
curation, supervision, and writing—review and editing. X.H. and J.W.
(Jianfeng Wang): funding acquisition. All authors have read and agreed
to the published version of the manuscript.
Institutional Review Board Statement
Not applicable.
Data Availability Statement
The raw sequence data have been submitted to the NCBI Short Read
Archive (SRA) with accession number with accession number
. The metabolomics raw data has been uploaded to the
MetaboLights project number
[124]www.ebi.ac.uk/metabolights/MTBLS10402 (accessed on 24 July 2024)
[[125]33]. The original contributions presented in the study are
included in the article, further inquiries can be directed to the
corresponding authors.
Conflicts of Interest
The authors declare no conflicts of interest.
Funding Statement
This research was supported by the open project titled “Identification
of Anti-Damage Functional Factors in Anoectochilus roxburghii and Their
Mechanisms of Action” (2021DG700024-KF202407) funded by the State Key
Laboratory for Managing Biotic and Chemical Threats to the Quality and
Safety of Agro-Products, and the key project “Research and Application
of Standardized Cultivation of Anoectochilus roxburghii Using Spent
Mushroom Substrate from Industrial Shiitake Cultivation” (2021-2-001b)
funded by Jinhua Science and Technology Bureau, Zhejiang province,
China.
Footnotes
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References