Fucoxanthin Shields Vero Cells from H₂O₂-Induced Oxidative Damage by Potentially Activating the Nrf2 Signalling Pathway

Smita Kumbhar¹*, Manish Bhatia², Prafulla Choudhari², Vinod Gaikwad³, Mohini Salunke⁴, Balaji Wakure⁵

¹Department of Pharmaceutical Chemistry, Sanjivani College of Pharmaceutical Education and Research (Autonomous), Kopargaon - 423603, Maharashtra, India.

²Department of Pharmaceutical Chemistry, Bharati Vidyapeeth College of Pharmacy, Kolhapur, India.

³Department of Pharmaceutics, The National Institute of Pharmaceutical Education and Research, Hajipur, India.

⁴Department of Pharmacognosy, Vilasrao Deshmukh Foundation, Group of Institutions,

VDF School of Pharmacy, Latur - 413531, Maharashtra, India.

⁵Department of Pharmaceutics, Vilasrao Deshmukh Foundation, Group of Institutions,

VDF School of Pharmacy, Latur - 413531, Maharashtra, India.

*Corresponding Author E-mail: smitakumbhar@gmail.com

ABSTRACT:

Background: Traditional herbal medicine has long made use of fucoxanthin, an important pigment present in edible brown seaweed. It is critical for immune system support and the regulation of reactive oxygen species (ROS) metabolism, both of which are necessary for preserving human health. The purpose of this work is to investigate the protective mechanisms of fucoxanthin in Vero cells against oxidative stress and cytotoxicity induced by hydrogen peroxide (H₂O₂), with a specific focus on the Nrf2 signalling pathway and mitochondrial integrity. Methods: The sulforhodamine B (SRB) assay was used in the study to evaluate the cytotoxic effects of fucoxanthin on Vero cells. It investigated the effects of fucoxanthin on protein-related cytoprotective genes (including Nrf2), intracellular reduced glutathione, lactate dehydrogenase (LDH) leakage, ROS levels, and cell survival under H2O2-induced oxidative stress. Variations in cell viability, oxidative stress, and ROS levels were also investigated. Results: When Vero cells were treated with 2,7-dichlorodihydrofluorescein diacetate (DCFDA), fucoxanthin dramatically decreased the formation of intracellular ROS and increased cell survival. The pigment acted by blocking the mitochondrial-mediated apoptosis pathway and stimulating the Nrf2 signalling pathway to successfully alleviate H₂O₂-induced mitochondrial depolarisation and apoptosis. Fucoxanthin also seems to enhance H₂O₂ production in the culture medium, suggesting that it serves a dual purpose in enhancing cellular antioxidant responses and counteracting oxidative agents. In conclusion, this novel study shows that fucoxanthin provides Vero cells with a variety of protective and neutralising effects against oxidative stress. We verified that fucoxanthin's complex chemical composition shields human kidney cells from H2O2-induced oxidative damage through processes including Nrf2 pathway activation and mitochondrial protection using a special in vitro oxidative stress model. This study creates new opportunities to investigate the therapeutic potential of fucoxanthin in oxidative stress-related illnesses.

KEYWORDS: Fucoxanthin, ROS, SRB assay, Vero cells, Nrf2 signalling pathway.

INTRODUCTION:

Approximately 10% of all carotenoids are naturally occurring fucoxanthin, which is derived from brown seaweed such as Laminaria Japonica. This orange pigment is a component of the algal light-harvesting complex, together with chlorophylls a and c and β-carotene. Fucoxanthin, long utilised in herbal therapy to treat fever, bloating, haemorrhoids, and urinary problems, is now known to possess antioxidant, anti-apoptotic, anti-obesity, and anti-diabetic qualities. It has been demonstrated to shield Vero cells, which are fibroblasts found in humans and monkeys, from UVB and H2O2-induced damage^1-3.

Reactive oxygen species (ROS) are molecules formed from oxygen that are vital to cell signalling and homeostasis. Examples of ROS include H2O2, singlet oxygen, superoxide anions, and hydroxyl radicals. On the other hand, high ROS levels can harm or even kill cells, which is why they are linked to conditions including cancer, atherosclerosis, diabetes, inflammation, and ageing. Oxidative stress is caused by H₂O₂, in particular, which diffuses across cell membranes and interferes with mitochondrial activity. Because they may cause cancer, synthetic antioxidants like BHA and BHT are only occasionally used in food and medicine to stop oxidation^4,5.

Because of its susceptibility to oxidative stress, the Vero cell line—derived from African green monkey kidney tissue—is widely employed in biomedical research and vaccine production. A popular technique for assessing cytotoxicity and cell proliferation, the sulforhodamine B (SRB) assay attaches itself to protein residues in trichloroacetic acid-fixed cells to give an accurate assessment of the viability of cells in an oxidative environment^6–10.

The purpose of this work is to evaluate the antioxidant efficacy of fucoxanthin against H₂O₂-induced oxidative damage in Vero cells. Fucoxanthin is thought to lessen oxidative stress by blocking the mitochondrial death pathway and stimulating the Nrf2/HO-1 signalling pathway. This study provides fresh perspectives on the protective mechanisms of fucoxanthin as well as possible therapeutic uses for oxidative stress-related illnesses^11–14.

MATERIALS AND METHODS:

Materials:

Vero cells were obtained from NCCS, Pune. They are generated from renal fibroblasts of African green monkeys. The supplier of fucoxanthin was Sristi Bio Science Pvt. Ltd. in Hyderabad, Telangana. We bought assay kits for measuring glutathione and LDH activity from Clementia Biotech in New Delhi.

Cell Culture and Treatment:

The Vero cells were planted with 1 mL of DMEM media supplemented with 10% FBS (HIMEDIA-RM10432) and 1% antibiotic solution in six-well plates, with a density of 50,000 to 100,000 cells per well. The plates were incubated with 5% CO₂ at 37°C for a duration of 24 hours. The medium was changed after the first incubation, and the cells were exposed to the test drug for an additional 24 hours at a concentration of 115 µg/ml.

Evaluating the Impact of Fucoxanthin on Vero Cell Viability:

Cell Viability Assessment: The SRB assay, which gauges a substance's cytotoxicity, was used to ascertain the impact of fucoxanthin on Vero cell viability¹⁵. In a 96-well plate, Vero cells were seeded at a density of 8,000 cells per well in DMEM media (HIMEDIA-AT149-1L), with 1% antibiotic solution and 10% FBS (HIMEDIA-RM10432-500M) added as supplements. For twenty-four hours, the plates were incubated at 37°C with 5% CO2.

Treatment and Fixation: Different amounts of fucoxanthin (1–1000µM) produced in an incomplete medium were applied to the cells following the initial incubation. To fix the cells, 100µL of 10% trichloroacetic acid (TCA, Fisher Scientific-28444) was applied to each well after a further 24hours of incubation. After that, distilled water was used to rinse the plate and it was left to air dry.at room temperature.

Staining and Measuring: The cells were then stained by adding 0.04% SRB solution (Ottokemi-3520-42-1) to each well and incubating the plate for an hour. After removing the unbound dye, the wells were cleaned with 1% (v/v) acetic acid (SRL Chem-64-19-7) and allowed to air dry. A tris base solution (pH 10.5) was added to each well in order to solubilise the protein-bound dye, and the plate was shaken on an orbital shaker for ten minutes. 510nm was the absorbance obtained with an ELISA plate reader (iMark, Biorad, USA).

Detection of intracellular ROS by Flow Cytometry:

Using the DCFDA test, the amounts of intracellular ROS generated by H₂O₂ were determined. Fucoxanthin (115µg/ml) was added to Vero cells and cultured for a full day. The liquid was removed after incubation, and trypsin-EDTA was used to collect the cells. After that, the cells were put into 1.5 ml tubes, cleaned with 500µl of cold PBS, and the pellet that was left over was put back into 100 µl of PBS with 2µM DCFDA. The samples were examined right away with a flow cytometer (BD FACS Calibur, USA) to check for the generation of ROS.

Lactate Dehydrogenase (LDH) Leakage Assay:

After being seeded at a density of 1×10⁵ cells/mL in 96-well plates, vero cells were incubated for a full day. After that, they received either 50µM fucoxanthin or 50 µM vitamin E for two hours. Subsequently, 200µM H2O2 was introduced into every well, and the cells underwent a 24hour incubation period at 37°C. The samples were incubated at 25°C after the reaction solution from the LDH test kit was added. A microplate reader (PowerWave XS, Bio-Tek, Winooski, VT, USA) was used to quantify LDH activity at 450nm. The percentage of LDH leakage was calculated in relation to the maximal LDH activity of the control group¹⁷.

Detection of Intracellular Reduced GSH:

After plating 1×10⁵ cells/mL in 96-well plates, the Vero cells were cultured for a full day. After that, cells were treated to either vitamin E or 50µM fucoxanthin for two hours. After treatment, the cells were incubated for a further 24hours at 37°C with the addition of 200µM H2O2. The cells were then homogenised with an ultrasonic instrument, trypsinised, and cleaned with PBS. Using a glutathione determination kit (Clementia Biotech, New Delhi) and the manufacturer's instructions, intracellular GSH levels were determined. Using an excitation wavelength of 350nm, absorbance at 420nm was measured using a fluorescent microplate reader (Spectra Max M5, MD, CA, USA) to determine the GSH concentration¹⁸.

RESULTS AND DISCUSSION:

Fucoxanthin was applied to Vero cells at doses ranging from 1 to 1000µM, whereas solvent alone was applied to the control group. The SRB experiment showed a dose-dependent reduction in cell viability, with 1000µM showing the greatest inhibition and strong cytotoxicity. Fucoxanthin's cytotoxic effects are probably influenced by its high local concentration close to the cell membrane. Fucoxanthin's strong cytotoxicity was demonstrated by the 351.9µM IC50 it discovered in Vero cells¹⁹.

Furthermore, ROS levels were evaluated by SRB staining, which demonstrated that fucoxanthin successfully decreased ROS production. This reduction was associated with a decrease in comet tail formation and phospho-histone H2A.X expression, indicating protection against DNA damage. Additionally, as demonstrated by a decrease in apoptotic bodies labelled with DCFDA, fucoxanthin attenuated the apoptosis generated by hydrogen peroxide. The potential of the mitochondrial membrane was maintained, while apoptotic indicators like Bax, caspase-9, and caspase-3 were reduced.

These findings highlight the protective function of fucoxanthin against oxidative stress, which increases cell lifespan by scavenging reactive oxygen species and preventing apoptosis. Figure 1: SRB Assay of Cell Viability

Figure 2: Cytoprotective Effects of Fucoxanthin on Vero Cells Against H₂O₂-Induced Oxidative Stress.

ROS Estimation with Flow Cytometry -Vero Cell line:

The fluorescent dye 2,7-dichlorodihydrofluorescein diacetate (DCFDA) was used to measure the ROS-scavenging activity of fucoxanthin in Vero cells treated with H₂O₂ (Figure 3) ^20–22. According to the SRB experiment, fucoxanthin demonstrated considerable intracellular ROS scavenging activity at the following concentrations: 97.14% at 1µM, 84.96% at 10µM, 73.51% at 50µM, 58.71% at 100µM, 54.89% at 250µM, 49.16% at 500µM, and 47.49% at 1000µM.

Interestingly, the SRB assay results (Figure 4) demonstrated that fucoxanthin alone did not exhibit toxicity to Vero cells at concentrations up to 100 µM. This finding underscores fucoxanthin's potential as a protective agent, capable of significantly reducing ROS levels without causing harm to the cells at therapeutic concentrations.

The study’s novel insights reveal that fucoxanthin's ROS-scavenging efficiency decreases with increasing concentration, which suggests a saturation effect at higher doses. Moreover, the lack of toxicity at 100 µM indicates a favorable therapeutic window for fucoxanthin, highlighting its potential for applications where oxidative stress mitigation is crucial. This makes fucoxanthin a promising candidate for further research into its protective mechanisms and potential therapeutic uses in oxidative stress-related conditions.

Effects of Fucoxanthin on H₂O₂-Induced LDH Leakage in Vero Cells:

LDH leakage in Vero cells exposed to H2O2-induced oxidative stress was assessed in order to evaluate the protective effects of fucoxanthin. As shown in Figure 5, the highest LDH leakage group's leakage level was standardised to 100%. When compared to the control group, which had an LDH leakage rate of 16.71±4.48% (P<0.01), the model group, which had received H₂O₂, had an LDH leakage rate of 28.45±5.13%.

Among the treated groups, the F5 group demonstrated the most substantial reduction in LDH leakage (P< 0.05). The group treated with vitamin E (VE) also showed a significant decrease in LDH leakage, with a rate of 18.70±4.98% (P<0.05) compared to the model group. This data suggests that both fucoxanthin and vitamin E effectively mitigate cellular damage induced by oxidative stress, with fucoxanthin showing a particularly notable protective effect.

Effects of Fucoxanthin on Intracellular GSH Content in H₂O₂-Treated Vero Cells:

Figure 6 illustrates that the intracellular glutathione (GSH) content in the control group was set at 100%. In the model group treated with H₂O₂, GSH content decreased significantly to 62.29±6.92% compared to the control (P<0.01). Treatment with vitamin E (50µM) increased GSH levels to 106.94±5.70% relative to the model group (P<0.01). Fucoxanthin pre-treatment at various concentrations yielded GSH contents of 110.69 ±4.39%, 120.98±6.72%, 103.97±7.12%, and 96.05± 5.59%, respectively, demonstrating a significant improvement compared to the model group (P<0.01). Fucoxanthin effectively mitigated H₂O₂-induced cytotoxicity in Vero cells. The SRB assay confirmed that fucoxanthin pre-treatment (at 1, 10, 50, 100, 250, 500, and 1000µM) provided significant protection against oxidative damage induced by H₂O₂, with 1000 µM of fucoxanthin showing the highest cell survival rates (Figure 1). This concentration was therefore used in subsequent experiments.

Moreover, fucoxanthin pre-treatment notably reduced levels of procaspase-3 protein, indicating that fucoxanthin can inhibit apoptosis caused by oxidative stress. The SRB assay further validated fucoxanthin's cytoprotective effects against oxidative damage.

Additional analyses revealed that fucoxanthin treatment effectively reversed the rise in ROS levels caused by H₂O₂, as demonstrated by the DCFDA-based ROS generation assay. Furthermore, measurements of mitochondrial membrane potential showed that fucoxanthin preserved membrane integrity, with treated cells exhibiting fluorescence intensities comparable to untreated controls, unlike the significant reduction observed in H₂O₂-treated cells. This highlights fucoxanthin's ability to protect against mitochondrial dysfunction induced by oxidative stress.

Figure 5: Impact of Fucoxanthin on LDH Leakage Induced by H2O2 in Vero Cells

Figure 6: Impact of Fucoxanthin on Intracellular GSH Levels in H2O2-Treated Vero Cells

Oxidative stress is linked to a number of health problems, such as ageing, cancer, metabolic disorders, cardiovascular illnesses, and inflammation. It arises from an imbalance between the generation of reactive oxygen species (ROS) and antioxidant defence. Strong antioxidant fucoxanthin is well known for its capacity to break down singlet oxygen and neutralise free radicals. The effectiveness of fucoxanthin in reducing H₂O₂-induced oxidative damage in Vero cells was examined in this work. According to our research, fucoxanthin efficiently quenched hydroxyl radicals in a cell-free Fenton reaction system in addition to scavenging intracellular ROS in Vero cells treated with H₂O₂^23,24.

Dietary phenols, such as fucoxanthin, reduce oxidative stress by scavenging ROS and activating the Nrf2 pathway, a key regulator of redox homeostasis in eukaryotic cells. The highly sensitive 2,7-dichlorofluorescein (DCF) assay, which detects ROS by converting 2,7-dichlorodihydrofluorescein (DCFH) into a fluorescent product, demonstrated increased intracellular ROS levels following H₂O₂ exposure, leading to heightened oxidative stress. However, fucoxanthin showed significant antioxidant and protective effects, as evidenced by SRB and DCF-DA assays.

Previous research highlights DNA as a major target of ROS-induced oxidative stress, with severe ROS damage leading to apoptosis. Our SRB assay results indicate that fucoxanthin protects cellular DNA from oxidative damage, potentially preventing apoptosis. This protection is further supported by evidence of fucoxanthin's role in maintaining mitochondrial membrane integrity and inhibiting the mitochondrial apoptotic pathway, which involves Bcl-2 family proteins and caspase signaling²⁵.

Additionally, fucoxanthin significantly reduced H₂O₂-induced intracellular LDH leakage, suggesting it helps preserve cell membrane integrity. This protective effect could be attributed to fucoxanthin's two hydroxyl groups, which are linked to reduced ROS production. Pre-treatment with fucoxanthin also reversed H₂O₂-induced reductions in intracellular GSH levels, outperforming VE at 50µM. Fucoxanthin’s antioxidant activity is likely enhanced by its hydroxyl group, epoxide group, and allenic link.

Overall, fucoxanthin demonstrated high efficacy in mitigating oxidative stress without notable adverse effects, supporting its potential as a therapeutic agent to protect against oxidative damage and related skin issues.

CONCLUSION:

Fucoxanthin's strong cytotoxic effects and enhanced cellular viability make it a highly effective combatant of reactive oxygen species (ROS). Apart from its antioxidant characteristics, fucoxanthin also gains from testing technology developments, which makes the SRB assay a perfect instrument for evaluating drug-induced cytotoxicity, especially in large-scale applications.

In our work, we treated the medium with fucoxanthin after adding hydrogen peroxide (H₂O₂) to cause oxidative stress. Intracellular ROS increased with the addition of H₂O₂, but were significantly decreased by fucoxanthin therapy. Notably, in the setting of H₂O₂-induced oxidative stress, fucoxanthin demonstrated a dose-dependent reduction in cell damage and a marked improvement in cell viability.

Our findings suggest that fucoxanthin offers protective benefits to Vero cells against H₂O₂-induced oxidative damage, potentially through the activation of the Nrf2 signaling pathway, with a mechanism possibly involving PI3K-dependent pathways.

CONSENT FOR PUBLICATION:

The authors have approved Consent for publication.

COMPETING INTERESTS:

The authors have no competing interests to declare that are relevant to the content of this article.

REFERENCE:

1. Suja C. Shuhaib B. Muhammed Abdurahman. Hunaida Khathoom. Simi K. Review on Dietary Antioxidants. Research Journal of Pharmacy and Technology. 2016; 9(2): 196-202. doi:10.5958/0974-360X.2016.00035.4

2. Din NAS. Mohd Alayudin S. Sofian-Seng NS. et al. Brown Algae as Functional Food Source of Fucoxanthin: Review. Foods. 2022; 11(15): 2235. doi:10.3390/foods11152235

3. Zheng J. Piao MJ. Keum YS. Kim HS. Hyun JW. Fucoxanthin Protects Human Keratinocytes Against Oxidative Stress by Blocking Free Radicals and Inhibiting Apoptosis. Biomolecules and Therapeutics (Seoul). 2013; 21(4): 270-276. doi:10.4062/biomolther.2013.030

4. Nita M. Grzybowski A. Role of Reactive Oxygen Species and Oxidative Stress in Age-Related Ocular Diseases and Other Pathologies of Eye Segments. Oxidative Medicine and Cellular Longevity. 2016; 2016:3164734. doi:10.1155/2016/3164734

5. Jang EJ. Kim SC. Lee JH. et al. Fucoxanthin, a constituent of Laminaria japonica, triggers AMPK-mediated cytoprotection and autophagy in hepatocytes under oxidative stress. BMC Complementary and Alternative Medicine. 2018; 18(1): 97. doi:10.1186/s12906-018-2164-2

6. Rodríguez-Luna A. Ávila-Román J. Oliveira H. Motilva V. Talero E. Fucoxanthin and Rosmarinic Acid Combination Has Anti-Inflammatory Effects through Regulation of NLRP3 Inflammasome in UVB-Exposed HaCaT Keratinocytes. Marine Drugs. 2019; 17(8):451. doi:10.3390/md17080451

7. Remya R. Radhika Rajasree S. A Study on Bioactive Compounds Derived from Brown Seaweeds and Their Therapeutic Applications Towards Various Diseases. Research Journal of Pharmacy and Technology. 2016; 9(4): 369-372. doi:10.5958/0974-360X.2016.00066.4

8. Zhang Y.L. Chen G.L. Liu Y. Zhuang X.C. Guo M.Q. Stimulation of ROS Generation by Extract of Warburgia ugandensis Leading to G0/G1 Cell Cycle Arrest and Antiproliferation in A549 Cells. Antioxidants. 2021; 10(10): 1559. doi:10.3390/antiox10101559

9. Seo MJ. Seo YJ. Pan CH. Lee OH. Kim KJ. Lee BY. Fucoxanthin Suppresses Lipid Accumulation and ROS Production During Differentiation in 3T3-L1 Adipocytes. Phytotherapy Research. 2016; 30(11): 1802-1808. doi:10.1002/ptr.5683

10. Foo SC. Yusoff FM. Ismail M. et al. Antioxidant Capacities of Fucoxanthin-Producing Algae as Influenced by Their Carotenoid and Phenolic Contents. Journal of Biotechnology. 2017; 241: 175-183. doi:10.1016/j.jbiotec.2016.11.026

11. Ferdous KA. Burnett G. Scott M. Amjad E. Bannerman S. Park HA. Neuroprotective Function of Fucoxanthin in Oxidative Stress-Mediated Mitochondrial Dysfunction. Current Developments in Nutrition. 2022; 6(Suppl 1): 787. doi:10.1093/cdn/nzac064.006

12. Kim S. Kim M. Kang MC. et al. Antioxidant Effects of Turmeric Leaf Extract Against Hydrogen Peroxide-Induced Oxidative Stress In Vitro in Vero Cells and In Vivo in Zebrafish. Antioxidants (Basel). 2021; 10(1): 112. doi:10.3390/antiox10010112

13. Kirindage KGIS. Fernando IPS. Jayasinghe AMK. et al. Moringa oleifera Hot Water Extract Protects Vero Cells from Hydrogen Peroxide-Induced Oxidative Stress by Regulating Mitochondria-Mediated Apoptotic Pathway and Nrf2/HO-1 Signaling. Foods. 2022; 11(3): 420. doi:10.3390/foods11030420

14. Vijayraja D. Jeyaprakash K. Preliminary Phytochemical Analysis, In Vitro Antioxidant and Anti-Inflammatory Activity of Turbinaria ornata (Turner) J. Agardh. Research Journal of Pharmacy and Technology. 2017; 10(7): 2243-2248. doi:10.5958/0974-360X.2017.00397.3

15. Lourenço-Lopes C. Fraga-Corral M. Jimenez-Lopez C. Carpena M. Pereira AG. Garcia-Oliveira P. Prieto MA. Simal-Gandara J. Biological Action Mechanisms of Fucoxanthin Extracted from Algae for Application in Food and Cosmetic Industries. Trends in Food Science and Technology. 2021; 117:163-181. doi:10.1016/j.tifs.2021.03.012

16. Adan A. Kiraz Y. Baran Y. Cell Proliferation and Cytotoxicity Assays. Current Pharmaceutical Biotechnology. 2016; 17(14): 1213-1221. doi:10.2174/1389201017666160808160513

17. Zhou Y. Zhu L. Li H. et al. In Vivo and In Vitro Neuroprotective Effects of Maca Polysaccharide. Frontiers in Bioscience (Landmark Edition). 2022; 27(1): 8. doi:10.31083/j.fbl2701008

18. Selvam R. Anandhi D. Saravanan D. Revathi K. Antioxidant Properties of Punica granatum Fruit Rind Extract Against Liver Hepatocellular Carcinoma Studied in HEPG2 Cell Line. Research Journal of Pharmacy and Technology. 2019; 12(10): 4719-4723. doi:10.5958/0974-360X.2019.00813.8

19. Hansel CM. Diaz JM. Production of Extracellular Reactive Oxygen Species by Marine Biota. Annual Review of Marine Science. 2021; 13:177-200. doi:10.1146/annurev-marine-041320-102550

20. Maecker HT. McCoy JP. Nussenblatt R. Standardizing Immunophenotyping for the Human Immunology Project. Nature Reviews Immunology. 2012; 12(3):191-200. doi:10.1038/nri3158

21. Sutariya S. Pansuriya V. Sharma M. Bajpai AB. Gupta A. Antimicrobial, Antioxidant and Anti-Inflammatory Potential of Flavonoid and Terpenoid from Gymnosporia montana. Research Journal of Pharmacy and Technology. 2023; 16(4): 1945-1950. doi:10.52711/0974-360X.2023.00319

22. Surh YJ. Kundu JK. Na HK. Nrf2 as a Master Redox Switch in Turning on the Cellular Signaling Involved in the Induction of Cytoprotective Genes by Some Chemopreventive Phytochemicals. Planta Medica. 2008; 74(13): 1526-1539. doi:10.1055/s-0028-1088302

23. Giudice A. Arra C. Turco MC. Review of Molecular Mechanisms Involved in the Activation of the Nrf2-ARE Signaling Pathway by Chemopreventive Agents. Methods in Molecular Biology. 2010; 647: 37-74. doi:10.1007/978-1-60761-738-9_3

24. Maecker HT. McCoy JP. Nussenblatt R. Standardizing Immunophenotyping for the Human Immunology Project. Nature Reviews Immunology. 2012; 12(3): 191-200. doi:10.1038/nri3158

25. Kirindage KGIS. Fernando IPS. Jayasinghe AMK. et al. Moringa oleifera Hot Water Extract Protects Vero Cells from Hydrogen Peroxide-Induced Oxidative Stress by Regulating Mitochondria-Mediated Apoptotic Pathway and Nrf2/HO-1 Signaling. Foods. 2022; 11(3): 420. doi:10.3390/foods11030420

Received on 22.02.2024 Revised on 12.07.2024

Accepted on 20.10.2024 Published on 27.03.2025

Available online from March 27, 2025

Research J. Pharmacy and Technology. 2025;18(3):1140-1146.

DOI: 10.52711/0974-360X.2025.00164

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.