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ORIGINAL ARTICLE
Year : 2019  |  Volume : 9  |  Issue : 4  |  Page : 156-164

Preventive Effect of Opuntiol, Isolated from Opuntia ficus indica (L. Mill), Extract Against Ultraviolet A Radiation-Induced Oxidative Damages in NIH/3T3 Cells


1 Department of Biochemistry & Biotechnology, Annamalai University, Annamalainagar 608002, Tamil Nadu, India
2 Department of Biochemistry, Dharumapurm Gnanambigai Government Arts College for Women, Mayiladuthurai 609001, Tamil Nadu, India

Date of Submission14-Oct-2019
Date of Decision20-Oct-2019
Date of Acceptance05-Nov-2019
Date of Web Publication28-Nov-2019

Correspondence Address:
Nagarajan Rajendra Prasad
Department of Biochemistry & Biotechnology, Annamalai University, Annamalainagar 608 002, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijnpnd.ijnpnd_69_19

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   Abstract 


Aim: We investigated the role of opuntiol, isolated from Opuntia ficus indica, against ultraviolet A waveband-mediated oxidative damages in the mouse embryonic fibroblast cell lines (NIH‐3T3). Materials and Methods: The antioxidant potential of opuntiol was carried out by hydroxyl radical, superoxide anion and DPPH radical scavenging assays. The preventive effect of opuntiol against UV-mediated cytotoxicity was revealed by MTT assay. Further, the oxidative end points during UV-exposure, in the presence and absence of opuntiol, was analyzed by DCFH-DA staining, rhodamine-123 staining and alkaline comet assay. Results: Opuntiol significantly neutralizes hydroxyl (OH), superoxide anion (O2•–), hydrogen peroxide (H2O2), and 2,2-diphenyl-2-picrylhydrazyl (DPPH•) radicals in a concentration-dependent manner. In this study, the NIH-3T3 cells were treated with UVA-waveband in the presence and absence of opuntiol and oxidative damage markers were analyzed. We observed that opuntiol pretreatment (5 μM-20 μM) prevented 10 mJ/cm2UVA radiation-induced cytotoxicity in NIH-3T3 cells. Further, single UVA-radiation induces reactive oxygen species (ROS) through intracellular photosensitizers. Conversely, opuntiol pretreatment prevented UVA-mediated ROS generation and subsequent lipid peroxidation and loss of enzymatic antioxidants (superoxide dismutase [SOD], catalase, and glutathione peroxidase) in the NIH-3T3 cells. It has also been observed that the UVA-mediated ROS subsequently induces DNA damage and alters mitochondrial transmembrane potential (MMP). We noticed that opuntiol prevents UVA-radiation-mediated DNA single-strand breaks. Further, it prevents loss of MMPs and apoptotic morphological changes in the NIH-3T3 cells. Conclusion: Thus, these findings illustrate that opuntiol prevents UVA-radiation-mediated oxidative stress-related biochemical changes in the cellular system.

Keywords: Antioxidants, DNA damage, opuntiol, oxidative stress, ultraviolet-A radiation


How to cite this article:
Ponniresan Vk, Maqbool I, Thangaiyan R, Govindasamy K, Prasad NR. Preventive Effect of Opuntiol, Isolated from Opuntia ficus indica (L. Mill), Extract Against Ultraviolet A Radiation-Induced Oxidative Damages in NIH/3T3 Cells. Int J Nutr Pharmacol Neurol Dis 2019;9:156-64

How to cite this URL:
Ponniresan Vk, Maqbool I, Thangaiyan R, Govindasamy K, Prasad NR. Preventive Effect of Opuntiol, Isolated from Opuntia ficus indica (L. Mill), Extract Against Ultraviolet A Radiation-Induced Oxidative Damages in NIH/3T3 Cells. Int J Nutr Pharmacol Neurol Dis [serial online] 2019 [cited 2019 Dec 6];9:156-64. Available from: http://www.ijnpnd.com/text.asp?2019/9/4/156/271857




   Introduction Top


The sun emits different wavebands of ultraviolet radiation. Among different wavebands, the ultraviolet-A waveband (320–400 nm) has been linked with oxidative damages. The cellular molecules like porphyrins, cytochromes, flavins, etc., absorb UVA-waveband and generate reactive oxygen species (ROS) and singlet oxygen through Joblonski reactions[1]. The UVA-radiation-induced ROS causes several oxidative damage related events in the cellular system and ultimately induces premature photoaging in the human skin. Therefore, the UVA-radiation has generally been named as “aging rays”.[2] Hence, it is important to identify potential countermeasures against UVA-radiation-mediated oxidative damages.

Exogenous antioxidants have been used to prevent UVA-induced photodamage.[3] Several studies illustrate that plant-derived phytochemicals can absorb UVA photons and possess the potential to modulate UVA-mediated oxidative damages. Further, phytochemicals work at the molecular level and prevent UVA-mediated stress-activated signaling events.[4] Cactus Opuntia ficus indica (Opuntiol) is a family of Cactaceae present in the dry and desert area, which is a livestock for arid and semiarid regions of the earth[5]. Opuntiol is one of the active compounds from O. ficus indica and it belongs to the group of flavonoids in nature. We recently isolated and characterized opuntiol from O. ficus indica [data not shown]. O. ficus indica receive high levels of UV ambiance and hence they might have developed defenses against the detrimental effects of UV light due to the presence of antioxidants with strong UV-absorbing properties.[6],[7] Opuntiol has already been reported as a major flavonoid in O. ficus indica.[8] As O. ficus indica is a desert plant and survives extreme sunlight environment we hypothesize that the major active constituent of this plant opuntiol might protect human cells from UVA-radiation effects. Therefore, in this study, we investigated the effect of opuntiol against UVA-radiation-induced oxidative stress and DNA damage in NIH‐3T3 cells.


   Materials and methods Top


Chemicals

Phenazine methosulfate (PMS), 2,2-diphenyl- 2-picrylhydrazyl (DPPH•), Thiobarbituric acid reactive substances (TBARS), nitroblue tetrazolium (NBT), 5,5-ditiobis 2-nitrobenzoic acid (DTNB), 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT), 2,7-diacetyl dichlorofluorescein (DCFH-DA), rhodamine 123, low melting agarose (LMPA), normal melting agarose (NMPA), phosphate-buffered saline (PBS) were purchased from Sigma Chemicals Co., St. Louis, USA.

Free radical scavenging methods

Hydroxyl radical scavenging activity was measured by studying the competition between deoxyribose and opuntiol by Fe3+–Ascorbate–EDTA–H2O2 system according to the method described by Elizabeth and Rao.[9] The formation of hydroxyl radicals was measured by the degradation of deoxyribose which on treating with TBA forms a pink-colored product. The absorbance of the product resulted from the formation of hydroxyl radical was read in a spectrophotometer at 535 nm. Superoxide anion (O2•–) scavenging activity of opuntiol was determined by the method of Liu et al.[10] Superoxide anion formed from using PMS/NADH coupling reaction reduces NBT and reaction endpoints were measured at 560 nm. The DPPH is a stable radical that accepts an electron to become a stable molecule. The effect of opuntiol on DPPH radical scavenging was assayed using the method of Williams et al.[11] Decreased absorbance of the reaction mixture in all the assays indicated increased radical scavenging activity. The percent of scavenging or inhibition was calculated according to the following formula, the efficiency of opuntiol was compared with vitamin C as standard:

% of scavenging or inhibition = [(Absorbance control − Absorbance sample) / Abs control] ×100

Cell lines and conditions

This work was conducted in a mouse embryonic cell line (NIH/3T3). The NIH/3T3 cells were procured from National Centre for Cell Science (NCCS), Pune, India, NIH‐3T3 cells were cultured at 37°C in 5% CO2 in DMEM medium supplemented with 10% fetal bovine serum, 1 µg/mL hydrocortisone, 10 mg/mL human epidermal growth factor, 10 µg/mL heparin, and antibiotics. The cells were treated as follows:
  • Group I: Control cells
  • Group II: Opuntiol (20 μM) alone treated cells
  • Group III: UVA (10 J/cm2) alone irradiated cells
  • Group IV: Opuntiol (20 μM) pretreated and UVA-irradiated cells


UVA-irradiation procedure

Cells were seeded in six well plates at a density of 2×105 cells per well and cultured for 24 h. UVA-irradiation (10 J/cm2) was performed as described previously.[12] A fresh medium was added after irradiation and all the experiments were carried out after 24 h incubation unless otherwise mentioned.

Cell viability by MTT assay

The preventive effect of opuntiol against UVA-induced cytotoxicity was assessed by MTT assay as described previously.[13],[12] To understand the preventive effect of opuntiol against UVA-induced cytotoxicity, the NIH/3T3 cells were incubated with the selected concentrations of opuntiol (5–50 µM) for 30 min before each UVA exposure. Then, the cells were cultured for another 24 h. Then, 100 µL of MTT solution was added and the incubation was extended for another 4 h. Finally, 100 µL of DMSO was added and the absorbance was measured using a multimode plate reader (Tecan, Austria) at 570 nm.

Quantification of intracellular ROS

The intracellular ROS levels were measured by DCFH-DA method as described previously.[14],[15] Opuntiol pretreated and/or UVA-treated fibroblasts in six well plates were incubated for 15 min with 10 µM DCFH-DA in PBS, washed three times with PBS. Fluorescence was determined at 488/525 nm by spectrofluorometer.

Changes in mitochondrial transmembrane potential (Δψm)

Mitochondrial membrane potential (MMP) was analyzed using rhodamine-123 fluorescent staining.[16],[12] Mouse embryonic fibroblast cell lines (NIH‐3T3) (1×106 cells/well) were seeded in were well plates and treated with various concentrations (5–50 µM) of opuntiol as per the experimental procedure. After the treatment, cells were incubated with rhodamine-123 (1 mg/mL) for 30 min. The cells were then washed with PBS and observed under a fluorescence microscope (450–490 nm). Images were acquired with a fluorescence microscope (Life technologies, USA).

Biochemical estimations

The cells were harvested by trypsinization, and the cell pellet obtained was suspended in PBS. The suspension was taken for lipid peroxidative markers and antioxidants analysis. The lipid peroxidation level was estimated by analyzing thiobarbituric acid reactive substances (TBARS) by reacting with thiobarbituric acid in an acidic condition to generate a pink color chromophore, which was read at 535 nm.[17] SOD activity was assayed.[18] Catalase activity was assayed,[19] by quantifying the amount of hydrogen peroxide after the reaction with dichromate in the presence of acetic acid. The activity of glutathione peroxidase was assayed by the method described by Rotuk et al.[20] The GSH content was measured according to the method of Ellman.[21]

Apoptotic morphological changes by acridine orange–ethidium bromide dual staining

Ethidium bromide/acridine orange (EtBr/AO) staining was carried out to detect morphological evidence of apoptosis in the Opuntiol and UVA-irradiated cells.[22],[23] The cells were labeled with a 1:1 ratio of AO (100 µg/mL) and EB (100 µg/mL) in PBS and incubated for 5 min. Then the excess dye was removed by washing with PBS. The apoptotic cells, characterized by shrinking of cells, nuclear fragmentation, brightly fluorescent, and apoptotic nuclei, were easily detected and the percentage apoptotic cells were calculated.

Alkaline single-cell gel electrophoresis

The DNA damage was estimated by alkaline single-cell gel electrophoresis (comet assay) according to the method as described by Singh et al.[24] and Kanimozhi et al.[23] A layer of 1% NMPA was prepared on microscope slides. After UVA-irradiation, NIH/3T3 cells (50 µL) were mixed with 200 µL of 0.5% LMPA. The suspension was pipetted onto the pre-coated slides. Slides were immersed in cold lysis solution (pH 10) and kept at 4°C for 60 min. Then, the slides were placed in alkaline electrophoresis buffer (pH 13) and left for 25 min. Next, slides were transferred to fresh alkaline electrophoresis buffer and electrophoresis was performed at 25 V for 25 min at 4°C. Slides were neutralized in 0.4 M Tris (pH 7.5) for 5 min and stained with 20 µg/mL ethidium bromide. The images were captured and analyzed by CASP software.

Statistical analysis

All the values were expressed as means ± SD. The data were statistically analyzed by Duncan’s multiple range test (DMRT) using a statistical package program (SPSS). p‐value (≤ 0.05) was considered statistically significant.


   Results Top


Effect of opuntiol on free radical scavenging assay

[Figure 1] shows (A) hydroxyl radical, (B) DPPH•, and (C) superoxide anion scavenging activity of opuntiol. Opuntiol inhibits the radical formation and the percentage of inhibition was observed in a concentration-dependent manner. The IC50 (inhibitory concentration 50) values of opuntiol in hydroxyl radical, DPPH and superoxide anion were found to be 0.078, 0.003, 0.625 μM, respectively and the free radical scavenging property was comparable to standard vitamin C.
Figure 1 Effect of opuntiol on (A) hydroxyl radical scavenging (B) DPPH scavenging (B) superoxide anion scavenging activity. Values are given as mean ± S.D. of experiments in each group.

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Effect of opuntiol and/or UVA-induced cytotoxicity in NIH‐3T3 cells by MTT assay

UVA exposure significantly reduced cell viability when compared with non-irradiated cells. We observed that opuntiol treatment prevented UVA-induced loss of cell viability in a concentration-dependent manner. Further, we noticed that 20 µM of opuntiol possess a better cytoprotective effect than other concentrations studied [Figure 2]. Hence, we have chosen 20 µM of opuntiol for further photoprotective experiments.
Figure 2 Protective effects of opuntiol on UVA-induced cytotoxicity. Values are given as means ± S.D. of six experiments in each group. Values not sharing a common superscript differ significantly at p < 0.05 (DMRT).

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Effect of opuntiol on UVA-induced intracellular ROS generation in NIH‐3T3 cells

In this study, the intracellular ROS production was significantly increased in UVA-exposed cells when compared to the control cells. Opuntiol pretreatment significantly prevented the intracellular ROS production in UVA-induced NIH-3T3 cells [[Figure 3]A and 3B].
Figure 3 (A). Effect of opuntiol and/or UVA radiation on intracellular ROS generation analyzed by DCFH-DA staining. Photomicrograph shows the green fluorescence under a green lamp (original magnification, 20X). (B) Bar diagram representing percent of fluorescence intensity analyzed using a multimode reader (Teccan, Austria). Values are given as means ± S.D. of six experiments in each group. Values not sharing a common marking (a, b, c, d) differ significantly at p ≤ 0.05 (DMRT).

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Opuntiol on UVA-induced lipid peroxidation and antioxidant status

In this study, UVA-exposed cells showed a dramatic rise in the levels of TBRAS and LHP when compared to control cells. Treatment with opuntiol before UVA-irradiation significantly decreased TBARS and LHP levels [Figure 4]. Furthermore, reduced glutathione (GSH) level was decreased in UVA-induced NIH-3T3 cells. Opuntiol pretreatment significantly restored GSH levels in UVA-induced NIH-3T3 cells. Furthermore, UVA-irradiation significantly decreased the activities of SOD, CAT, and GPx in NIH-3T3 cells. Opuntiol pretreatment prevented UVA-induced loss of activities of SOD, CAT, and GPx activities in NIH-3T3 cells [Table 1].
Figure 4 Effect of opuntiol on UVA-induced TBARS level in NIH-3T3 cells. Values are given as means ± S.D. of six experiments in each group. Values not sharing a common marking (a,b,c…) differ significantly at p ≤ 0.05 (DMRT).

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Table 1 Effect of Opuntiol on UVA-induced SOD, CAT, GPx and GSH in NIH‐3T3 cells

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Opuntiol on UVA-induced MMP alteration, apoptotic changes and DNA damage in NIH-3T3 cells

UVA-irradiated cells showed decreased Rhodamine-123 fluorescence due to depolarized mitochondria. Opuntiol treatment before UVA-induction significantly prevented the loss of UVA-mediated Δψm in NIH-3T3 cells [[Figure 5]A and 5B]. Apoptotic morphological changes and nuclear condensation were analyzed by ethidium bromide/acridine orange (EB/AO) staining. UVA-exposure caused significant apoptotic morphological changes in NIH-3T3 cells [[Figure 6]A and 6B]. In contrast, opuntiol pretreatment prevented UVA-mediated apoptotic cell death and morphological changes. Further, we found that UVA-exposure caused comet tails due to DNA damage. Opuntiol pretreatment prevented UVA-induced comet tail formation when compared to UVA-alone treated cells [[Figure 7]A and 7B].
Figure 5 (A). Effect of opuntiol on UVA-induced loss of mitochondrial membrane potential in NIH-3T3 cells. Photomicrograph shows the fluorescence observed under a green lamp (original magnification, 20X). (B) Bar diagram representing percent of fluorescence intensity using a multimode reader (Tecan, Austria). Values are given as means ± S.D. of six experiments in each group. Values not sharing a common marking (a,b,c...) differ significantly at p ≤ 0.05 (DMRT).

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Figure 6 (A) Effect of opuntiol on UVA-irradiation-induced apoptotic morphological changes by AO/EtBr staining. UVA-exposed cells show EtBr staining due to membrane damage and apoptotic morphological changes. Images were taken using a fluorescence microscope (original magnification, 20X). (B) Bar diagram representing percent apoptotic cell death in different experimental groups. Values are given as means ± S.D. of six experiments in each group. Values not sharing a common marking (a,b,c…) differ significantly at p ≤ 0.05 (DMRT).

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Figure 7 (A) Effect of opuntiol and/or UVA-induced DNA single-strand breaks analyzed by single-cell gel electrophoresis. Photomicrographs showed DNA damage evidenced by the increased tail formation in UVA-exposed NIH-3T3 cells (original magnification, 20X). (B) Percent Tail DNA and Comet attributes were analyzed using CASP software. Values are given as means ± S.D. of six experiments in each group. Values not sharing a common marking (a,b,c) differ significantly at p ≤ 0.05 (DMRT).

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   Discussion Top


There is a continuous search for new therapeutic agents to treat cancer with better efficiency and lesser side effects. Approximately, 60% of clinically approved anticancer drugs are derived from the secondary metabolites found in nature.[25] According to the reports obtained from several epidemiological and case-control studies, the plant-based phytochemicals exhibit the potential to be considered as anticancer drugs against diverse types of cancers.[26] O. ficus indica is a species of cactus that has long been a domesticated crop plant grown in agricultural economies throughout arid and semiarid parts of the world.[27] The fruits and stems of O. ficus indica have been conventionally employed in folk medicine in many nations across the globe for several medicinal purposes.[28] Therefore clinical pharmacologic interest in the efficacy and safety of the phytochemicals present in genus Opuntia has grown in recent years.

In this study, we studied the preventive effect of opuntiol against ultraviolet-A radiation-induced oxidative damages in NIH/3T3 cells. We observed that opuntiol treatment showed significant free radical activities against hydroxyl, DPPH, and superoxide free radicals [Figure 1]. The IC50 values for hydroxyl radical, DPPH, and superoxide radical scavenging activity were found to be 0.078, 0.003, 0.625 μM, respectively. The free radical activity of opuntiol followed a concentration-dependent manner. A list of antioxidant phytochemicals present in O. ficus indica (L.) has already been reported.[7] Loganayagi et al.[29] reported that opuntiol behaves as an inhibitor of corrosion of mild steel in the acid medium due to its antioxidant potential. Opuntiol has been found to display appreciable antioxidant properties.[30] Opuntiol possesses 6-(hydroxymethyl)-4-methoxy groups. These functional groups donate electrons to the free radicals and responsible for their inherent antioxidant potential.

As opuntiol behaves as a potent antioxidant it could be used to prevent ultraviolet radiation-induced oxidative damages. The cytotoxicity assay showed that opuntiol treatment prevented UVA-induced loss of cell viability in a concentration-dependent manner in NIH/3T3 cells [Figure 2]. This might be due to its preventive effect against UVA-mediated oxidative cell death. To understand its preventive potential against ROS mediated cell death we performed a series of oxidative damages related experiments. The formation of intracellular ROS is a hallmark of oxidative stress.[31] We found that opuntiol pretreatment significantly prevented the intracellular ROS production in UVA-induced NIH-3T3 cells in a concentration-dependent manner [Figure 3]. It has been reported that the O. ficus indica extract neutralizes the aflatoxin B1-mediated generation of ROS.[32] The UVA-induced ROS causes membrane lipid peroxidation in the intact living cells. Elevated lipid peroxidation products have been linked to serious effects such as loss of fluidity, inactivation of membrane enzymes and increases in the permeability of ions, which may lead to disruption of cell membrane integrity.[5] We noticed that opuntiol pretreatment significantly decreased UVA-induced TBARS levels [Figure 4].

The cellular antioxidant enzymes behave in a coordinated manner to maintain the normal cellular redox homeostasis.[33] The UVA-exposure generates higher intracellular ROS levels which frequently overwhelms the endogenous antioxidant capacity of the cells. This causes redox imbalance in the cellular system and induces severe oxidative damages to cellular macromolecules. Reports show that both acute as well as chronic UVA exposure depletes the enzymatic and non-enzymatic antioxidant levels via the production of free radicals through photosensitizers.[34] The supplementation of exogenous antioxidants possesses the capacity to improve cellular redox homeostasis which has been affected by oxidants like UVA radiation. We found that opuntiol prevent UVA-mediated loss of cellular antioxidants system [Table 1]. The flavonols present in O. ficus indica has been reported to protect skin antioxidant systems in rat models.[35] The SOD activity has been protected by O. ficus indica upon nickel induced toxicity in rats.[36] Further, O. ficus indica possesses the capacity to enhance the endogenous antioxidants activities in ethanol intoxicated rats.[37]

Loss of mitochondrial membrane potential is an early step in apoptotic cell death, which causes excessive ROS buildup and consequently triggers cytochrome c resulting in the execution of cell death via activation of caspases.[38] We noticed that opuntiol pretreatment significantly prevented UVA mediated loss of mitochondrial membrane potential in NIH/3T3 cells in a concentration-dependent manner [Figure 5]. It has been reported earlier that plant-derived phytochemicals can prevent the loss of mitochondrial membrane potential.[39] The UVA-induced oxidative damages lead to apoptotic cell death. In agreement with this, we observed the protective effect of opuntiol on UVA-radiation induced apoptotic and morphological changes in NIH/3T3 cells [Figure 6]. The DNA strand breaks observed in this study may be due to ROS attacks at the deoxyribose moiety of DNA, oxidative base damages (8-deoxy guanosine) and the formation of cyclobutane pyrimidine dimers. HPLC-ESI-MS/MS studies showed that T <> T and T <> C dimers are generated upon UVA-irradiation in human skin cells.[40],[41] The generation of CPDs may be due to direct UVA-photon absorbance and excitation to novel charge-transfer states.[42] Furthermore, the formation of 8-deoxy guanosine by the singlet oxygen produced by UVA-radiation through cellular photosensitizers has also been documented for UVA-mediated DNA strand breaks.[43] Opuntiol prevention on UVA-induced DNA damages may be due to several reasons: it may absorb UVA photons and serves as sunscreens; it may scavenge UVA-induced free radicals through its electron-donating antioxidant property; further, it could modulate the cellular redox signaling and DNA repair pathways thereby minimize UVA-mediated DNA strand breaks. The O. ficus indica has been reported earlier to prevent oxidative and DNA damage induced by the mycotoxin zearalenone in Balb/c mice.[44] The ethanolic extract of O. ficus indica is reported to act as an active scavenger of hydroxyl radicals and thereby significantly prevents DNA nicking.[45] Brahmi et al.[32] also reported that O. ficus indica exhibits preventive genotoxic effects in mice models. Further, Brahmi et al.[46] reported a protective effect of O. ficus indica (cactus) extract against cisplatin-induced oxidative stress, genotoxicity, and apoptosis in mice experimental models.


   Conclusion Top


This study showed that opuntiol pretreatment prevents UVA-radiation mediated oxidative damage indices like lipid peroxidation, DNA strand breaks, and apoptotic incidence in NIH 3T3 cells. Opuntiol behaves as an excellent antioxidant in different free radical scavenging system. Therefore, it could be postulated that the antioxidant property of opuntiol might be the reasons for its photoprotection property.

Acknowledgements

The author greatly acknowledges the Indian Council of Medical Research, India, for providing financial support (ICMR Letter: No. 45/21/2018/BMS/TRM) in the form of Senior Research Fellow to Mr. P. Veermani kandan.

Financial support and sponsorship

The financial support is provided by Indian Council of Medical Research, India (ICMR Letter: No. 45/21/2018/BMS/TRM).

Conflicts of interest

There are no conflicts of interest.



 
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