Abstract
Dark tea, rich in nutricines including tea polyphenols and free amino
acids, is a kind of post-fermented tea. The potential application of
nutricines against oxidative damage and senescence, which drives animal
health maintenance and disease prevention, has attracted considerable
interest. In this study, the effect of dark tea and its effects on
longevity and defense against oxidative stress was investigated in the
Caenorhabditis elegans (C. elegans) model. Under normal conditions,
dark tea extended the lifespan without significant impairment of
propagation. It also improved the motility, alleviated the fat
accumulation and apoptosis. Additionally, orally administered dark tea
could significantly decrease the level of reactive oxygen species (ROS)
and resulted in a superior lifespan in H[2]O[2]-induced oxidative
stressed C. elegans. In antioxidant assays in vitro, dark tea was found
to be rich in strong hydroxyl, DPPH and ABTS+ free radical scavenging
capacity. Interestingly, mRNA sequence analyses further revealed that
dark tea may catalyze intracellular relevant oxidative substrates and
synthesize antioxidants through synthetic and metabolic pathways. These
results suggest that dark tea is worth further exploration as a
potential dietary supplement for the maintenance of animal health and
the prevention of related diseases.
Keywords: dark tea, oxidative stress, longevity, mRNA sequencing
analysis, Caenorhabditis elegans
1. Introduction
The health of farm animals is critical to production and profitability.
Alterations in energy metabolism can adversely affect their health.
Elevated metabolic demands can lead to a significantly increased oxygen
demand which can result in an upsurge in reactive oxygen species (ROS)
([31]1). This, in turn, disrupts the equilibrium between the production
and elimination of free radicals, contributing to a heightened
inflammatory response ([32]2). Disruptions in the redox balance have
been associated with common diseases such as enteritis and mastitis in
pigs and cattle, and recurrent airway obstruction in horses ([33]3).
However, the currently used chemically synthesized antioxidants have
been under suspicion for their association with organic damage ([34]4,
[35]5). The efficient production of foods at a low cost is of great
importance for good health. Therefore, researchers have developed a
keen interest on the potential application of natural nutrients against
oxidative damage and for longevity.
Tea is a commonly consumed functional beverage. It has high levels of
safety and significant therapeutic effects, together with low toxicity
and minimal side effects. According to the different production
methods, tea can be classified into white tea, green tea, yellow tea,
oolong tea, black tea, and dark tea. The quality characteristics of tea
are associated with a combination of various active substances,
including polyphenolic compounds for astringency, free amino acids for
the sense of newness, and volatile substances for aromas ([36]6,
[37]7). Tea has a variety of health properties, such as antioxidant,
anti-inflammatory, immunomodulatory, anti-cancer, cardio-protective,
anti-diabetic, weight loss and hepatoprotective effects ([38]8). The
available data suggest that green tea polyphenol supplementation at
postpartum improved the milk yield and health status in cows with
hyperketonemia during early lactation ([39]9). Green tea extracts could
improve intestinal microflora balance, contributing to the prevention
of digestive and respiratory organ diseases in calves ([40]10). The
catechins components in green tea have been found to relieve oxidative
stress and fatty liver disease in dairy cows during the periparturient
phase and transition period ([41]11, [42]12). Hence, tea can be used as
a functional substance in livestock feed.
Dark tea is a tree belonging to the Camellia sinensis family,
distinguished by the oily black or black-brown color of its leaves
([43]13). The process of production of dark tea involves
microorganisms; hence, dark tea is the only post-fermented tea among
the six major tea types ([44]14). Compared to the widely studied green,
black, and oolong teas, dark tea is often overlooked as a unique
post-fermented tea. Dark tea contains phytochemicals and
macronutrients, also believed to be beneficial to animals ([45]15). As
a natural and harmless nutraceutical, it can be used as a nutritional
supplement, food additive and medicinal ingredient ([46]16, [47]17).
Caenorhabditis elegans (C. elegans) is the first multicellular organism
to have its genome sequenced fully. The conservation of
illnesses-related pathways between C. elegans and higher organisms,
along with the advantages of its short life cycle, simple culture and
high reproduction capacity ([48]18, [49]19), has made C. elegans a
favorable in vivo non-rodent model organism for mechanistic
explanations and high-throughput screening of drug candidates, being
screened for a range of oxidative stress, toxicity and related
conditions or diseases ([50]20).
Therefore, in this study, three types of Chinese dark tea, all of which
are commonly available in the market and have high sales volumes, were
selected as raw materials, namely Brick tea, Pu'er tea, and Liubao tea,
to investigate the effects of dark tea on the lifespan, propagation,
motility, fat deposition, apoptosis, and resistance to oxidative stress
in C. elegans. Additionally, the differences in the expression of genes
were explored by the mRNA sequence analyses.
2. Materials and methods
2.1. Preparation of dark tea extract
Brick tea (Anhua, China), Pu'er tea (Xishuangbanna, China), or Liubao
tea (Wuzhou, China), were mixed with water in a 1:15 ratio of tea to
water, and heated in a water bath at 85°C for 1 h, cooled and filtered,
and then subjected to rotary evaporation at 58°C for 1 h, respectively
([51]21).
2.2. Active ingredients in dark tea extract
The total polyphenol contents of dark tea were determined through the
iron tartrate colorimetric method ([52]22). Free amino acids were
detected through ninhydrin colorimetry ([53]23). The active ingredients
in dark tea extract are listed in [54]Supplementary Table 1.
2.3. Free radical scavenging ability of dark tea
Determination of the free radical scavenging ability of Brick, Pu'er,
and Liubao teas at concentrations of 200, 400, 600 and 800 μg/mL,
respectively.
2.3.1. Hydroxyl radical scavenging ability of dark tea
A sample solution (0.5 mL) of the dark tea extracts was added to 0.5 mL
of salicylic acid ethanol solution (9 mmol/L; LABGO, China), 0.5 mL of
H[2]O[2] (9 mmol/L; Guangfu, China) solution, and 0.5 mL of FeSO[4]
(9 mmol/L; BEIJINGSHIJI, China) solution. The mixtures were allowed to
react for 30 min in a 37°C water bath, and the absorbance of the
resulting solution was measured at 510 nm, and the values were assessed
against a blank ([55]24). The hydroxyl radical scavenging ability was
calculated by the following formula:
[MATH: Hydroxylradicalscavengingactivity%=1−A1−A2A0×100% :MATH]
where A1 is the absorbance of the reaction solution with the sample, A2
is the absorbance of the solution without salicylic acid and A0 is the
control group where the sample was replaced with distilled water.
2.3.2. DPPH free radical scavenging ability of dark tea
Dark tea extracts (2 mL) were added to 2 mL of DPPH (Aladdn, China)
ethanol solution (5 mg/mL). The mixtures were allowed to react for
30 min in a dark place, and the absorbance of the resulting solution
was measured at 517 nm, and the values were calculated against a blank
([56]25). The DPPH radical scavenging ability was calculated by the
following formula:
[MATH: DPPHradicalscavengingactivity%=1−A1−A2A0×100% :MATH]
where A1 is the absorbance of the reaction solution with the sample, A2
is the absorbance of anhydrous ethanol in place of DPPH and A0 is the
absorbance of anhydrous ethanol in place of sample.
2.3.3. ABTS+ free radical scavenging ability of dark tea
Dark tea extracts (3 mL) were added to 1 mL of
2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonate) (ABTS; Rhawn, Chian)
solution. The mixtures were allowed to react for 6 min in a 30°C water
bath, and the absorbance of the resulting solution was measured at
734 nm, and the values were assessed against a blank ([57]26). The
ABTS+ radical scavenging ability was calculated by the following
formula:
[MATH: ABTS+radicalscavengingactivity%=1−A1A2×100% :MATH]
where A1 is the absorbance of the reaction solution with the sample and
A2 is the control group where the sample was replaced with distilled
water.
2.4. Caenorhabditis elegans strains and maintenance
In this research, the wild-type N2 strain of C. elegans was used. All
C. elegans strains were grown in the nematode growth medium (NGM) at
20°C, and all C. elegans feed on Escherichia coli OP50. All C. elegans
strains and the Escherichia coli OP50 were purchased from the
Caenorhabditis Genetics Centre, University of Minnesota (Minneapolis,
MN, USA). C. elegans was transferred by cutting a small portion of the
nematode-containing NGM using a sterile scalpel, placing it upside down
in OP50-coated NGM, and incubating it at 20°C to allow C. elegans to
crawl onto the new NGM. At the time of synchronization during the
spawning period, C. elegans were washed down with M9 buffer, the lysate
was added, and the precipitate was taken by centrifugation at low
speed.
2.5. Lifespan assay
The synchronized N2 C. elegans were placed on NGM containing Brick tea
extract (600, 700, 800 μg/mL), Pu’er tea extract (600, 700, 800 μg/mL),
and Liubao tea extract (600, 700, 800 μg/mL) for incubation, which was
set up as the experimental group, and the one with no dark tea extract
as the control group. To block C. elegans reproduction, 5-Fluorouracil
(FUDR; Rhawn, China) was added. For each strain, at least 100
egg-synchronized C. elegans were grown at 20°C and transferred daily to
a fresh plate ([58]27). The number of C. elegans surviving, dying and
lost was recorded every day until all C. elegans were dead.
2.6. Lifespan assay in the oxidative damage state
The synchronized N2 C. elegans were placed on NGM containing 600 μg/mL
Brick tea extract, 700 μg/mL Pu’er tea extract, and 700 μg/mL Liubao
tea extract for cultivation. They were allowed to grow until reaching
the L4 stage after which cultivation was continued for an additional
4 days and compared with the control group. Oxidative damage to C.
elegans was induced by adding 200 μL of H[2]O[2] (0.5 mmol/L, Rhawn,
China) solution to a 96-well plate ([59]28). Then 15 C. elegans were
placed into each well, and the number of C. elegans that died was
observed at hourly intervals until all of the C. elegans were dead, and
the survival rate was estimated.
2.7. Progeny assay
The method was the same as mentioned in Section 2.6. During the
reproductive period (approximately days 1–6), C. elegans were
transferred every day to new NGM plates and allowed to deposit progeny.
Record the number of eggs laid per day ([60]29).
2.8. Motility assay
The same method as mentioned in Section 2.6 was followed. FUDR (Rhawn,
China) was added to block C. elegans reproduction and then incubated at
20°C until days 2, 4, 8, 12, and 14. C. elegans were then picked and
placed onto agar spacers coated with OP50. The frequency of head
bobbing in 1 min and the number of body bending within 20 s were
observed using a body microscope ([61]30).
2.9. Fat deposition assay
For this, the protocol mentioned in Section 2.6 was followed.
Synchronized C. elegans were grown for 4 days after which they were
washed with M9 buffer, and the precipitate was collected after
low-speed centrifugation. Subsequently, 100 μL of 1% paraformaldehyde
(Skyho, China) was added, and the sample was kept at 4°C for 15 min
before being snap-frozen and stored at −80°C. After 1 h, the samples
were removed, thawed, centrifuged, and rinsed with M9 buffer. Next, a
mixture of equal volumes of 2% Triton X-100 (Applygen, China) and 1%
Oil Red O (Solarbio, China) was prepared, and 100 μL of this mixture
was added for precipitation. The mixture was then incubated in a 37°C
temperature-controlled shaker for 30 min ([62]31). Finally, C. elegans
were picked and placed on an agar spacer using a picker, and images
were captured under a fluorescence microscope.
2.10. Reactive oxygen species (ROS) accumulation assay
The DCFH-DA method ([63]32) was used to detect the level of ROS in
vivo. ROS levels in C. elegans were measured under both normal
conditions and following induction of oxidative damage by H[2]O[2].
Same method as Section 2.6. Subsequently, the C. elegans were exposed
to H[2]O[2] (0.5 mmol/L) for 2 h at 20°C. The OP50 around C. elegans
was washed using M9 buffer; from this, 100 μL of precipitate was taken
and 1 μL of 10 mM H2DCF-DA (Chemstan, China) was added, and placed in a
constant temperature shaker at 37°C in dark for 30 min. C. elegans was
then transferred to a 2% agar spacer, and observed and photographed
using a fluorescence microscope at the excitation wavelength of 485 nm
and the emission wavelength of 528 nm.
2.11. Apoptosis assay
Apoptosis was experimentally detected in vivo using acridine orange
staining ([64]33), following the same method mentioned in Section 2.6.
After three rinses C. elegans with M9 buffer, the supernatant was
aspirated, and 100 μL of acridine orange (25 μg/mL, Klamar, China)
staining solution was added and placed on a constant temperature shaker
at 37°C, in the dark for 2 h. Subsequently, C. elegans were placed in
blank NGM for 10 min to allow recovery from the staining. Finally, they
were transferred to 2% agar pads and observed under a fluorescence
microscope at an excitation wavelength of 488 nm and an emission
wavelength of 515 nm.
2.12. Transcriptomics analysis
After 4 days of feeding on dark tea, C. elegans was rinsed, placed in
microfuge tubes, and left to settle, after which the precipitate was
taken. RNA-seq was performed on a sequencing platform at Beijing BMK
Biotechnology Co., Ltd. (Beijing, China), to obtain insights into
aggregate gene transcription in subject C. elegans cells.
2.13. Statistical analysis
Statistical analysis was performed using GraphPad Prism 8, and
expressed as mean ± standard deviation (SD). One-way ANOVA was used for
multiple group comparisons. Values with p < 0.05 were considered
statistically significant. The intensity of quantified fluorograms was
analyzed using ImageJ software. Each experiment was repeated three
times.
3. Results
3.1. Dark tea extends the lifespan of Caenorhabditis elegans
The C. elegans were successively treated with Brick, Pu'er, and Liubao
teas under normal culture conditions. As shown in [65]Figure 1 and
[66]Table 1, all these treatments affected the normal C. elegans
lifespan. The mean lifespan of control C. elegans was 14 days.
Treatment with the different teas was found to prolong the lifespan of
C. elegans, resulting in increased average lifespan of 21.4, 14.3, and
14.3% after treatment with Brick tea at concentrations of 600, 700, and
800 μg/mL, respectively, 7.1, 21.4, and 14.3% after exposure to the
same concentrations of Pu’er tea, and 21.4, 28.4, and 21.4% for Liubao
tea, respectively, all relative to the control group. In addition, 600
μg/mL Brick tea, 700 μg/mL Pu'er tea, and 700 μg/mL Liubao tea had the
most significant effect on the increase in average and maximum lifespan
([67]Table 1). Therefore, the above-mentioned concentrations were
selected for subsequent experiments.
Figure 1.
[68]Figure 1
[69]Open in a new tab
Effects of various concentrations of dark tea (A, Brick Tea; B, Pu’er
Tea; C, Liubao Tea) on the lifespan in C. elegans.
Table 1.
Mean and maximum lifespan in C. elegans (Mean ± SD).
Group Mean lifespan (d) Maximum lifespan (d) Lifespan improvement
rate%[70]^a
Control 13.76 ± 1.67 20 ± 2.05
600 μg/ml Brick Tea 17.2 ± 1.70 22 ± 2.48 20
700 μg/ml Brick Tea 16.24 ± 1.77 22 ± 1.7 15.3
800 μg/ml Brick Tea 16.14 ± 1.60 22 ± 2.05 14.7
600 μg/ml Pu’er Tea 15.7 ± 1.85 20 ± 1.63 12.4
700 μg/ml Pu’er Tea 17.44 ± 1.05 26 ± 2.05 21.1
800 μg/ml Pu’er Tea 16.5 ± 1.62 24 ± 1.63 16.6
600 μg/ml Liubao Tea 17.08 ± 1.37 24 ± 2.24 19.4
700 μg/ml Liubao Tea 18.7 ± 1.51 30 ± 1.63 26.4
800 μg/ml Liubao Tea 17.42 ± 1.57 26 ± 1.64 21
[71]Open in a new tab
^a
Lifespan improvement rate%: Maximum life increase/Maximum lifespan of
the control.
3.2. Dark tea enhances the oxidative stress resistance in Caenorhabditis
elegans
3.2.1. Dark tea extends the lifespan of Caenorhabditis elegans under
oxidative damage conditions
Lifespan involves the process of gradual aging, and the oxidative
damage pathway accelerates aging ([72]34). Under oxidative stress, the
lifespan of dark tea-treated C. elegans increased significantly,
especially in those treated with Brick tea or Liubao tea ([73]Figure
2).
Figure 2.
Figure 2
[74]Open in a new tab
Effects of dark tea on the lifespan in C. elegans under oxidative
damage conditions.
3.2.2. Dark tea decreases the ROS level of Caenorhabditis elegans
Under both normal conditions and oxidative stress, the control group
had the highest ROS level and this effect was minimized by dark tea
treatments ([75]Figure 3). The fluorescence intensity decreased by 41,
68, and 37% after treatment with Brick, Pu’er, and Liubao teas,
respectively ([76]Figure 3C), without H[2]O[2] treatment, and decreased
by 28.4, 35.4, and 22.2%, respectively ([77]Figure 3D), in the presence
of H[2]O[2] compared with the control group. These results demonstrated
that dark tea markedly reduced the levels of ROS in C. elegans,
indicating that dark tea has strong antioxidant activity in vivo.
Figure 3.
[78]Figure 3
[79]Open in a new tab
Effects of dark tea on the ROS levels in C. elegans. (A) ROS
fluorescence plot in the normal state. (B) ROS fluorescence plot in the
oxidative damage state. (C) Quantitative analysis plot of ROS
fluorescence intensity in the normal state. (D) Quantitative analysis
plot of ROS fluorescence intensity in the oxidative damage state. ****
indicates p < 0.0001.
3.2.3. Dark tea scavenges hydroxyl, DPPH, and ABTS+ free radical in vitro
To further assess the effects of dark tea on rendering stress
resistance in C. elegans, the free radicals scavenging capacity of dark
tea was investigated. As shown in [80]Figure 4, the scavenging capacity
of 600 μg/mL of Brick tea for hydroxyl, DPPH, and ABTS+ radicals was
maximized. The scavenging ability of Pu’er tea and Liubao tea for DPPH,
and ABTS+ radicals increased with increasing concentration; however,
the scavenging ability for hydroxyl radicals reached a maximum at
600 μg/mL. These results suggest the strong free radicals scavenging
capacity of dark tea.
Figure 4.
[81]Figure 4
[82]Open in a new tab
Effects of dark tea on the free radicals scavenging capacity. (A) -OH.
(B) DPPH. (C) ABTS+.
3.3. Dark tea exerts no toxicity on the fertility of Caenorhabditis elegans
The spawning period of C. elegans was about 6–7 days, and mainly
concentrated in the first 3 days ([83]Figure 5A). There was no
significant difference between the experimental groups in terms of the
total number of offspring ([84]Figure 5B). The results indicated that
the reproductive capacity of C. elegans is not impaired by dark tea and
provided evidence for the safety of dark tea.
Figure 5.
[85]Figure 5
[86]Open in a new tab
Effects of dark tea on the reproductive capacity in C. elegans. (A) The
number of offspring daily in each groups in the pawning period. (B) The
total of number of offspring in each group in the pawning period.
3.4. Dark tea promotes the motility of Caenorhabditis elegans
With aging, muscle function gradually declines, which in turn slows
down mobility. The effect of dark tea on motility was recorded on days
2, 4, 8, 12 and 14 post-dosing, and the results are presented in
[87]Figure 6. On day 12, the motility of C. elegans decreased
substantially. The dark tea significantly affected the frequency of
head bobbing in C. elegans in the pre-preliminary stage, among these,
Liubao tea had the most significant effect ([88]Figure 6A). Dark tea
substantially enhanced the body bending frequency of C. elegans,
whereas the effect of Pu’er tea on C. elegans in the later stages of
the process was not significant ([89]Figure 6B). Thus, dark tea can
alleviate the aging-related decrease in the energy transportation
capacity of C. elegans.
Figure 6.
[90]Figure 6
[91]Open in a new tab
Effects of dark tea on the motility in C. elegans. (A) Frequency of
head bobbing in 1 min. (B) Degree of body bending in 20 s. * indicates
p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001.
3.5. Dark tea alleviates the accumulation of fat of Caenorhabditis elegans
Fat is the root cause of inflammation due to aging ([92]35). Staining
with Oil Red O (Solarbio, China) showed a significantly lower number of
fat cells in the experimental group than those in the control group
([93]Figure 7A), and C. elegans fed Brick tea had the lowest body fat
content. Further analysis revealed that the body fat content of C.
elegans fed with Brick tea, Pu’er tea, or Liubao tea was reduced by 9,
4, and 8%, respectively ([94]Figure 7B). These results suggest that
dark tea significantly ameliorated age-associated physiological
characteristics by reducing fat deposition in C. elegans by reducing
fat accumulation.
Figure 7.
Figure 7
[95]Open in a new tab
Effects of dark tea on the fat content in C. elegans. (A) Plot of fat
staining. (B) Plot of quantitative analysis of staining intensity. **
indicates p < 0.01, **** indicates p < 0.0001.
3.6. Dark tea reduced the apoptosis of Caenorhabditis elegans
Caenorhabditis elegans treated with Brick tea, Pu'er tea, and Liubao
tea had significantly lower apoptosis than the control group
([96]Figure 8A). Quantitative analysis of the fluorescence intensity in
C. elegans fed Brick tea, Pu’er tea, and Liubao tea decreased by 15,
24, and 14%, respectively ([97]Figure 8B). The above results show that
dark tea could achieve anti-aging effects by inhibiting the degree of
apoptosis.
Figure 8.
Figure 8
[98]Open in a new tab
Effects of dark tea on the apoptosis in C. elegans. (A) Apoptosis
fluorescence graph. (B) Quantitative analysis graph of apoptosis
fluorescence intensity. **** indicates p < 0.0001.
3.7. Transcriptomics analysis
3.7.1. Gene expression differences
Genetic data of dark tea-treated three groups are shown in [99]Figure
9. The volcano plot (red, up-regulated; blue, down-regulated) revealed
that the number of down-regulated or up-regulated genes in the Brick
tea, Pu’er tea, and Liubao tea groups overlapped, while each group also
exhibited unique genes ([100]Figure 9A). Interestingly, the number of
down-regulated genes was the highest in the Liubao tea group, while the
number of up-regulated genes was the most in the Brick tea group was
the most ([101]Figure 9B). Compared to the control group, the same 344
genes were identified in the Brick tea, Pu’er tea, and Liubao tea
groups ([102]Figure 9C).
Figure 9.
[103]Figure 9
[104]Open in a new tab
Distribution of genes identified in dark tea. (A) Volcano plot of
differentially expressed genes. The horizontal coordinate indicates the
logarithmic value of the fold difference in expression of a gene in the
two samples and the vertical coordinate indicates the negative
logarithmic value of the statistical significance of the change in gene
expression. Red dots indicate up-regulation and blue dots indicate
down-regulation. (B) Up-regulated and down-regulated genes. (C) Venn
diagram of the set of differential genes (fold change≥2).
3.7.2. GO enrichment of differentially quantified genes
The gene products were analyzed for GO enrichment in terms of
biological process (BP), cellular component (CC), and molecular
function (MF), as shown in [105]Figure 10. A few of the same
up-regulated and down-regulated genes with functional annotations were
identified in the Brick tea, Pu’er tea, and Liubao tea groups, which
included cellular process and metabolic process in BP, cellular
anatomical entity in CC, and catalytic activity and binding in MF. The
alterations in the above-mentioned biological functions have all been
shown to affect lifespan ([106]36). Thus, dark tea mainly affects
endocrine system-related pathways, especially catalysis-related
bio-metabolism within cells.
Figure 10.
Figure 10
[107]Open in a new tab
GO annotation categorization statistical chart. (A) Brick Tea. (B)
Pu’er Tea (C) Liubao Tea. The horizontal coordinate is the GO
classification, the vertical coordinate is the number of genes, and
different colors represent the different primary classifications to
which they belong.
3.7.3. Specific regulation pathways of dark tea
To further explore the regulation pathways of dark tea, a KEGG pathway
enrichment analysis was conducted ([108]Figure 11). In the Brick tea,
Pu’er tea, and Liubao tea groups, the ubiquitin mediated proteolysis,
spliceosome and mannose type O-glycan biosynthesis pathways were
significantly up-regulated, while fatty acid metabolism and fatty acid
degradation pathways were significantly down-regulated. Among the
annotated pathways, a majority were closely linked to glucose and lipid
metabolism. These pathways suggested the metabolism and biosynthesis
are significantly involved in the redox reaction, which were important
to improve antioxidant defense system.
Figure 11.
[109]Figure 11
[110]Open in a new tab
Bubble chart of genes enriched in KEGG pathways. (A) Brick Tea. (B)
Pu’er Tea (C) Liubao Tea. The bubble size represents the number of
genes in the enriched pathway terms, and the bubble color represents
the p value.
A total of 2, 1, and 2 genes, respectively, were significantly
up-regulated (p < 0.05) in the ubiquitin mediated proteolysis, mannose
type O-glycan biosynthesis, and fatty acid metabolism and degradation
pathways, respectively ([111]Table 2). Specifically, the expression of
PRKN and SKP1 in the ubiquitin mediated proteolysis was significantly
up-regulated (p < 0.05). After dark tea treatment, the expression of
K12H6.3 in the mannose type O-glycan biosynthesis was significantly
up-regulated (p < 0.05); in addition, the expression of ACADM, CPT1 in
the fatty acid metabolism and degradation pathways was significantly
up-regulated (p < 0.05).
Table 2.
Genes after dark tea intervention in the ubiquitin mediated
proteolysis, mannose type O-glycan biosynthesis, fatty acid metabolism
and fatty acid degradation pathways in C. elegans.
Pathways No Gene name Regulated Log2FC[112]^a
Ubiquitin mediated proteolysis 1 PRKN Up 1.43
2 SKP1 Up 2.63
Mannose type O-glycan biosynthesis 1 K12H6.3 Up 3.32
Fatty acid metabolism and degradation 1 ACADM Down −1.17
2 CPT1 Down −1.48
[113]Open in a new tab
^a
Log2FC indicates differences in gene expression in the comparison of
two samples.
4. Discussion
As the only post-fermented tea, many biochemical reactions of the
fermentation process in the production of dark tea result in
differences that set dark tea and other five types of tea. Twenty-two
macronutrient chemical composition indicators in 65 Chinese dark teas
have been reported, with the richer ones being polyphenols and free
amino acids ([114]9). In the present study, we first determined the
content of the main two nutrients in the three represented dark teas.
The contents of tea polyphenols and L-theanine in dark tea were
maintained at around 10 and 1.5% ([115]Supplementary Table 1).
Free radical production is essential in normal metabolism, and
excessive free radical formation may lead to oxidative stress and other
related diseases ([116]37). Xu et al. ([117]38) found that tea
polyphenols increased SOD, CAT, T-AOC and GSH-Px contents along with a
reduction in MAD and ROS in crows. In this study, we found that orally
supplied dark tea could significantly decrease the ROS levels and
exhibited a superior lifespan in H[2]O[2]-induced oxidative stress C.
elegans. Similarly, we observed an improvement in-OH, DPPH, and ABTS+
free radicals scavenging capacity in antioxidant assays in vitro,
indicating the scavenging ability of dark tea.
The relationship between the activity of antioxidants and longevity
promotion has long been noticed. Although many chemically synthesized
antioxidants are available, long-term use may have potential side
effects ([118]2, [119]3). We found that dark tea extended the lifespan
and motility without significantly impairing propagation in C. elegans.
Obesity due to excessive fat accumulation is associated with
accelerated onset of diseases occurring in old age, while fat ablation
increases life span ([120]39). Apoptosis is also considered an
effective immune defense mechanism when the body is subjected to
various harmful stimuli ([121]40, [122]41). Our results indicated that
the lipid oxidation products and apoptosis in C. elegans could be
reduced after dark tea treatment. Therefore, several conclusions and
speculations can be drawn, which suggest that the longevity extension
of dark tea might be attributed to the inhibition of lipid oxidation
and the extent of generation of apoptosis-related products to protect
the health of the body. Noticeably, no significant toxic effects of
dark tea were observed in our study.
GO analysis allows for a standardized description of the gene products
in terms of the biological pathways involved and cellular localization
([123]42). To elucidate the roles of the identified differentially
expressed genes, GO analysis revealed significant enrichment of BP, CC,
and MF, namely, cellular process, metabolic process, catalytic
activity, and binding, which play independent roles in antioxidant and
anti-aging traits. According to KEGG enrichment analysis the number of
genes annotated to a metabolic pathway in a differentially expressed
gene is significantly greater than the proportion of background genes
among all such genes ([124]43). Among the annotated pathways, most are
related to glucose and lipid metabolism. Four common enriched signaling
pathways identified in three dark tea groups included mannose type
O-glycan biosynthesis, fatty acid metabolism, fatty acid degradation,
ubiquitin mediated proteolysis, and spliceosome. Further comparison of
the differentially expressed genes with the functional annotations of
the genes in the database, revealed that most of the genes with more
significant changes in gene up-regulation and down-regulation because
of dark tea treatment were regulated by SKP1, K12H6.3, ACADM, and CPT1.
SKP1 was up-regulated in the ubiquitin mediated proteolysis pathway,
which connects cell cycle regulators to the ubiquitin proteolysis
machinery to thus improve cellular growth ([125]44). K12H6.3 was found
to be involved in the mannose type O-glycan biosynthesis pathway; the
gene encodes the relevant core α1,3-fucosyltransferase, whose
expression is promoted during development ([126]45). ACADM and CPT1 are
key genes catalyzing mitochondrial fatty acid metabolism and
degradation. They catalyze the rate-limiting step of the conversion of
acyl-coenzyme. As into acyl-carnitines, which can cross membranes to
enter the mitochondria, and elevate triglyceride, phospholipid, and
droplet levels of cellular lipids to regulate genetic, epigenetic,
physiological, and nutritional modulators ([127]46).
Overall, it may be safely concluded that dark tea can independently
reduce oxidative stress, thus preventing oxidative injury. As a natural
and non-toxic antioxidant, dark tea can be used as a nutritional
supplement for livestock and as a functional component of livestock
feed, thus acting as a defense against various diseases caused by
oxidative damage. It thus has great promise for animal health and
disease prevention. Further exploration of dark tea as a potential
dietary supplement for animal health maintenance and disease prevention
is worth exploring.
5. Conclusion
In conclusion, the safety of dark tea was demonstrated in C. elegans
and dark tea was found to mitigate oxidative stress-induced damage by
promoting the clearance of free radicals. Additionally, it was observed
that dark tea modulated metabolic processes, including carbohydrates,
lipids, and proteins, catalyzing the synthesis of antioxidants from
specific oxidative substrates within the cell.
Data availability statement
The datasets presented in this study can be found in online
repositories. The names of the repository/repositories and accession
number(s) can be found at: [128]https://figshare.com/,
[129]10.6084/m9.figshare.24609774.
Ethics statement
The animal study was approved by Animal Care and Use Committee,
Changchun University of Science and Technology. The study was conducted
in accordance with the local legislation and institutional
requirements.
Author contributions
JW: Writing – original draft, Writing – review & editing. KZ: Writing –
original draft. YZ: Writing – original draft. SG: Writing – review &
editing. SZ: Writing – review & editing.
Acknowledgments