Exploring the Neuroprotective Potential of Triphala Against Oxidative Stress-Induced Neurotoxicity

In this report we will discuss about protective effect of triphala against oxidative and below are the summaries point:- Oxidative stress and neurotoxicity are key factors in neurological diseases, and Triphala, an Ayurvedic medicine, shows potential neuroprotective effects. The study used cell lines and zebrafish models to test Triphala's protective effects against oxidative stress-induced damage. Triphala increased cell viability and proliferation, inhibited apoptosis, and upregulated proteins associated with oxidative stress defense.

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Journal Critique: Protective Effect of Triphala against Oxidative Stress Induced
Neurotoxicity
Oxidative stress is a key pathophysiological mechanism in many neurological
diseases. When in a state of oxidative stress, the body is overwhelmed by the
excessive production of reactive oxygen species (ROS). These unstable molecules
have the potential to induce DNA damage and trigger apoptotic processes. Of the
components of ROS, it is hydrogen peroxide (H 2O2) that possesses neurotoxic effects.
As such, neuroprotective mechanisms are the hallmark of treatment for aforementioned
neurological conditions. In this regard, the neuroprotective potential of Ayurvedic
medicine has been the subject of recent efforts to explore its effectiveness in combating
oxidative stress-induced damage and neurodegenerative diseases. The research
material of this critique focuses on the neuroprotective effect of Triphala, a
well-recognized Ayurvedic concoction of indian gooseberry (Emblica officinalis), baheda
(Terminalia bellirica), and myrobalan (Terminalia chebula).
To test the protective effect of Triphala, the study utilized the neuroblastoma
SH-SY5Y cell line and zebrafish model as subjects. MTT assay was performed to test
cell viability after neurotoxicity is induced using H 2O2. The study set up the following
design: the experimental model consisted of H2O2-challenged cells administered with
Triphala, the blank model consisted of cells administered with neither Triphala or H 2O2,
while the model control consisted of purely H 2O2-challenged cells. ELISA Assay was
used to determine the anti-apoptotic activity of Triphala, while cell proliferation was
analyzed using flow cytometry. The study analyzed the expression levels of
inflammatory markers and oxidative stress-associated proteins (such as MAPK, SOD1,
etc.) using Western Blot analysis. Finally, neuroprotective and antioxidative capacity
was tested on zebrafish models and was compared with glutathione, an established
antioxidant.
The study found that groups treated with intermediate concentrations of Triphala
had increased cell viability and proliferation rates. Triphala was also able to inhibit

apoptosis in H 2O2 treated cells. Furthermore, proteins associated with oxidative stress
defense, catalase and SOD1, were found to be upregulated in cells treated with
Triphala. Downregulation of apoptosis-related proteins, such as MAPK proteins, were
also observed in the experimental model. Finally, tests using zebrafish models showed
that Triphala exhibited comparable neuroprotective and antioxidative capacity with
glutathione.
As evident from the preceding discussion, the authors used cell viability, cell
proliferation, anti-apoptotic activity, the upregulation and downregulation of proteins
associated with oxidative stress, specific neuroprotective activity, and antioxidative
capacity as parameters in assessing the neuroprotective potential of Triphala. Thus, to
accomplish this, the researchers used multiple techniques such as MTT assay, ELISA
assay, flow cytometry, and in vivo fluorescence imaging in zebrafish models. While most
of these tests are gold standards in assessing specific parameters, they are not without
limitations. For instance, the study claims that cell viability, measured by the MTT assay,
is a good indicator of cell health. However, while MTT assay measures cell viability, it
does not always mean that viable cells are healthy cells. The only qualifier for viable
cells is that it is metabolically active, as measured by the reduction of tetrazolium salt
(MTT) by mitochondrial enzymes, which forms a colored formazan product in these
assays (van Merloo et al., 2011). Furthermore, MTT assays may give a false image of
the total viable cells in a sample; non-viable cells can still retain metabolic activity. For
instance, cells undergoing apoptosis may remain metabolically active, and may be
detected by MTT assays as viable (Ghasemi et al., 2021).
Furthermore, another technique that has significant limitations is Western blot
analysis, which was used by authors to gain insight into expression levels of specific
proteins associated with oxidative stress defense and apoptosis. Western blot analysis
is only a semi-quantitative technique in that it only indirectly detects the presence of the
specific protein through primary and secondary antibodies (Mahmood and Chang Yang,
2012). Often, the results of this analysis is quantified using image analysis softwares
like ImageJ which measures the intensity of bands generated by samples loaded onto
the gel. However, because it uses antibodies to detect protein presence, Western Blot
analysis may tend to have reduced specificity due to the chances of the antibodies

cross-reacting with non-target protein. Furthermore, the identification of proteins using
Western blot analysis is only done by comparing the position of protein bands against
the molecular weight marker, rendering it a non-specific technique. Thus, the
researchers cannot confidently say that the proteins they have detected are indeed the
oxidative stress and apoptosis-related proteins they intend to detect. To do so, they
would need to perform highly specific identification techniques such as n-termini
sequencing (Deng et al., 2016).
Nonetheless, despite these limitations, the paper remains to be commendable in
its effort to map out the neuroprotective potential of Triphala. It was able to find positive
results in both in vitro and in vivo tests, suggesting the need to further investigate the
therapeutic effects of Triphala in neurological diseases.

References used
Deng, J., Zhang, G., Huang, F.-K., & Neubert, T. A. (2015). Identification of protein
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https://doi.org/10.1007/978-1-4939-2550-6_19
Ghasemi, M., Turnbull, T., Sebastian, S., & Kempson, I. (2021). The MTT assay: Utility,
limitations, pitfalls, and interpretation in bulk and single-cell analysis. International
Journal of Molecular Sciences, 22(23), 12827.
https://doi.org/10.3390/ijms222312827
Mahmood, T., & Yang, P.-C. (2012). Western blot: technique, theory, and trouble
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