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Research Article
Protective Effect of Triphala against Oxidative Stress-
Induced Neurotoxicity
Wanchen Ning ,
1
Simin Li ,
2
Jokyab Tsering ,
3
Yihong Ma ,
4
Honghong Li ,
3
Yuezhu Ma ,
5
Anthony Chukwunonso Ogbuehi ,
6
Hongying Pan ,
7
Hanluo Li ,
8
Shaonan Hu ,
9
Xiangqiong Liu ,
3
Yupei Deng ,
3
Jianlin Zhang ,
10
and Xianda Hu
3
1
Department of Conservative Dentistry and Periodontology, Ludwig Maximilian University of Munich, Goethestrasse 70,
Munich 80336, Germany
2
Stomatological Hospital, Southern Medical University, Guangzhou 510280, China
3
Laboratory of Cell and Molecular Biology, Beijing Tibetan Hospital, China Tibetology Research Center,
218 Anwaixiaoguanbeili Street, Chaoyang, Beijing 100029, China
4
Department of Neurology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan
5
Peking University Third Yanqing Hospital, 28 East Shuncheng St., Yanqing District, Beijing 110229, China
6
Faculty of Physics, University of Münster, Wilhelm-Klemm-Straße 9, Münster 48149, Germany
7
School of Dentistry, University of Michigan, 1011 N University Ave, Ann Arbor, MI 48109, USA
8
Department of Cranio/Maxillofacial Surgery, University Clinic Leipzig, Liebigstr. 12, Leipzig 04103, Germany
9
Innovation Center Computer Assisted Surgery (ICCAS), Leipzig University, Semmelweisstraße 14, Leipzig 04103, Germany
10
Department of Neurosurgery, Taian Central Hospital, Taian City, Shandong Province 271000, China
Correspondence should be addressed to Xianda Hu; hellocean@hotmail.com
Received 25 November 2020; Revised 4 March 2021; Accepted 27 March 2021; Published 9 April 2021
Academic Editor: Andrea Scribante
Copyright © 2021 Wanchen Ning et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background. Oxidative stress is implicated in the progression of many neurological diseases, which could be induced by various
chemicals, such as hydrogen peroxide (H
2
O
2
) and acrylamide. Triphala is a well-recognized Ayurvedic medicine that possesses
different therapeutic properties (e.g., antihistamine, antioxidant, anticancer, anti-inflammatory, antibacterial, and anticariogenic
effects). However, little information is available regarding the neuroprotective effect of Triphala on oxidative stress. Materials
and Methods.Anin vitro H
2
O
2
-induced SH-SY5Y cell model and an in vivo acrylamide-induced zebrafish model were
established. Cell viability, apoptosis, and proliferation were examined by MTT assay, ELISA, and flow cytometric analysis,
respectively. The molecular mechanism underlying the antioxidant activity of Triphala against H
2
O
2
was investigated dose
dependently by Western blotting. The in vivo neuroprotective effect of Triphala on acrylamide-induced oxidative injury in
Danio rerio was determined using immunofluorescence staining. Results. The results indicated that Triphala plays a
neuroprotective role against H
2
O
2
toxicity in inhibiting cell apoptosis and promoting cell proliferation. Furthermore, Triphala
pretreatment suppressed the phosphorylation of the mitogen-activated protein kinase (MARK) signal pathway (p-Erk1/2, p-
JNK1/2, and p-p38), whereas it restored the activities of antioxidant enzymes (superoxide dismutase 1 (SOD1) and catalase) in
the H
2
O
2
-treated SH-SY5Y cells. Consistently, similar protective effects of Triphala were observed in declining neuroapoptosis
and scavenging free radicals in the zebrafish central neural system, possessing a critical neuroprotective property against
acrylamide-induced oxidative stress. Conclusion. In summary, Triphala is a promising neuroprotective agent against oxidative
stress in SH-SY5Y cells and zebrafishes with significant antiapoptosis and antioxidant activities.
Hindawi
BioMed Research International
Volume 2021, Article ID 6674988, 11 pages
https://doi.org/10.1155/2021/6674988
1. Introduction
Oxidative stress is implicated in the progression of many
inflammatory and malignant diseases, including neurodegen-
erative disease (such as Alzheimer’s disease [1], Parkinson’s
disease [2], mild cognitive impairment [3, 4], and vascular
dementia [5]), as well as oral disorders like dental caries, peri-
odontal disease [6, 7], oral mucositis [8], and oral cancer [9].
The severe or prolonged oxidative stress is mainly induced
by the excessive production of reactive oxygen species
(ROS), which causes DNA damage, oxidative proteins, and
peroxidation of lipids and thereby triggers cell apoptosis
[10, 11]. ROS is composed of superoxide anion (
·
O
2
−
), hydro-
gen peroxide (H
2
O
2
), and hydroxyl radicals (OH
·
) [12], of
which H
2
O
2
is a culprit, destroying neurons,and thus is widely
used as a stimulant of neutral cells to establish an in vitro oxi-
dative injury model [13, 14]. In addition, acrylamide, a vinyl
monomer formed in high-temperature foods [15], is applied
as a neurotoxin in various cell and animal models for its over-
production of ROS. For instance, acrylamide was suggested to
induce cell apoptosis in human neuroblastoma SH-SY5Y cells
[16] and promote neurotoxicity in the zebrafish model [17].
Triphala, or ‘Bras Bu gSum Thang’in Tibetan, is a well-
recognized Ayurvedic medicine consisting of dried fruits of
three plant species, including Emblica officinalis (family
Euphorbiaceae), Terminalia bellirica (family Combretaceae),
and Terminalia chebula (family Combretaceae). Modern stud-
ies have shown that Triphala has many properties, including
antihistamine, antioxidant, antitumor, anti-inflammatory,
antibacterial, antiviral, antifungal, and anticariogenic effects
[18, 19]. In addition, Triphala has been investigated to be
effective in treating many types of cancers and oral diseases,
including prostate cancer [20], pancreatic cancer [21], colon
cancer [22], and gynecological cancers [23], as well as peri-
odontal diseases [24]. Regarding Triphala’s neuroprotective
ability, a research group from Taiwan has revealed the protec-
tive effect of Terminalia chebula extracts on a neuron-like rat
pheochromocytoma (PC12) cell line [25, 26]. Thus, it is of
note that Triphala has many possibilities to possess neuropro-
tective property. However, despite the recent report, current
information regarding the neuroprotective effect of Triphala
is still limited. Thus, the present study is aimed at investigating
Triphala’s neuroprotective property against oxidative stress-
induced damage in vitro and in vivo.
2. Materials and Methods
2.1. Cell Culture. Human neuroblastoma (SH-SY5Y) cells
were provided by China Infrastructure of Cell Line
Resource. SH-SY5Y cells were cultured in Dulbecco’s mod-
ified Eagle medium (DMEM) (Corning), supplemented with
10% fetal bovine serum (FBS) (Corning) and 1% penicillin-
streptomycin (Beyotime Biotechnology Inc., Nantong, China).
The cells were incubated at 37
°
C under a humidified atmo-
sphere of 5% carbon dioxide.
2.2. MTT Assay for Cell Viability Analysis. SH-SY5Y cells
were seeded in 96-well plates (CoStar, USA) separately at a
density of 4,000 cells per well. Triphala extract power
(AL1675; Dabur India Ltd., Alwar, India) was prepared and
diluted in the same way as our previous study [23, 27].
Firstly, to determine the optimal concentrations of Triphala
and H
2
O
2
, higher concentrations (0.08, 0.4, 2, 10, 50, and
250 μg/mL) and lower concentrations (0.014, 0.041, 0.12,
0.37, 1.11, 3.33, and 10 μg/mL) of Triphala were applied to
treat SH-SY5Y cells for 48 h. Meanwhile, cells were treated
with 300, 400, 500, 600, 700, and 800 μmol/L of hydrogen
peroxide (H
2
O
2
) for 20 h, separately. Then, cell viability was
measured using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-
tetrazolium bromide (MTT) assay (Boster Biological Tech-
nology Company, Wuhan, Hubei, China). Briefly, each well
was incubated with 10 μL of MTT solution for 4 h at 37
°
C,
added with 110 μL formazan solvent, and the absorption at
490 nm was measured using a microplate reader (Beijing
Pulang New Technology Co., Beijing, China).
Next, the neuroprotective effect of Triphala on H
2
O
2
tox-
icity was estimated by preincubating cells with increasing
concentrations (0.014, 0.041, 0.12, 0.37, 1.11, 3.33, and
10 μg/mL) of Triphala for 24 h and then challengin g the cells
with 400 μmol/L H
2
O
2
for an additional 20 h. Cells treated
without either H
2
O
2
or Triphala were considered as blank
control, while the H
2
O
2
-induced injury cell model, in which
only H
2
O
2
was added, was regarded as model control. After
the drug intervention, cell viability was determined using the
MTT assay as described above and the percentage of cell viabil-
ity was calculated as follows: viability ð%Þ=ðODexperiment −
ODblank Þ/ðODmodel −ODblank Þ×100%. The percentage of
the protective effects was calculated; thus, protective effects
ð%Þ=1−viability (%).
2.3. ELISA Assay for Cell Apoptosis Analysis. The Cell Death
Detection Enzyme-Linked Immunosorbent Assay (ELISA)
Plus Kit (Roche Diagnostics, Basel, Switzerland) was applied
to examine the apoptosis inhibition activity of Triphala as
our previous study [23]. Briefly, the cells were seeded and cul-
tured using the same method described above. SH-SY5Y cells
in the logarithmic growth phase were seeded in 96-well plates
and pretreated with different concentrations (0.37, 1.1, and
3.3 μg/mL) of Triphala for 24 h. Then, cells were treated with
400 μmol/L H
2
O
2
for an additional 20 h. The setup of the
blank control and model control was the same as above. After
the drug intervention, the cells were collected and resus-
pended in lysis buffer and the lysate was centrifuged to
remove the intact nuclei. The supernatant containing cyto-
plasmic histone-associated DNA fragments was transferred
into a streptavidin-coated microplate and was then incubated
with immunoreagent and substrate for quantitative immuno-
assay. The OD value was determined at 405 nm using a
microplate reader, and the fold increase of DNA fragmenta-
tion, reflecting the number of programmed cell deaths, was
calculated as absorbance of treated cells/absorbance of nega-
tive control cells.
2.4. Flow Cytometric Analysis. Flow cytometry assays were
processed using monoclonal antibody Ki-67 and Annexin
V/propidium iodide (PI) double staining, to evaluate the
degree of proliferation apoptosis, as in our previous study
[23]. Briefly, SH-SY5Y cells were seeded into 6-well plates
2 BioMed Research International
(Nest Biotech., China) at a density of 1×10
5cells per well.
After pretreatment with different concentrations (0.37, 1.1,
and 3.3 μg/mL) of Triphala for 24 h, the cells were treated
with 400 μmol/L H
2
O
2
for an additional 20 h. Then, the cells
were harvested by trypsinization, incubated with −20
°
Cin
absolute ethanol, washed, and resuspended in cell staining
buffer. Subsequently, cells were fixed and the nuclear mem-
brane was permeabilized using Foxp3/Transcription Factor
Staining Buffer Set (eBioscience Inc., San Diego, CA) before
staining with anti-Ki67 antibody (Miltenyi Biotec, Bergisch
Gladbach, Germany) at 4
°
C for 1 h. To detect the apoptosis,
cells were incubated using the FITC Annexin V Apoptosis
Detection Kit with PI (Dojindo Molecular Technologies
Inc.), at room temperature for 15 min. Afterwards, the fluo-
rescent staining was detected and analyzed using a flow
cytometer (BD Biosciences, San Jose, CA). The mean fluores-
cent intensity for Ki-67 was calculated using FlowJo VX soft-
ware (Tree Star Inc., Ashland, OR).
2.5. Western Blot Analysis. SH-SY5Y cells were seeded in the
10 cm Petri dishes with a density of 5×10
5cells per dish.
Firstly, the cells were pretreated with increasing concentra-
tions (0.37, 1.1, and 3.3 μg/mL) of Triphala for 24 h and stim-
ulated with 400 μmol/L H
2
O
2
. The blank control and model
control were established as described above. Then, the cul-
tured cells were harvested by centrifugation and fractionated
using the Nuclear and Cytoplasmic Protein Extraction Kit
(Beyotime Biotechnology Inc., Nantong, China) following
the manufacturer’s instruction with the supplement of prote-
ase inhibitor cocktail and phosphatase inhibitor cocktail,
offered by Sigma-Aldrich Corp. (St. Louis, MO). The protein
concentrations were determined using the Pierce BCA Pro-
tein Assay Kit (Thermo Fisher Scientific, Waltham, MA).
The total or nuclear proteins were separated by SDS-PAGE
electrophoresis, transferred to a nitrocellulose membrane,
and then incubated with monoclonal antibodies, including
phospho-p44/42 mitogen-activated protein kinase (MAPK)
extracellular signal-related kinase (ERK)1/2. (Thermo Fisher),
phospho-c-Jun amino-terminal kinases (JNK)1/2 (Thermo
Fisher), and phospho-p38 MAPK, superoxide dismutase 1
(SOD1), and catalase, which were purchased from Cell Signal-
ing Technology (Danvers, USA). Afterwards, rabbit anti-
mouse secondary antibody (Abcam, Cambridge, UK) and goat
anti-rabbit secondary antibody (Abcam) were attached and
expression levels of proteins were detected by chemilumines-
cence using a Pierce ECL Plus Western Blotting Substrate
(Thermo Fisher).
2.6. Zebrafish Embryos. The wild-type AB strain of zebra-
fishes (Danio rerio) were obtained from Hunter Biotechnol-
ogy (Hangzhou, China). The zebrafishes were maintained
in the 28
°
C reverse osmosis water, incubating with
200 mg/L instant sea salt and 50–100 mg/L CaCO3, at an
electrical conductivity of 450~550 μS/cm and a pH of 6.5–
8.5. The embryos used in this study were generated by natu-
ral pairwise mating.
2.7. In Vivo Maximum Tolerated Concentration (MTC) of
Triphala. A total of 270 wild-type AB strain zebrafishes were
randomly selected at 1 day postfertilization (dpf), and 30 zeb-
rafishes per well were plated in 6-well plates (Nest Biotech.,
China). Acrylamide stock (L2028046; Aladdin, Shanghai,
China) was dissolved with DMSO into 1 mol/L, 30 μLofwhich
was then added into 3 mL fish water, reaching the final con-
centration (10 mmol/L). Triphala was prepared as described
above. Prepared acrylamide was applied to establish a zebra-
fish neuroapoptosis model. Then, to explore the maximum
tolerated concentration (MTC) of Triphala, we treated the
acrylamide-stimulated zebrafishes together with different con-
centrations of 3mL Triphala (0.123, 0.370, 1.11, 3.33, 10.0,
30.0, and 90.0 μg/mL) at the same time, for 24h. The control
group was incubated with neither Triphala nor acrylamide,
while 10mM acrylamide only stimulated the model group.
2.8. In Vivo Neuroprotective Effect of Triphala. A total of 240
wild-type AB strain zebrafishes were randomly selected at
1 dpf, and 30 zebrafishes per well were plated in 6-well plates.
Control and model groups were established as described
above. Five different concen trations of 3 mL water-soluble
Triphala (0.123, 0.370, 1.11, 3.33, and 10.0 μg/mL) were
applied to treat the 10 mM acrylamide-induced fishes at the
same time. Glutathione (GSH) powder (SLCF2362; Sigma-
Aldrich, Shanghai, China) was diluted into 615 μg/mL, and
3 mL GSH was used to stimul ate the acrylamide-induced
fishes together, regarded as a positive control group to com-
pare the effects of Triphala. Then, the eight groups of fishes
were incubated at 28
°
C for 24 h. Then, the fishes were stained
with acridine orange (AO) (494-38-2, Sigma, China). After
that, ten zebrafishes from each group were randomly
selected, which were observed and imaged using a fluores-
cence microscope (VertA1; Shanghai Tucson Vision Tech-
nology Co., China). The mean fluorescence intensity was
analyzed using NIS-Elements D 3.20 image software. The sta-
tistical analysis of the fluorescence intensity was used to eval-
uate the neuroprotective efficacy of Triphala.
2.9. In Vivo Antioxidant Effect of Triphala. A total of 240
wild-type AB strain zebrafishes were randomly selected at
1 dpf, and 30 zebrafishes per well were plated in a 12-well
plate (Nest Biotech., China). Control and model groups were
established as described above. Five different concentrations
of 3 mL water-soluble Triphala (0.123, 0.370, 1.11, 3.33, and
10.0 μg/mL) and 615 μg/mL GSH were applied to treat the
acrylamide-induced fishes together. Meanwhile, 2 mL E3
medium with ROS-specificfluorescent substrate (CM-
H2DCFDA) (C6827/2146733; Invitrogen, USA) was added
into each well. Subsequently, the fishes and medium were
transferred into a 96-well plate (CoStar, USA) and incubated
for 20 h at 28
°
C (protected from light). Next, the plate was
scanned with the multifunctional enzyme marker (Spark;
Tecan, Switzerland) and each group’sfluorescence was mea-
sured. Finally, the fluorescence value of each group was col-
lected and analyzed using SPARKCON TROL Dashboard
software. The fluorescence value was used to evaluate the
ability of Triphala in scavenging free radicals.
2.10. Statistical Analysis. All in vitro data were represented as
means ± SDs of a minimum of 3 independent experiments.
3BioMed Research International
Statistical analyses were carried out by one-way ANOVA,
with Bonferroni correction, via SPSS Statistics 26.0 software.
Apvalue <0.05 was considered to be statistically significant.
3. Results
3.1. Optimal Concentration of Triphala and Hydrogen
Peroxide (H
2
O
2
) in SH-SY5Y Cells. In order to determine
the optimal concentration of Triphala applied in the subse-
quent experiments, the proliferation rate of various concen-
trations of Triphala was firstly measured using MTT assay.
Among the higher concentrations (from 0.08 to 250 μg/mL)
of Triphala, only 50 and 250 μg/mL Triphala significantly
decreased cell viability, with the proliferation rates of
−17.84% and −56.38% (p<0:05), compared to blank control
(see Figure 1(a)). However, lower concentrations (from 0.014
to 10 μg/mL) of Triphala promoted cell viability in SH-SY5Y
cells, of which 0.37, 1.11, and 3.33 μg/mL Triphala dramati-
cally enhanced the proliferation rates to 142.55%, 183.12%,
and 226.80% (p<0:05) (see Figure 1(b)). By virtue of these
results, pretreatment with low concentrations of Triphala
(from 0.014 to 10 μg/mL), especially 0.37, 1.11, and
3.33 μg/mL, was considered as optimal to examine its neuro-
protective effect against H
2
O
2
-induced damage.
Aimed at determining the optimal concentration of H
2
O
2
to exert neurotoxicity, the apoptosis rate of various concen-
trations of H
2
O
2
was measured using an MTT assay. Relative
to 300, 400, 500, 600, 700, and 800 μmol/L (μM) of the H
2
O
2
apoptosis rate was upregulated to 30.14%, 47.65%, 63.53%,
80.41%, 80.36%, and 79.78%, respectively (see Figure 1(c)).
Based on the above, the half-maximal inhibitory concentra-
tion (IC50) of H
2
O
2
was 400 μmol/L; thereby, stimulating
400 μmol/L H
2
O
2
for 20 h was considered as optimal and
applied in subsequent experiments.
–80
0.08
0.40
2.00
10.00
50.00
250.00
–60
–40
–20
0
Proliferation rate (%)
20
Concentration (𝜇g/mL)
Cell viability promotion effect of triphala on
on SH-SY5Y cells
(a)
–100
0.013
0.041
0.120
0.370
1.110
3.330
10.000
0
100
200
300
400
Proliferation rate (%)
500
Concentration (𝜇g/mL)
Proliferative effect of triphala on
SH-SY5Y cells
⁎
⁎
⁎
(b)
Concentration (𝜇mol/L)
Cytotoxic effect of H2O2 on SH-SY5Y
cells
0
300
20
40
60
80
Apoptosis rate (%)
100
400
500
600
700
800
(c)
Protective effect of triphala on H2O2–
induced apoptosis in SH-SY5Y cells
⁎
⁎
⁎
⁎
⁎
⁎
Concentration (𝜇g/mL)
–50
0.014
0
50
100
150
Protective rate (%)
200
0.041
0.120
0.370
1.110
3.330
10.000
(d)
Figure 1: Effects of Triphala and hydrogen peroxide (H
2
O
2
) on cell viability in SH-SY5Y cells. (a) Cell viability promotion effect of Triphala
on SH-SY5Y cells. SH-SY5Y cells were treated with higher concentrations (0.08, 0.4, 2, 10, 50, and 250 μg/mL) of Triphala for 48 h. (b)
Proliferative effect of Triphala on SH-SY5Y cells. SH-SY5Y cells were treated with lower concentrations (0.014, 0.041, 0.12, 0.37, 1.11,
3.33, and 10 μg/mL) of Triphala for 48 h. (c) Cytotoxic effect of H
2
O
2
on SH-SY5Y cells. SH-SY5Y cells were treated with different
concentrations (300, 400, 500, 600, 700, and 800 μmol/L) of H
2
O
2
for 20 h. (d) Protective effect of Triphala on H
2
O
2
-induced apoptosis in
SH-SY5Y cells. SH-SY5Y cells were pretreated with increasing concentrations (0.014, 0.041, 0.12, 0.37, 1.11, 3.33, and 10 μg/mL) of
Triphala for 24 h and then exposed to 400 μmol/L of H
2
O
2
for 20 h. The data are expressed as mean ± SD (n=3). ∗p<0:05 as compared
to control.
4 BioMed Research International
3.2. Neuroprotective Effect of Triphala on SH-SY5Y Cells.
Aimed at determining Triphala’s neuroprotective effect on
H
2
O
2
-induced SH-SY5Y cells, cell viability was measured
by the MTT assay. In a dose-dependent experiment, increas-
ing concentrations of Triphala were applied to preincubate
the SH-SY5Y cells which were then treated with H
2
O
2
. Cor-
responding to 0.014, 0.041, 0.12, 0.37, 1.11, 3.33, and
10 μg/mL Triphala pretreatment, the calculated protective
rates were 17.82%, 54.98%, 61.08%, 109.22%, 125.65%,
108.34%, and 61.96%, respectively (see Figure 1(d)). Except
the lowest concentration (0.014 μg/mL), other concentra-
tions of Triphala markly (0.041, 0.12, 0.37, 1.11, 3.33, and
10 μg/mL) upregulated the protective rates in the H
2
O
2
-
induced SH-SY5Y cells (p<0:05), compared to the model
control (see Figure 1(d)). Combined, Triphala played a pro-
tective role in attenuating the cell viability loss in SH-SY5Y
cells treated with H
2
O
2
.
3.3. Inhibition of Triphala on H
2
O
2
-Induced Apoptosis in SH-
SY5Y Cells. ELISA was used to determine the effect of Tri-
phala on H
2
O
2
-induced apoptosis in SH-SY5Y cells. Com-
pared to the model control, the apoptosis inhibitory rates of
H
2
O
2
-induced SH-SY5Y cells were significantly upregulated
to 22.53%, 23.66%, and 35.45%, by 0.37, 1.11, and 3.33 μg/mL
Triphala, respectively (p<0:05) (see Figure 2(a)). The above
results suggested an inhibitory effect of Triphala in suppress-
ing the apoptosis stimulated by H
2
O
2
.
3.4. Proliferative Effect of Triphala on H
2
O
2
-Induced SH-
SY5Y Cells. The cell proliferation rate was evaluated by flow
cytometry analysis using the FITC Annexin V Apoptosis
Detection Kit. As shown in Figure 2(b), the results revealed
a significant reduction of 20.60% in the proliferation rate in
SH-SY5Y cells exposed to H
2
O
2
, compared to the blank con-
trol. In contrast, 0.37, 1.11, and 3.33 μg/mL Triphala preincu-
bation increased the proliferation rates by 17.93%, 15.11%,
and 22.81%, in the H
2
O
2
-induced SH-SY5Y cells (see
Figure 2(b)). The results suggested that H
2
O
2
is an inhibitor,
but Triphala is a promoter in the SH-SY5Y cell proliferation.
3.5. Investigation of Molecular Pathways. To further deter-
mine the neuroprotective mechanisms of Triphala, the
expression of p-ERK1/2, p-JNK1/2, p-p38 MAPK, SOD1,
and catalase was examined in H
2
O
2
-induced SH-SY5Y cells
by Western blotting. Higher levels of p-Erk1/2, p-JNK1/2,
and p-p38 and lower levels of SOD1 and catalase were
expressed in the model control, relative to blank control (see
Figure 2(c)). Furthermore, the degrees of p-Erk1/2 and p-
p38 in the H
2
O
2
-induced SH-SY5Y cells were decreased by
0.37 and 1.1 μg/mL Triphala pretreatment (see Figure 2(c)).
On the contrary, in comparison with the model control,
3.3 μg/mL Triphala promoted the expression of p-Erk1/2, as
well as p-p38 (see Figure 2(c)). As to the p-JNK1/2 content,
upregulation of JNK protein was inhibited by Triphala prein-
cubation (0.37, 1.1, and 3.3μg/mL) in the SH-SY5Y cells
exposed to H
2
O
2
and p-JNK1/2 expression was dramatically
attenuated to the lowest level, which is similar to that
expressed in blank control (see Figure 2(c)).
Significant upregulation of SOD1 was stimulated by 0.37,
1.1, and 3 μg/mL Triphala pretreatment in the H
2
O
2
H2O2-
induced SH-SY5Y cells, relative to model control (see
Figure 2(c)). Moreover, 3 μg/mL increased the SOD1 degree
to the highest point that is even higher than blank control
(see Figure 2(c)). Similarly, higher protein levels of catalase
were stimulated by Triphala preincubation (0.37, 1.1, and
3.3 μg/mL) in SH-SY5Y cells exposed to H
2
O
2
, compared to
both model and blank controls (see Figure 2(c)). Combined,
the three concentrations (0.37 , 1.1, and 3 μg/mL) of Triphala
applied in the experiment were efficient in restoring the levels
of neuroprotective-related proteins in the SH-SY5Y cells
exposed to H
2
O
2
.
3.6. MTC of Triphala in the Zebrafish Model. In our study, the
MTC of Triphala was determined firstly according to the fish
(mammalian) toxicology [28]. The threshold of Triphala
should be, on the one hand, high enough to maximize the
neuroprotective effect. On the other hand, the preferred con-
centration would not be too high to induce neurotoxicity [17,
29]. As Table 1 showed, three engagements, 1.11 μM (1/9
MTC), 3.33 μM (1/3 MTC), and 10.0 μM (MTC), were
selected as the preferred concentration of Triphala in the fol-
lowing experiment, which promoted better biological pheno-
types of the zebrafishes, compared to the model fishes.
Otherwise, Triphala of 30.0 and 90.0 μg/mL exerted toxicity
and induced worse biological phenotypes of the zebrafishes,
leading to a 3.33% (1/30th) mortality of the fishes. Zebra-
fishes treated with 0.123 and 0.370 μg/mL Triphala per-
formed similarly to the model fishes. Thus, the MTC of
Triphala in vivo was determined as 10.0 μg/mL.
3.7. Triphala Suppressed Acrylamide-Induced Neurotoxicity
in the Zebrafish Model. According to the fluorescence inten-
sity, Triphala’sin vivo neuroprotective effects were observed
in the zebrafish model injured by acrylamide. The fluores-
cence intensity of the control group (4153043 pixels) was sig-
nificantly lower than that of the model group (10860891
pixels) (p<0:05) (see Figures 3 and 4(a)), indicating that
the zebrafish model was successfully established. Also, the
fluorescence intensity of the GSH-treated-positive group
(4517110 pixels) was significantly lower than that of the
model group (p<0:05) (see Figures 3 and 4(a)), suggesting
the neuroprotective effects of the GSH on acrylamide. The
fluorescence intensity rates of the increasing concentration
(0.123, 0.370, 1.11, 3.33, and 10.0 μg/mL) of Triphala were
8611230, 6392234, 4382534, 4313911, and 4155579, respec-
tively. Triphala of 0.123 and 0.370 μg/mL exerted no signifi-
cant protective effects on zebrafishes exposed to acrylamide
(p>0:05) (see Figures 3 and 4(a)). However, decreasing
pixels of 1.11, 3.33, and 10.0 μg/mL Triphala were detected,
compared to the model group (p<0:05) (see Figures 3 and
4(a)). The findings supported that Triphala, a comparable
neuroprotective agent as GSH, inhibited the acrylamide-
induced neuroapoptosis in zebrafishes.
3.8. Triphala Scavenged Free Radicals in the Zebrafish Model
Exposed to Acrylamide. Based on the fluorescence value, the
antioxidative effects of Triphala in vivo were evaluated in
5BioMed Research International
0
0.37 1.11 3.33
20
40
Apoptotic inhibition rate (%)
60
Concentration (𝜇g/mL)
Apoptosis inhibitory effect of triphala on
H2O2-induced apoptosis in SH-SY5Y cells
⁎
⁎
⁎
(a)
Cell proliferation rate of H2O2-induced
SH-SY5Y cells with/without triphala
101
0
100
200
300
400
Count
500
562
102103
M3
20.60%
104
APC-H
0 𝜇g/mL
105106107.2 101
0
100
200
300
400
Count
500
562
102103
M3
17.93%
104
APC-H
0.37 𝜇g/mL
105106107.2
101
0
100
200
300
400
Count
500
562
102103
M3
15.11%
104
APC-H
1.11 𝜇g/mL
105106107.2
M3
17.93%
101
0
100
200
300
400
Count
500
562
102103
M3
22.81%
104
APC-H
3.33 𝜇g/mL
105106107.2
(b)
Expression degree of neuroprotective proteins in the
H2O2-induced SH-SY5Y cells with/without triphala
Tri pha la (𝜇g/mL)
Control
p-ERK1/2
p-p38
p-JNK 1/2
SOD 1
Catalase
𝛽-Actin
Model 0.33 1 3
(c)
Figure 2: Effect of Triphala on hydrogen peroxide (H
2
O
2
)-induced apoptosis in human neuroblastoma SH-SY5Y cells. SH-SY5Y cells were
preincubated by 0.37, 1.11, and 3.33 μg/mL Triphala for 24 h and then exposed to 400 μMH
2
O
2
for 20 h. In the control group, cells were not
treated with either H
2
O
2
or Triphala. In the model group, only H
2
O
2
was added into cells, without Triphala incubation. (a) ELISA kit was used
to determine the apoptosis inhibitory effect of Triphala on H
2
O
2
-induced SH-SY5Y cells. (b) Flow cytometry analysis was applied to evaluate
the cell proliferation rate. (c) Western blotting was performed to determine the degrees of the neuroprotective proteins (p-Erk1/2, p-p38, p-
JNK1/2, SOD, and catalase) in the H
2
O
2
-induced SH-SY5Y cells with/without Triphala pretreatment. The blots shown are representative of
three independent experiments. β-Actin was used as a loading control. Data are expressed as mean ± SD (n=3). ∗p<0:05 as compared to
control.
6 BioMed Research International
the zebrafish model exposed to acrylamide. The fluorescence
value of the control group was 1667, which was significantly
lower than that of the model group (2363) (p<0:05) (see
Figure 4(b)), supporting the practicability of the zebrafish
model. Besides, compared to that of the model group, the
reduced fluorescence value of the GSH-treated-positive
group (124) was measured (p<0:05) (see Figure 4(b)),
indicating that GSH is an efficient antioxidant agent in scav-
enging the free radicals produced by acrylamide. The fluores-
cence intensity rates of the increasing concentration (0.123,
0.370, 1.11, 3.33, and 10.0 μg/mL) of Triphala were 858,
816, 716, 746, and 1168, respectively. Except for 10.0 μg/mL
Triphala, other concentrations of Triphala (0.123, 0.370,
1.11, and 3.33 μg/mL) significantly decreased the level of free
radical, relative to that of the model group (p<0:05) (see
Figure 4(b)). The findings suggested Triphala’s comparable
antioxidant efficiency as GSH in scavenging the free radicals
in zebrafish exposed to acrylamide.
4. Discussion
Herewith, we investigated the antioxidant and neuroprotec-
tive aspects of Triphala using an in vitro H
2
O
2
-induced SH-
SY5Y cell model and an in vivo acrylamide-induced zebrafish
model. Our MTT, ELISA, and flow cytometric results indi-
cated that Triphala could attenuate the cytotoxic effects of
H
2
O
2
by promoting cell proliferation and inhibiting cell apo-
ptosis. Moreover, the underlying mechanisms were analyzed
Table 1: MTC exploration of Triphala in vivo.
Group (n=30)Triphala concentration
(μg/mL)
Number of deaths
(tails)
Mortality
(%) Biological phenotypes
Control / 0 0.00 Normal
Model / 0 0.00 Pericardial oedema; slight bending of the bodies
Triphala+acrylamide
0.123 0 0.00 Similar to the model
0.370 0 0.00 Similar to the model
1.11 0 0.00 Better than the model
3.33 0 0.00 Better than the model
10.0 0 0.00 Better than the model
30.0 1 3.33 Worse than the model
90.0 1 3.33 Worse than the model
Control Model GSH 615 𝜇g/mL
Triphala 1.11 𝜇g/mLTriphala 0.370 𝜇g/mL
Triphala 10.0 𝜇g/mLTriphala 3.33 𝜇g/mL
Triphala 0.123 𝜇g/mL
Figure 3: Representative photographs of the brain area (central nervous system) of the zebrafish model. The control group was incubated
with neither Triphala nor acrylamide. The model group was only exposed to 10 mM acrylamide for 24h. Glutathione (GSH) of 615 μg/mL
was applied to treat the acrylamide-induced zebrafishes together for 24 h (positive control group). Increasing concentrations (0.123, 0.370,
1.11, 3.33, and 10.0 μg/mL) of Triphala were applied to treat the acrylamide-induced fishes for 24 h at the same time (experiment group).
The in vivo neuroapoptosis is stained in green and detected with the fluorescence microscope.
7BioMed Research International
by Western blotting, demonstrating that pretreatment of Tri-
phala upregulated antioxidant enzymes (SOD1 and catalase)
and suppressed MAPK (p-Erk1/2, p-JNK1/2, and p-p38)
activation, which in turn exerted its neuroprotective effects
on inhibiting cellular apoptosis against oxidative damage.
Consistently, in vivo immunofluorescence staining evidenced
the neuroprotective role of Triphala against the neurotoxicity
induced by acrylamide in zebrafishes.
The cell viability level and proliferation rate are recog-
nized as good indicators of cell health [30]. In our study,
H
2
O
2
exposure induced apoptosis of SH-SY5Y cells by
decreasing cell viability and inhibiting cell proliferation, con-
sistent with previous reports [14, 31]. Possessing antioxidant
properties, our previous study suggested the antitumor effect
of Triphala on inhibiting the proliferation and increasing the
apoptosis in many cancer cell lines [23]. In contrast, one fruit
of Triphala (Terminalia chebula extract) was investigated by
another two studies, suggesting its neuroprotective effects on
enhancing cell viability in H
2
O
2
-treated PC12 cells [25, 26].
Our experiment first evaluated Triphala’s neuroprotective
effect on H
2
O
2
-induced SH-SY5Y cells to the best of our
knowledge. Triphala dose-dependently increased the prolif-
erative and protective rates of SH-SY5Y cells exposed to
H
2
O
2
, while Triphala markly attenuated the H
2
O
2
-induced
increase in apoptosis. Consistently, Triphala’s protective
activity is similar to the neuroprotective peptide Orexin-A
in increased proliferation in SH-SY5Y cells induced by
H
2
O
2
[14]. In general, the findings overall indicated that Tri-
phala plays a neuroprotective role in alleviating the H
2
O
2
-
induced neurotoxicity.
Notably, based on immunofluorescence staining results,
Triphala was evidenced as a comparable neuroprotective
potent as GSH, decreasing the acrylamide-induced toxicity
in the central neural system of zebrafish. In line with the pre-
vious reports, our study confirmed that acrylamide could
contribute to neuroapoptosis [32] and promote the free rad-
icals in zebrafishes. GSH is determined as a known antioxi-
dant that plays an essential role in balancing the oxidative
stress in brain cells [32]. Besides, reduced GSH has been
implicated in many neurological diseases, such as Parkin-
son’s disease [33], Alzheimer’s disease [34], epilepsy [35],
and Huntington’s disease [36]. In consistant with these
reports, GSH application in the current study inhibited the
neuroapoptosis and production of free radicals in zebrafish
exposed to acrylamide. Similarly, Triphala possessed equiva-
lent neuroprotective and antioxidant effects as GSH. Our
findings first demonstrated that Triphala (especially 1.11
and 3.33 μg/mL) could decline neuroinjury and scavenge
the free radicals in the zebrafish central neural system, exert-
ing a critical neuroprotective property against acrylamide-
induced oxidative stress.
Additionally, Triphala’s underlying antioxidant mecha-
nisms were also determined in a dose-dependent experiment
by Western blotting through measuring neuroprotective-
related proteins’expressions. Two major cellular antioxidant
enzymes, SOD1 and catalase, can scavenge the ROS products
to control the antioxidant system [37]. Recent studies
reported that Triphala effectively attenuated oxidative stress
via SOD1/catalase restoration both in vitro and in vivo [38].
For instance, Triphala extracts were able to quench free rad-
icals by inducing SOD and catalase against bacteria [39].
Meanwhile, Triphala could increase the SOD and catalase
expressions in the selenite-induced cataract model [40]. Fur-
thermore, due to its antioxidant potential, Triphala restored
the levels of SOD and CAT in a rat model of colitis [41]
and arthritis [42]. In line with the above evidence, our finding
Triphala suppressed acrylamide-induced
neurotoxicity in zebrafish model
Tri pha la (𝜇g/mL)
Control
Model
GSH 615 𝜇g/mL
0.123
0.370
1.11
3.33
10.0
Fluorescence intensity (pixels)
5×1006
1×1007
2×1007
⁎⁎⁎
⁎
⁎
(a)
Triphala scavenged free radicals in zebrafish
model exposed to acrylamide
Tri pha la (𝜇g/mL)
Control
Model
GSH 615 𝜇g/mL
0.123
0.370
1.11
3.33
10.0
Fluorescence value
1000
0
2000
3000
⁎
⁎
⁎⁎⁎
⁎
(b)
Figure 4: Effects of Triphala and acrylamide on zebrafishes in vivo. (a) The neuroprotective effects of Triphala in vivo were evaluated in the
zebrafish model injured by acrylamide, according to the fluorescence intensity. Increasing concentrations (0.123, 0.370, 1.11, 3.33, and
10.0 μg/mL) of Triphala, as well as 615 μg/mL glutathione (GSH), were applied to treat the acrylamide-induced zebrafishes together for
24 h. (b) The antioxidative effects of Triphala in vivo were evaluated in the zebrafish model exposed to acrylamide, according to the
fluorescence value. Increasing concentrations (0.123, 0.370, 1.11, 3.33, and 10.0 μg/mL) of Triphala, as well as 615 μg/mL glutathione
(GSH), were applied to treat the acrylamide-induced zebrafishes together for 20 h. Control: zebrafishes incubated with fish water; model:
zebrafishes only exposed to acrylamide. The data are expressed as mean ± SD (n=3). ∗p<0:05 as compared to control.
8 BioMed Research International
revealed that H
2
O
2
exposure decreased expressions of SOD
and catalase in SH-SY5Y cells, whereas the reductions were
enhanced by Triphala (0.37, 1.1, and 3 μg/mL) pretreatment.
Moreover, higher concentrations of Triphala tend to be more
efficient as it promoted the expression to a level even higher
than the blank control. The restoring activities of SOD and
catalase in the H
2
O
2
injury model indicated the neuroprotec-
tive role of Triphala in antioxidant processes via activating
the SOD1/catalase clearance pathway.
Furthermore, ERKs, p38 MAPKs (p38), and JNKs the
three well-known MAPK signal pathways, were investigated
in the Triphala-induced antioxidant activities [43]. Recog-
nized as the essential mediators, MAPK signaling pathways
underlining the neural diseases are attracting more and more
attention. Several studies have validated oxidative stress
results in neural damage via MAPK signal cascades [43,
44]. For example, a significant increase in the phosphoryla-
tion of ERK1/2, JNK1/2, and p38 MAPK protein was
detected in the ischemic penumbra rat model with middle
cerebral artery occlusion and reperfusion [45]. Consistent
with the previous findings, the present study has proven that
H
2
O
2
promoted apoptosis through upregulating the phos-
phorylation of p-Erk1/2, p-JNK1/2, and p-p38 in SH-SY5Y
cells. More importantly, our finding firstly demonstrated that
Triphala attenuated the levels of p-Erk1/2, p-JNK1/2, and p-
p38 in the H
2
O
2
-induced SH-SY5Y cells, indicating its anti-
oxidant and neuroprotective properties against oxidative
stress. Interestingly, in a dose-dependent experiment, unlike
the fact that 0.37 and 1.1 μg/mL Triphala significantly sup-
pressed the activation of p-Erk1/2, p-JNK1/2, and p-p38,
3μg/mL Triphala downregulated the p-JNK1/2 level. In con-
trast, it upregulated the contents of p-Erk1/2 and p-p38. As
mentioned, it is worth noting that Triphala is meanwhile
proposed to be prooxidant in promoting the apoptosis of
cancer cells by releasing ROS productions [38]. Oral admin-
istration of 50/100 mg/kg Triphala significantly induced apo-
ptosis in Capan-2 cancer cells through ROS generation,
associated with increased expression of p53 and ERK [21].
In the normal cells, ROS usually is low; however, the level
rises in the cancer cells, which crosses the threshold and
forces the cell into apoptosis [46]. Considering that SH-
SY5Y cell is a human neuroblastoma cell line, Triphala’s pro-
oxidant activity in our experiment is a possibility, being
supported by our investigation indicating that 50 and
250 μg/mL Triphala suppressed cell proliferation.
However, some limitations still existed in this study. First,
the major focus is put on the antioxidant activities of Tri-
phala, whereas, as mentioned above, the application of high
or low concentrations of Triphala is likely to exert opposite
effects on the proliferation rate of cancer cells, accounting
for its prooxidant ability in combating tumors. These obser-
vations led to the hypothesis that the pleiotropic functions of
Triphala on regulating oxidative stress may depend on vari-
ous conditions. Furthermore, as a multi-ingredient formula-
tion, Triphala consists of three fruit herbal medicines and
hundreds of components, the majority of which are polyphe-
nols and their bioactive metabolites, such as gallic acid, qui-
nic acid, teresautalic acid, chebulinic acid, corilagin, salicin,
ethyl gallate, and methyl gallate [47–50]. Therefore, one
point of further study should explore pharmacological mech-
anisms of the critical compounds with various concentra-
tions in regulating antioxidant activities using different cell
models. Second, few reports on the neuroprotective effects
of Triphala are cognitive and only major antioxidant genes
and vital signaling pathways were investigated in this study.
Thus, the identification of the compound-target gene net-
work is of great importance. Bioinformatics analysis, such
as gene ontology enrichment analysis, functional pathways
analysis, and target protein/miRNA interaction analysis,
could facilitate a better understanding of the possible ingredi-
ents, targets, and mechanisms. Third, up to now, although a
few clinical trials on Triphala have been done, such as gingivi-
tis (NCT01898000) and periodontal disease (NCT01900535),
most of the studies are done in animals and in vitro models;
thus, more in vivo and clinical studies are needed in multiple
conditions prior to its applicability.
It is important to note that this present research’sfind-
ings also have a transfer value for future clinical use.
Recently, there is an upsurge in the areas related to applying
Triphala as an alternative therapy in various diseases due to
its gifted therapeutic activity and minimal or no side effects.
Neutralizing oxidative stress is core in the treatment of both
neurodegenerative diseases and oral disorders. By that, Tri-
phala’s antioxidant and protective effects revealed in this
study will also be pertinent to examine how Triphala plays
a vital role in dentistry. For instance, Triphala was identified
as an antibacterial and anti-inflammatory agent in treating
periodontal diseases, with comparable chlorhexidine efficacy
but without any detected side effect [24]. Moreover, Triphala
could be applied as a root canal irrigant in treating primary
endodontic infections, considering the high toxicity of the
efficient irrigant (sodium hypochlorite). In addition, posses-
sing the inhibitory activity against PMN-type collagenase,
Triphala could be an alternative medicine of doxycycline in
healing periodontal destruction, without side effects. Fur-
thermore, other potential applications of Triphala in the oral
cavity are noteworthy, such as wound healing of oral mucosa
and regeneration in hard and soft tissues. Nevertheless, the
detailed molecule mechanisms under these benefits of Tri-
phala also necessitate investigations in the context of den-
tistry in well-designed preclinical studies.
5. Conclusion
In summary, the current in vitro study firstly indicated the
neuroprotective effect of Triphala on attenuating H
2
O
2
-
induced apoptosis in SH-SY5Y cells via restoration of the
antioxidant enzymes (SOD1 and catalase) and suppression
of the MAPK (p-Erk1/2, p-JNK1/2, and p-p38) activation.
Furthermore, Triphala was evidenced as a comparable
neuroprotective potent as GSH in vivo, which declined neu-
roapoptosis and scavenged free radicals in the zebrafish cen-
tral neural system, possessing a critical neuroprotective
property against acrylamide-induced oxidative stress. Con-
sequently, Triphala might be a potential therapeutic agent
to treat neurodegenerative diseases associated with oxidative
stress.
9BioMed Research International
Data Availability
The data used to support the findings of this study are avail-
able from the corresponding author upon request.
Conflicts of Interest
The authors declare that they have no conflicts of interest.
Authors’Contributions
Wanchen Ning (wanchenning0627@gmail.com) and Simin
Li (simin.li.dentist@gmail.com) are equally the co-first
authors. Prof. Dr. Xianda Hu is the senior author of this
research work. Dr. Jianlin Zhang (zhangjl2979@sina.com)
and Prof. Dr. Xianda Hu (hellocean@hotmail.com) are
equally the corresponding authors.
Acknowledgments
This work was funded by the National Natural Science Foun-
dation of China (Grant no. 81801635), which was provided
to support the research work supervised by Prof. Min Wang
at the Chinese Academy of Medical Sciences & Peking Union
Medical College (CAMS & PUMC).
References
[1] A. M. Swomley and D. A. Butterfield, “Oxidative stress in Alz-
heimer disease and mild cognitive impairment: evidence from
human data provided by redox proteomics,”Archives of Toxi-
cology, vol. 89, no. 10, pp. 1669–1680, 2015.
[2] E. Deas, N. Cremades, P. R. Angelova et al., “Alpha-synuclein
oligomers interact with metal ions to induce oxidative stress
and neuronal death in Parkinson’s disease,”Antioxidants &
Redox Signaling, vol. 24, no. 7, pp. 376–391, 2016.
[3] S. I. Mota, R. O. Costa, I. L. Ferreira et al., “Oxidative stress
involving changes in Nrf2 and ER stress in early stages of Alz-
heimer's disease,”Biochimica et Biophysica Acta (BBA) -
Molecular Basis of Disease, vol. 1852, no. 7, pp. 1428–1441,
2015.
[4] Y. Xu, Q. Wang, Z. Wu et al., “The effect of lithium chloride on
the attenuation of cognitive impairment in experimental hypo-
glycemic rats,”Brain Research Bulletin, vol. 149, pp. 168–174,
2019.
[5] Q. Wang, W. Yang, J. Zhang, Y. Zhao, and Y. Xu, “TREM2
overexpression attenuates cognitive deficits in experimental
models of vascular dementia,”Neural Plasticity, vol. 2020, 10
pages, 2020.
[6] J. Kumar, S. L. Teoh, S. Das, and P. Mahakknaukrauh, “Oxida-
tive stress in oral diseases: understanding its relation with
other systemic diseases,”Frontiers in Physiology, vol. 8,
p. 693, 2017.
[7] Y. Wang, O. Andrukhov, and X. Rausch-Fan, “Oxidative stress
and antioxidant system in periodontitis,”Frontiers in Physiol-
ogy, vol. 8, p. 910, 2017.
[8] N. Sardaro, F. Della Vella, M. A. Incalza, D. Di Stasio, and
A. Lucchese, “Oxidative stress and oral mucosal diseases: an
overview,”In Vivo, vol. 33, no. 2, pp. 289–296, 2019.
[9] B.-J. Lee, M.-Y. Chan, H.-Y. Hsiao, C.-H. Chang, L.-P. Hsu,
and P.-T. Lin, “Relationship of oxidative stress, inflammation,
and the risk of metabolic syndrome in patients with oral can-
cer,”Oxidative Medicine and Cellular Longevity, vol. 2018, 7
pages, 2018.
[10] B. Uttara, A. Singh, P. Zamboni, and R. Mahajan, “Oxidative
stress and neurodegenerative diseases: a review of upstream
and downstream antioxidant therapeutic options,”Current
Neuropharmacology, vol. 7, no. 1, pp. 65–74, 2009.
[11] Y. Xu, Q. Wang, D. Li et al., “Protective effect of lithium chlo-
ride against hypoglycemia-induced apoptosis in neuronal
PC12 cell,”Neuroscience, vol. 330, pp. 100–108, 2016.
[12] G. Aliev, M. A. Smith, D. Seyidova et al., “The role of oxidative
stress in the pathophysiology of cerebrovascular lesions in Alz-
heimer’s disease,”Brain Pathology, vol. 12, no. 1, pp. 21–35,
2002.
[13] J.-F. Pei, X.-K. Li, W.-Q. Li et al., “Diurnal oscillations of endog-
enous H
2
O
2
sustained by p66
Shc
regulate circadian clocks,”
Nature Cell Biology, vol. 21, no. 12, pp. 1553–1564, 2019.
[14] C.-M. Wang, C.-Q. Yang, B.-H. Cheng, J. Chen, and B. Bai,
“Orexin-A protects SH-SY5Y cells against H2O2-induced oxi-
dative damage via the PI3K/MEK1/2/ERK1/2 signaling path-
way,”International Journal of Immunopathology and
Pharmacology, vol. 32, 2018.
[15] Y. Li, A. Zhou, X. Cui, Y. Zhang, and J. Xie, “6′′′-p-Coumar-
oylspinosin protects PC12 neuronal cells from acrylamide-
induced oxidative stress and apoptosis,”Journal of Food Bio-
chemistry, vol. 44, no. 9, article e13321, 2020.
[16] T. Sumizawa and H. Igisu, “Apoptosis induced by acrylamide
in SH-SY5Y cells,”Archives of Toxicology, vol. 81, no. 4,
pp. 279–282, 2007.
[17] M. Faria, T. Ziv, C. Gómez-Canela et al., “Acrylamide acute
neurotoxicity in adult zebrafish,”Scientific Reports, vol. 8,
no. 1, p. 7918, 2018.
[18] C. T. Peterson, K. Denniston, and D. Chopra, “Therapeutic
uses of Triphala in Ayurvedic medicine,”The Journal of Alter-
native and Complementary Medicine, vol. 23, no. 8, pp. 607–
614, 2017.
[19] M. S. Baliga, S. Meera, B. Mathai, M. P. Rai, V. Pawar, and P. L.
Palatty, “Scientific validation of the ethnomedicinal properties
of the Ayurvedic drug Triphala: a review,”Chinese Journal of
Integrative Medicine, vol. 18, no. 12, pp. 946–954, 2012.
[20] L. H. Russell, E. Mazzio, R. B. Badisa et al., “Differential cyto-
toxicity of Triphala and its phenolic constituent gallic acid on
human prostate cancer LNCap and normal cells,”Anticancer
Research, vol. 31, no. 11, pp. 3739–3745, 2011.
[21] Y. Shi, R. P. Sahu, and S. K. Srivastava, “Triphala inhibits both
in vitro and in vivo xenograft growth of pancreatic tumor cells
by inducing apoptosis,”BMC Cancer, vol. 8, no. 1, p. 294, 2008.
[22] R. Vadde, S. Radhakrishnan, L. Reddivari, and J. K. P. Vana-
mala, “Triphala extract suppresses proliferation and induces
apoptosis in human colon cancer stem cells via suppressing
c-Myc/cyclin D1 and elevation of Bax/Bcl-2 ratio,”BioMed
Research International, vol. 2015, Article ID 649263, 12 pages,
2015.
[23] Y. Zhao, M. Wang, J. Tsering et al., “An integrated study on the
antitumor effect and mechanism of Triphala against gyneco-
logical cancers based on network pharmacological prediction
and in vitro experimental validation,”Integrative Cancer Ther-
apies, vol. 17, no. 3, pp. 894–901, 2018.
[24] S. Prakash and A. U. Shelke, “Role of Triphala in dentistry,”
Journal of Indian Society of Periodontology, vol. 18, no. 2,
pp. 132–135, 2014.
10 BioMed Research International
[25] Y.-C. Shen, C.-W. Juan, C.-S. Lin, C.-C. Chen, and C.-
L. Chang, “Neuroprotective effect of Terminalia chebula
extracts and ellagic acid in pc 12 cells,”African Journal of Tra-
ditional, Complementary and Alternative Medicines, vol. 14,
no. 4, pp. 22–30, 2017.
[26] C. L. Chang and C. S. Lin, “Phytochemical composition, anti-
oxidant activity, and neuroprotective effect of Terminalia che-
bula Retzius extracts,”Evidence-Based Complementary and
Alternative Medicine, vol. 2012, 7 pages, 2012.
[27] J. Tsering and X. Hu, “Triphala suppresses growth and migra-
tion of human gastric carcinoma cells in vitro and in a zebra-
fish xenograft model,”BioMed Research International,
vol. 2018, Article ID 7046927, 6 pages, 2018.
[28] T. H. Hutchinson, C. Bogi, M. J. Winter, and J. W. Owens,
“Benefits of the maximum tolerated dose (MTD) and maxi-
mum tolerated concentration (MTC) concept in aquatic toxi-
cology,”Aquatic Toxicology, vol. 91, no. 3, pp. 197–202, 2009.
[29] J. R. Wheeler, G. H. Panter, L. Weltje, and K. L. Thorpe, “Test
concentration setting for fish _in vivo_ endocrine screening
assays,”Chemosphere, vol. 92, no. 9, pp. 1067–1076, 2013.
[30] Ö. Aslantürk, “In vitro cytotoxicity and cell viability assays:
principles, advantages, and disadvantages,”Genotoxicity - A
Predictable Risk to Our Actual World, vol. 2, pp. 64–80, 2018.
[31] H. R. Park, H. Lee, H. Park, J. W. Jeon, W.-K. Cho, and J. Y.
Ma, “Neuroprotective effects of Liriope platyphylla extract
against hydrogen peroxide-induced cytotoxicity in human
neuroblastoma SH-SY5Y cells,”BMC Complementary and
Alternative Medicine, vol. 15, no. 1, pp. 1–11, 2015.
[32] R. Dringen and J. Hirrlinger, “Glutathione pathways in the
brain,”Biological Chemistry, vol. 384, no. 4, pp. 505–516, 2003.
[33] J. Sian, D. T. Dexter, A. J. Lees et al., “Alterations in glutathione
levels in Parkinson’s disease and other neurodegenerative dis-
orders affecting basal ganglia,”Annals of Neurology, vol. 36,
no. 3, pp. 348–355, 1994.
[34] M. Gu, A. D. Owen, S. E. K. Toffa et al., “Mitochondrial func-
tion, GSH and iron in neurodegeneration and Lewy body dis-
eases,”Journal of the Neurological Sciences, vol. 158, no. 1,
pp. 24–29, 1998.
[35] S. G. Mueller, A. H. Trabesinger, P. Boesiger, and H. G. Wieser,
“Brain glutathione levels in patients with epilepsy measured by
in vivo (1) H-MRS,”Neurology, vol. 57, no. 8, pp. 1422–1427,
2001.
[36] I. Tkac, C. D. Keene, J. Pfeuffer, W. C. Low, and R. Gruetter,
“Metabolic changes in quinolinic acid-lesioned rat striatum
detected non-invasively by in vivo (1) H NMR spectroscopy,”
Journal of Neuroscience Research, vol. 66, no. 5, pp. 891–898,
2001.
[37] T. Sugawara and P. H. Chan, “Reactive oxygen radicals and
pathogenesis of neuronal death after cerebral ischemia,”Anti-
oxidants and Redox Signaling, vol. 5, no. 5, pp. 597–607, 2003.
[38] S. Prasad and S. K. Srivastava, “Oxidative stress and cancer:
chemopreventive and therapeutic role of Triphala,”Antioxi-
dants, vol. 9, no. 1, p. 72, 2020.
[39] R. Parveen, T. N. Shamsi, G. Singh, T. Athar, and S. Fatima,
“Phytochemical analysis and in-vitro biochemical characteri-
zation of aqueous and methanolic extract of Triphala, a con-
ventional herbal remedy,”Biotechnology Reports, vol. 17,
pp. 126–136, 2018.
[40] S. K. Gupta, V. Kalaiselvan, S. Srivastava, S. S. Agrawal, and
R. Saxena, “Evaluation of anticataract potential of Triphala in
selenite-induced cataract: in vitro and in vivo studies,”Journal
of Ayurveda and Integrative Medicine, vol. 1, no. 4, pp. 280–
286, 2010.
[41] V. Rayudu and A. B. Raju, “Effect of Triphala on dextran sul-
phate sodium-induced colitis in rats,”Ayu, vol. 35, no. 3,
pp. 333–338, 2014.
[42] S. Kalaiselvan and M. K. Rasool, “The anti-inflammatory effect
of Triphala in arthritic-induced rats,”Pharmaceutical Biology,
vol. 53, no. 1, pp. 51–60, 2015.
[43] T. Wang, Y. Liao, Q. Sun et al., “Upregulation of matrix
metalloproteinase-9 in primary cultured rat astrocytes induced
by 2-chloroethanol via MAPK signal pathways,”Frontiers in
Cellular Neuroscience, vol. 11, p. 218, 2017.
[44] M. Kovalska, L. Kovalska, M. Pavlikova et al., “Intracellular
signaling MAPK pathway after cerebral ischemia–reperfusion
injury,”Neurochemical Research, vol. 37, no. 7, pp. 1568–
1577, 2012.
[45] M. Jiang, J. Li, Q. Peng et al., “Neuroprotective effects of bilo-
balide on cerebral ischemia and reperfusion injury are associ-
ated with inhibition of pro-inflammatory mediator
production and down-regulation of JNK1/2 and p 38 MAPK
activation,”Journal of Neuroinflammation, vol. 11, no. 1,
p. 167, 2014.
[46] L. Wang, R. L. de Oliveira, S. Huijberts et al., “An acquired vul-
nerability of drug-resistant melanoma with therapeutic poten-
tial,”Cell, vol. 173, no. 6, pp. 1413–1425.e14, 2018.
[47] W. Wang, T. Liu, L. Yang et al., “Study on the multi-targets
mechanism of Triphala on cardio-cerebral vascular diseases
based on network pharmacology,”Biomedicine & Pharmaco-
therapy, vol. 116, p. 108994, 2019.
[48] D. N. Olennikov, N. I. Kashchenko, and N. K. Chirikova, “In
vitro bioaccessibility, human gut microbiota metabolites and
hepatoprotective potential of chebulic ellagitannins: a case of
Padma Hepaten® formulation,”Nutrients, vol. 7, no. 10,
pp. 8456–8477, 2015.
[49] M. Wang, Y. Li, and X. Hu, “Chebulinic acid derived from Tri-
phala is a promising antitumour agent in human colorectal
carcinoma cell lines,”BMC Complementary and Alternative
Medicine, vol. 18, no. 1, pp. 1–9, 2018.
[50] B. Avula, Y.-H. Wang, M. Wang, Y.-H. Shen, and I. A. Khan,
“Simultaneous determination and characterization of tannins
and triterpene saponins from the fruits of various species of
Terminalia and Phyllantus emblica using a UHPLC-UV-MS
method: application to Triphala,”Planta Medica, vol. 79,
no. 2, pp. 181–188, 2013.
11BioMed Research International
