ajst-2019-99_OA

Australian Journal of Science and Technology

ISSN Number (2208-6404)
Volume 3; Issue 5; December 2019

1 1
2 Original Article 2
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Protective effects of NAT-CLWTM on acetaminophen-induced 5
6
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8 oxidative stress-mediated apoptosis in hepatic HepG2 cells 7
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9 9
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11 Murugesan Jayaraman Bharathi1, Thomas Avoorathu Jacob2 11
12 12
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AQ2 FFF Bio Works LLP, IBAB, Electronic City, Bengaluru, Karnataka, India, 2FFF Bio Works LLP, Indiranagar, Bengaluru,
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Karnataka, India
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16 16
17 ABSTRACT 17
18 18
19 The hepatoprotective mechanism against drug-induced liver damage is the activity being searched to identify new therapeutic targets. Among 19
20 them, the detoxifying effect of NAT-CLWTM is a plant formulation, which exhibits several pharmacological properties such as antioxidant, 20
21 anti-inflammatory, anti-apoptotic activities, and inhibitory effect through its protective role in hepatic HepG2 cells. However, its therapeutic 21
22 mechanism of liver damage is still unclear. The aim of the present study, we employed that the hepatoprotective effect and underlying 22
23 mechanism of the NAT-CLWTM on acetaminophen (APAP) exposed hepatotoxicity were investigated in HepG2 cell line model. The results 23
24 24
showed that NAT-CLWTM pre-treatment dramatically increased APAP-induced cell viability and inhibit increased ROS generation. These
25 25
26 effects also showed strong antioxidants by an enhance in activity of GSH, SOD and by a diminished lipid peroxidation level compared to 26
27 APAP induced group. Furthermore, pre-treatment with NAT-CLWTM drastically prevented mitochondrial alteration and protected morphological 27
28 chances including (cell shrinkage, nuclear fragmentation, condensation, and cell blubbing) induced by APAP. These findings suggest that the 28
29 hepatoprotective activity of NAT-CLWTM on APAP-induced hepatotoxic was closely associated with suppression of APAP-induced oxidative 29
30 stress and mitochondrial dysfunction in HepG2 cells. These results strongly indicate that NAT-CLWTM has a significant protective effect against 30
31 APAP induced hepatotoxicity. 31
32 32
33 Keywords: Acetaminophen, apoptosis, NAT-CLWTM, oxidative stress, silymarin 33
34 34
AQ3 Submitted: ???, Accepted: ???, Published: ???
35 35
36 36
37 37
38 INTRODUCTION P450 enzymes including CYP2E1, CYP3A4, and CYP1A2.[4] 38
39 NAPQI depletes hepatic cellular glutathione (GSH) levels and 39
40 The liver is one of the important organs in the human body thereby adenosine triphosphate (ATP) causes mitochondrial 40
41 function its leads to cellular damage and finally causes 41
and actively involved in many metabolisms and major
42 42
43 function of the liver are protein, carbohydrates, lipids and DNA fragmentation and apoptosis. APAP also contributes to 43
44 also detoxification of powerful toxic drug and metabolites, enhanced ROS generation.[5,6] In addition, lipid peroxidation 44
45 chemical, and environmental pathogens.[1] However, it is resulting from oxidative stress has been demonstrated to 45
46 continuously and variedly to environmental toxins and contribute to the initiation and progression of APAP-induced 46
47 liver damage.[7] 47
abused by poor drug habits, alcohol, prescribed, and over
48 48
49 the counter drug which can eventually lead to various liver 49
50 diseases.[2,3] Acetaminophen (N-acetyl-p-aminophenol, APAP) The availability of synthetic drugs to treat several liver 50
51 is widely used as an analgesic and antipyretic agent is safe diseases in this state may further worsen the liver injury as 51
52 and effective at the therapeutic dose. However, APAP when well as need to get metabolized in the past liver damage.[8] 52
53 This increased load on liver function and required action of 53
taken high doses can cause serious liver injury, such as serve
54 54
56 hepatic necrosis, hepatic lesion, cirrhosis, total malfunction, the drug may not be observed. Vaccines, antiviral drugs, and 56
57 and death (). APAP-induced hepatotoxicity is metabolized to steroids used as a treatment for liver pathologies have possible 57
58 N-acetyl-p-benzoquinone imine (NAPQI) through cytochrome adverse effects, particularly if administrated chronically or 58
59 59
60 60
61 Address for correspondence: Thomas Avoorathu Jacob, FFF Bio Works LLP, Indiranagar, Bengaluru - 560 008, Karnataka, India. 61
62 E-mail: jacob. at@fffbioworks. com 62

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1 sub chronically.[9] To date, numerous compounds, including toxicity.[10] In brief, cells were seeded at a density of 3 × 1
2 polyphenols, flavonoids, quinines, coumarins, and carotenoids 105 cells/well and were cultured in 96 well plates followed 2
3 3
are derived from natural products widely distributed in by overnight incubation at 37°C. Followed by cells were
4 4
5 fruits, vegetables, and well-known medicinal plants. These treat with different concentration of NAT-CLWTM (3.9, 7.18, 5
6 compounds have been extensively thought to be adjuvant 15.62, 31.25, 62.50, 125, and 250 µg/ml) or APAP (20 mM) 6
7 and alternative medicines for the improvement of hepatic or silymarin (3.125, 12.5, 25, 50, 100, and 200 µg/ml) and 7
8 diseases by inhibited oxidative stress and mitochondrial incubated for 24 h. After incubation of respective time, 8
9 9
dysfunction through antioxidants. Hence, developing 100 µl of MTT reagent (5 mg/ml) was added to each well
10 10
11 pharmacology effective medicine from natural products and cells were incubated further 4 h at 37°C. The media were 11
12 has become an essential by the asset of its moderately low aspirated and the formazan crystals were dissolved in DMSO 12
13 toxicity or fewer side effects. NAT-CLWTM is a patent pending 100 µl/well and the absorbance was read with a microplate 13
14 formulated natural ingredients and developed by FFF BIO reader at 570 nm. 14
15 15
WORKS LLP as a hepatoprotective agent as used to treat
16 16
17 APAP-induced liver disease. This contains curcuminoids Lipid Peroxidation and Antioxidant Enzyme 17
18 and lutein combinations; these ingredients have established Activity Assay 18
19 hepatoprotective activities in isolation. Individually single The amount of TBARS, SOD, and GSH in the cell suspensions 19
20 ingredients of the formulation have been investigated to have was determined by measuring to the total lipid peroxidation, 20
21 21
protective activity against different models of experimental superoxide dismutase, glutathione levels, and the capability
22 22
23 hepatotoxicity. In the present investigation elaborated an to chelate ROS in HepG2 cells using MDA, SOD, and GSH 23
24 in vitro model, an efficient research was undertaken to assess assay kits obtained from Sigma-Aldrich, USA. In briefly, the 24
25 the potential effects of the formulation on the hepatotoxocity collected cells suspensions were measured the absorbance at 25
26 induced by APAP agent. The present communication 26
532 and 490 nm according to the manufactures instructions and
27 27
substantiates the therapeutic utility of the formulation as TBARS, SOD, and GSH content were calculated as nmol/mL
28 28
29 hepatoprotective agents. protein and U/mL. 29
30 30
31 Detection of Intracellular ROS Levels 31
32
MATERIALS AND METHODS 32
33
The levels of intracellular ROS generation were measured by 33
34 Chemicals DCFH-DA substrate as followed by according to the previous 34
35 Acetaminophen, curcuminoids, and lutein were obtained study with slight modifications.[11,12] In brief, HepG2 cells were 35
36 from Sigma-Aldrich, USA. NAT-CLW TM was developed seeded at a density of 1 × 106 cells per well in 6-well plates 36
37 under the standard conditions. Cells were treated with the 37
38
by FFF BIO WORKS LLP, Bengaluru, India. Dulbecco’s 38
39 modified eagle’s medium (DMEM), fetal bovine final concentration of 50 μg NAT-CLWTM and silymarin for 39
40 serum (FBS), trypsin-ethylenediaminetetra acetic 1 h, followed by APAP were exposed to HepG2 cells for 24 h. 40
41 acid, phosphate-buffered saline (PBS), and antibiotic/ After treatment cells were incubated with 5 µM DCFH-DA at 41
42 antimycotic reagent were purchased from HiMedia. 37°C for 30 min in the dark place. Cells were washed with 42
43 PBS buffer to remove excess dye and images were examined 43
44 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium 44
45 bromide (MTT), dichlorodihydrofluorescein diacetate under the fluorescence microscopy. 45
46 (DCFH-DA), Rhodamine 123 (Rho 123), Ethidium 46
47 bromide/acridine orange (EB/AO), and 6-diamidino-2- Determination of Mitochondrial Membrane 47
48 phenylindole (DAPI) were acquired from Sigma-Aldrich, Potential 48
49 A change in MMP was monitored by mitochondrial lipophilic 49
50 USA. All the other chemicals were used in analytical grade. 50
51 cation dye Rho-123, according to the previous study.[13] Briefly, 51
52 Cell Culture HepG2 cells were seeded at a density of 1 × 106 cells in six- 52
53 We purchased the HepG2 cell line from the National Centre for well plates with a medium containing 20 mM APAP in the 53
54 Cell Science (NCCS), Pune, India. Human HepG2 cells were presence or absence of 50 μg NAT-CLWTM for 24 h. Cells were 54
56 56
57 cultured in DMEM which is a basal medium supplemented incubated with Rho-123 (5 µM) in DMEM at room temperature 57
58 with 10% FBS, followed by 5% antibiotic/antimycotic reagents for 30 min and cells washed with PBS. Images were captured 58
59 were added to prevent bacterial and fungal contaminations. by fluorescence microscopy. 59
60 Cultured cells were maintained under the slandered condition 60
61 at 37°C in an atmosphere of 5% CO2. Ethidium Bromide/Acridine Orange (dual staining) 61
62 62
63
The cell morphology was evaluated using AO/EB fluorescence 63
64 Cell Viability and Cytotoxicity Assay staining was carried out by the method of Jimenez et al. 64
65 Cell viability assay was used to determine the cytotoxic (2008).[14] Briefly, the cells were plated at a density of 5 × 104 65
66 effects and protective effects against APAP-induced in six-well plates. They were grown at 37°C in humidifies 66

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1 CO2 incubator until they were reached at 70–80% confluent. tests (DMRT) using SPSS software. P ≤ 0.05 was considered 1
2 Then, the cells were treated with NAT-CLWTM/silymarin for to be significant. 2
3 3
24 h. The cell suspensions incubated with 100 µg/ml of dye
4 4
mixture AO/EB for 30 min in the dark. Then, the cells washed
5 RESULTS 5
6 once with PBS to remove excess dye and cells were visualized 6
7 immediately under a fluorescence microscope. 7
8 Cytotoxicity Effects of NAT-CLWTM and Silymarin 8
9 in HepG2 Cells 9
10
DNA Fragmentation by DAPI Staining 10
HepG2 cells were treated with different concentrations of NAT-
11 Nuclear morphology was measured by fluorescence 11
microscopy following DAPI sating. HepG2 cells were treated CLWTM and APAP-induced cytotoxicity was determined by
12 12
13 with APAP 20 mM 1 h before NAT-CLWTM/silymarin for 24 h. MTT assay for 24 h. Figure 1a and b shows the pretreatment 13
14 Then, the cells were washed with PBS (pH 7.4) and cells were with various concentrations of NAT-CLWTM and silymarin 14
15 alone for 24 h, did not show any toxic effect in HepG2 cells. 15
fixed with ice-cold 70% ethanol. Cells were incubated in DAPI
16 Further, the effect of NAT-CLW against APAP exposed 16
17 for 15 min at 37°C in wrapped in aluminum foil. Then, the 17
18 cells were washed with PBS and images viewed immediately HepG2 cells showed a significant percentage of growth 18
19 by fluorescence microscopy. in dose-dependent manner as compared to control cells. 19
20 Whereas, similar results were observed when pre-treatment 20
21 with silymarin in HepG2 cells was treat with APAP toxicity. 21
22
Statistical Analysis 22
23 All the values were analyzed as mean ± standard devotion (SD). From these results, we found significant lesser toxicity in 23
24 The significant variance all the data are subjected to one-way HepG2 cell (IC50 62.50 µg/ml), hence the results we taken for 24
25 analysis of variance (ANOVA) followed by Duncan multiple further experiments. 25
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64 Figure 1: Displaying the MTT cell viability analysis of silymarin and NAT-CLW in the APAP-induced HepG2 cells. (a and b) NAT-CLW 64
65 and silymarin were effectively increased the viability of hepatic HepG2 cells. Values are given as mean ± SD of three experiments in each 65
66 group. Values not sharing a common marking (*, #) differ significantly at P ≤ 0.05 (DMRT) 66

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1 Effects of NAT-CLWTM and Silymarin on TBARS An antioxidants act as the most important defense against 1
2 and Antioxidant Enzyme Activity on APAP-induced the free radical formation. Figure 2 shows APAP-induced 2
3 3
4
HepG2 Cells HepG2 cells significantly diminished in SOD activity and
4
5 The results showed that the significant increase in TBARS GSH levels due to excessive ROS production. On the other 5
6 level was observed in APAP-exposed HepG2 cells as hand, pretreatment with 62.50 μg of NAT-CLWTM significantly 6
7 compared to control cells. On NAT-CLWTM (62.50 μg/ml) and protected APAP-induced loss of antioxidant status (SOD 7
8 silymarin (50 μg/ml) pre-treatment significantly prevented activity and GSH levels) in HepG2 cells shows in Figure 2. 8
9 9
lipid peroxidation formation in APAP-induced HepG2 cells From these results, we find that NAT-CLW has more potent
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11 (Figure 2a). antioxidant than silymarin. 11
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62 c 62
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64 Figure 2: (a-c) Effect of NAT-CLW and silymarin on TBARS, GSH levels, and SOD enzyme activities in APAP-induced HepG2 cells. 64
65 Values are given as mean ± SD of three experiments in each group. Values not sharing a common marking (*, #) differ significantly at 65
66 P ≤ 0.05 (DMRT) 66

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1 Impact of NAT-CLWTM and Silymarin on Effect of NAT-CLWTM and Silymarin on 1

2 APAP-mediated Intracellular ROS Levels APAP-induced Nuclear Apoptosis 2
3 3
4
Figure 3 shows a significant increase an intracellular ROS The apoptotic morphological was observed in APAP-induced 4
5 formation in APAP (20 mM) induced in HepG2 cells as compared in HepG2 cells showed that the green nuclei fragmented 5
6 to control cells. Whereas, pre-treatment with NAT-CLWTM represented early apoptotic cells and yellow dots shows the 6
7 (62.50 µg/ml) significantly decline the ROS formation and condensed nuclei were of late apoptosis which was observed 7
8 as it indicates the lesser APAP-induced free radical release as in fluorescent microscopy (Figure 5). On the other hand, 8
9 9
10 compared with APAP alone treated group. Silymarin treated HepG2 cells pre-treated with NAT-CLWTM (62.50 µg/ml) 10
11 cells dramatically less prevention as compared to NAT-CLWTM. and silymarin 50 µg/ml showed increased green fluorescence 11
12 and prevented morphological changes were observed in 12
13 Effect of NAT-CLWTM and Silymarin on HepG2 cells treated with APAP, which indicate viable cells. 13
14 APAP-induced Reduction of MMP 14
15 15
16 In Figure 4, we examined that the APAP-induced cells are EFFECT OF NAT-CLWTM AND 16
17 indicated polarized mitochondrial membrane and decreased 17
18 green fluorescence as compared with untreated control cells. SILYMARIN ON APAP-INDUCED 18
19 HepG2 cells were pre-treated with NAT-CLWTM shows an NUCLEAR FRAGMENTATION 19
20 improved green fluorescence representing a depolarized state of 20
21 21
22 the mitochondrial membrane as compared to APAP alone treated Figure 6 shows HepG2 cells treated with APAP showed 22
23 cells. Observation made from our findings study strongly indicated characteristic changes associated with apoptosis, including 23
24 that NAT-CLWTM formulation exhibits more protective activity shrinkage, chromatin condensation, nuclear fragmentation, and 24
25 as compared to silymarin, it is indicated MMP protective activity. formation apoptotic bodies as compared to untreated control 25
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37 Figure 3: The NAT-CLW and silymarin reduce APAP-induced ROS generation in HepG2 cells. Intracellular ROS accumulation was 37
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39 measured using the fluorescence probe DCFH-DA staining 39
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Figure 4: The effect of NAT-CLW and silymarin reduces APAP-induced ROS generation in HepG2 cells. Intracellular ROS accumulation
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52 was measured using the fluorescence probe DCFH-DA staining 52
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Figure 5: The effect of NAT-CLW and silymarin on APAP-induced apoptosis morphological changes as stained by AO/EB staining in
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HepG2 cells

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1 1
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11 Figure 6: The effect of NAT-CLW and silymarin on APAP-induced apoptotic features in HepG2 cells by DAPI staining 11
12 12
13 cells. Whereas, treatment with NAT-CLWTM (62.50 µg/ml) is important to explore if NAT-CLWTM has been suppressed 13
14 14
showed inhibited condensation and fragmentation was observed oxidative stress these findings clearly indicated that NAT-
15 15
16 in APAP-induced in HepG2 cells. Silymarin when compared to CLWTM could reduce APAP-triggered ROS levels due to its 16
17 NAT-CLWTM had less prevention; these results clearly suggest potent antioxidant. It is generally recognized that increased 17
18 that NAT-CLWTM exhibits more potent than silymarin. lipid peroxidation formation and reduced cellular function 18
19 of enzymatic/non-enzymatic antioxidant defense systems are 19
20 20
the most important characteristics of hepatotoxicity induced
21 DISCUSSION 21
22 by APAP.[27] In the current study, we aimed to evaluate APAP 22
23 induced to HepG2 cells significantly decrease liver antioxidant 23
A significant number of plant extract and herbal formulations
24 capacity, as characterized by dramatically enhance in MDA 24
25 have been exhibit several biological properties to treat 25
content and diminish in hepatic levels of SOD and GSH, which
26 various life-threatening diseases.[15] To date, the available 26
27
are often regard as indicator of oxidative stress response[6,28] 27
experimental studies reveal plants extract and phytochemicals
28 However, pre-treatment with NAT-CLWTM markedly reversed 28
that exhibit hepatoprotective effects that the phytoconstituents
29 the changes in parameters of lipid peroxidation and antioxidant 29
30 including phenyl compounds, coumarins, essential oils, 30
status. Collectively, on the bases, our results demonstrated
31 monoterpenoids, diterpenoids, triterpenoids, steroids, 31
that the protective effect of NAT-CLWTM found more efficacy
32 alkaloids, and other nitrogenous compounds [16,17] that 32
33 than silymarin on APAP-induced liver damage. This study 33
representation of hepatoprotective effects against APAP coincident with the previous study demonstrated that the
34 34
35
exposed to protect the liver disease from the harmful sound of protective effects of curcumin against liver damage through 35
36 drug-exposed intoxication.[18] Various formulations containing attenuation of oxidative stress, inflammation, and cell death 36
37 plant extracts of single or multiple known polyherbal in both in vitro and in vivo. Further, NAT-CLWTM provides 37
38 formulations are available in the market for cure liver 38
39 protection against APAP was established in rat hepatocytes 39
disease.[19-21] NAT-CLWTM is a natural plant-based formulation mediated impaired lipid peroxidation, but no effect was found
40 40
41 it contains curcumin and lutein, which is shows number of on depletion of GSH and LDH and in time-dependent action 41
42 medicinal properties such as antioxidant, anti-apoptotic, and at low concentrations. However, overdoses have revealed the 42
43 it possesses hepatoprotective properties. Previous studies have protective effects against APAP.[29] 43
44 documented that the curcumin protects against APAP exposed 44
45 45
46 hepatotoxicity through its antioxidant properties.[22] Another Mitochondrial is a powerhouse of the cells for providing 46
47 study indicated that curcumin was found to protect against energy generating and also known as plays a key role in 47
48 genomic instability, cell death, and oxidative stress in liver.[23] cellular signaling pathways. Their inmost role in energy 48
49 Edakkadath et al. reported that the carotenoid lutein prevents 49
metabolism, as well as their high abundance in hepatocytes,
50 50
hepatic damage induced by paracetamol or ethanol induced. makes them important targets for drug-exposed hepatotoxicity.
51 51
52 Similarly, we find that HepG2 cells indicated cell death after Mitochondrial membrane potential and mitochondrial 52
53 induced overdose of APAP with a dose-response similarly permeability transition are biomarkers that have been used 53
54 that reported previously.[24,25] Our results strongly indicated to examine mitochondrial dysfunction and damage.[30,31] 54
56 that the pre-treatment of NAT-CLWTM formulation drastically 56
57
Loss of mitochondrial function, through NAPQI binds to 57
58 increased cell viability by APAP induced cell death; these result cellular protein as well as mitochondrial proteins and alters 58
59 suggesting that NAT-CLWTM is more potent than silymarin. the mitochondrial ATP syntheses, could lead to ineffective 59
60 ATP depletion, additionally opening mitochondrial membrane 60
61 The formation of ROS results in oxidative stress, lipid pore through which the release of cytochrome c into the 61
62 62
63
peroxidation, DNA damage, and loss of cellular function; cytosol, subsequently which forms apoptosome complex with 63
64 finally, it leads to apoptosis (). The major reactive species other mitochondrial factors such as Apaf-1 which activate 64
65 generated by oxidative stress which is responsible for the caspase.[32-34] APAP induced necrosis and apoptosis or both 65
66 occurrence and progression of APAP toxicity.[26] Therefore, it depending on the type of cells or other concentrations of APAP 66

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1 treatment.[33-35] In the current study, APAP-induced cells showed 8. Kumar A. A review on hepatoprotective herbal drugs. Int J Res 1
2 reduced mitochondrial function, DNA fragmentation; however, Pharm Chem 2012;2:92-102. 2
3 9. Seeff LB, Lindsay KL, Bacon BR, Kresina TF, Hoofnagle JH. 3
4
NAT-CLWTM pre-treatment before APAP-induced effectively 4
protected mitochondrial function and DNA fragmentation Complementary and alternative medicine in chronic liver disease.
5 Hepatology 2001;34:595-603. 5
6 in HepG2 cells. Several reports clearly demonstrated that 6
10. Mosmann T. Rapid colorimetric assay for cellular growth and
7 natural plant formulation occurring antioxidants could protect 7
8 survival: Application to proliferation and cytotoxicity assays. 8
APAP-induced DNA fragmentation as well as early event of J Immunol Methods 1983;65:55-63.
9 9
10 apoptotic formation (). Moreover, NAT-CLWTM prevented 11. Takanashi T, Ogura Y, Taguchi H, Hashizoe M, Honda Y. 10
11 APAP exposed apoptosis through modulation of mitochondrial Fluorophotometric quantitation of oxidative stress in the retina 11
12 apoptotic signaling molecules; likewise, NAT-CLWTM has been in vivo. Investig Ophthalmol Visual Sci 1997;38:2721-8. 12
13 reported to reduce apoptosis through restored mitochondrial 12. Yeligar SM, Harris FL, Hart CM, Brown LA. Ethanol induces 13
14 oxidative stress in alveolar macrophages via upregulation of 14
function. Hence, we conclude that NAT-CLWTM protects
15 NADPH oxidases. J Immunol 2012;188:3648-57. 15
16 APAP-induced apoptosis by preventing loss of MMP. 16
13. Satoh T, Enokido Y, Aoshima H, Uchiyama Y, Hatanaka H.
17 17
18 Changes in mitochondrial membrane potential during oxidative 18
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CONCLUSION stress-induced apoptosis in PC12 cells. J Neurosci Res 19
20 1997;50:413-20. 20
21 In this current study stated that NAT-CLWTM protects APAP- 14. Jimenez PC, Wilke DV, Takeara R, Lotufo TM, Pessoa C, de 21
22 exposed oxidative stress by inhibiting the ROS production Moraes MO, et al. Cytotoxic activity of a dichloromethane 22
23 and lipid peroxidation in HepG2 cells. Further, NAT-CLWTM extract and fractions obtained from Eudistoma vannamei 23
24 (Tunicata: Ascidiacea). Comp Biochem Physiol A Mol Integr 24
25 modulates mitochondrial function thereby protects NAT- 25
Physiol 2008;151:391-8.
26 CLWTM exposed DNA fragmentation. Further, we find that 26
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27 NAT-CLWTM treatment to have protected APAP-exposed 27
28 Phyllanthus niruri exhibit distinct in vitro anti-oxidant and in 28
apoptosis by modulated mitochondrial complex and helped vivo hepatoprotective activity against paracetamol-induced liver
29 29
to protect the mitochondrial function. Finally, the observation damage in mice. Food Chem 2008;111:845-51.
30 30
31 made from our findings clearly indicated that plant-derived 16. Sharma SK, Arogya SM, Bhaskarmurthy DH, Agarwal A, 31
32 formulation of NAT-CLWTM exhibits more potent activity than Velusami CC. Hepatoprotective activity of the Phyllanthus 32
33 silymarin administration. Further study needs to conventional species on tert-butyl hydroperoxide (t-BH)-induced cytotoxicity 33
34 medicinal formulation drug to treating various liver diseases. in HepG2 cells. Pharmacogn Mag 2011;7:229-33. 34
35 17. Valan M, Britto A, Venkataraman R. Phytoconstituents with 35
36 hepatoprotective activity. Int J Chem Sci 2010;8:1421-32. 36
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