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 Table of Contents  
ORIGINAL ARTICLE
Year : 2016  |  Volume : 8  |  Issue : 3  |  Page : 210-216  

Amelioration of oxidative DNA damage in mouse peritoneal macrophages by Hippophae salicifolia due to its proton (H+) donation capability: Ex vivo and in vivo studies


1 Department of Pharmaceutical Technology, Jadavpur University, Kolkata, West Bengal, India
2 Department of Pharmacy, Gurunanak Institute of Pharmaceutical Science and Technology, Sodepur, Kolkata, West Bengal, India

Date of Submission12-Sep-2015
Date of Decision29-Nov-2015
Date of Acceptance24-Nov-2015
Date of Web Publication22-Jun-2016

Correspondence Address:
Asis Bala
Department of Pharmacy, Gurunanak Institute of Pharmaceutical Science and Technology, Sodepur, Kolkata, West Bengal
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0975-7406.172663

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   Abstract 

Introduction: The present study evaluates the antioxidant effect of methanol extract of Hippophae salicifolia (MEHS) bark with special emphasis on its role on oxidative DNA damage in mouse peritoneal macrophages. Material and Methods: In vitro antioxidant activity was estimated by standard antioxidant assays whereas the antioxidant activity concluded the H+ donating capacity. Mouse erythrocytes' hemolysis and peritoneal macrophages' DNA damage were determined spectrophotometrically. In vivo antioxidant activity of MEHS was determined in carbon tetrachloride-induced mice by studying its effect on superoxide anion production in macrophages cells, superoxide dismutase in the cell lysate, DNA damage, lipid peroxidation, and reduces glutathione. Results: The extract showed good in vitro antioxidant activities whereas the inhibitory concentrations values ranged from 5.80 to 106.5 μg/ml. MEHS significantly (P < 0.05) attenuated the oxidative DNA damage. It also attenuated the oxidative conversion of hemoglobin to methemoglobin and elevation of enzymatic and nonenzymatic antioxidant in cells. Conclusion: The result indicates MEHS has good in vitro-in vivo antioxidant property as well as the protective effect on DNA and red blood cell may be due to its H+ donating property.

Keywords: Carbon tetrachloride, DNA, free radicals, Hippophae salicifolia, macrophage


How to cite this article:
Chakraborty M, Karmakar I, Haldar S, Das A, Bala A, Haldar PK. Amelioration of oxidative DNA damage in mouse peritoneal macrophages by Hippophae salicifolia due to its proton (H+) donation capability: Ex vivo and in vivo studies. J Pharm Bioall Sci 2016;8:210-6

How to cite this URL:
Chakraborty M, Karmakar I, Haldar S, Das A, Bala A, Haldar PK. Amelioration of oxidative DNA damage in mouse peritoneal macrophages by Hippophae salicifolia due to its proton (H+) donation capability: Ex vivo and in vivo studies. J Pharm Bioall Sci [serial online] 2016 [cited 2019 Aug 18];8:210-6. Available from: http://www.jpbsonline.org/text.asp?2016/8/3/210/172663

Oxygen is essential to life as living systems have evolved to survive in the presence of molecular oxygen. However oxygen has double-edged properties, being essential for life, it can also cause the damage within the cell by triggering oxidative events.[1] Mainly two types of reactive species are generated during oxidative stress. These are reactive oxygen species (ROS) and reactive nitrogen species (RNS). The most abundant ROS in the living cells are superoxide, hydrogen peroxide (H2O2), singlet oxygen, and highly toxic hydroxyl radicals. The RNS contains diverse nitrogen derivatives including nitric oxide and more toxic peroxynitrite that possesses a strong oxidative capacity.[2]

It is increasingly being realized that many of today's diseases are due to the “oxidative stress” that results from an imbalance between formation and neutralization of oxidants. Oxidative stress is initiated by free radicals, which seek stability through electron pairing with biological macromolecules such as proteins, lipids, and DNA in healthy human cells and cause protein, DNA, and lipid oxidation. These changes contribute to cancer, atherosclerosis, cardiovascular diseases, aging, and inflammatory diseases.[3] Antioxidants are our first line of defense against free radical damage and are critical for maintaining optimum health and well-being. Antioxidants are capable of stabilizing or deactivating free radicals before they attack cells.[4] All human cells protect themselves against free radical damage by enzymes such as superoxide dismutase (SOD) and catalase, or nonenzymatic compounds such as reduced glutathione (GSH).[5]

Plants are the rich sources of antioxidants. Certain phytoconstituents are capable of terminating free radical reactions thereby keeping oxidative damage at bay. Different herbal products are safer than synthetic medicines due to their lesser side effects and cost effectiveness. Therefore, it is in demand in the modern era to use such phytomedicines for therapy.[6]

Hippophaesalicifolia, commonly known as Sea-buckthorn, is a versatile plant with multipurpose uses. In India, two species of Sea buckthorn, namely, H.salicifolia D. Don and Hippophaerhamnoides L. are very common. H.salicifolia is a shrub-to-tree in nature and is restricted to the Himalayan region, whereas H.rhamnoides is bushy, growing at higher altitude in India, and widely distributed in Europe and Asia.[7]

The bark is traditionally used for its antidiarrheal, antitumor, and cosmetic purposes and also its ash has burned healing properties.[8] It has been reported that the plant has antibacterial and antifungal activities.[9] The hydroalcoholic extract of bark has also the antioxidant activity.[10]

The aim of the present study was to evaluate the antioxidant activity of H.salicifolia bark in mouse peritoneal macrophages and damage to the DNA. The molecular mechanism of action was also investigated by correlating its effect on antioxidant parameters through H + donating capability.


   Materials and Methods Top


Chemicals

1,1-diphenyl-2-picryl-hydrazyl (DPPH) was obtained from Sigma Chemicals, USA. Nitroblue tetrazolium (NBT), phenazine methosulphate (PMS), reduced nicotinamide adenine dinucleotide (NADH), sodium nitroprusside, napthyl ethylene diamine dihydrochloride, ascorbic acid, trichloroacetic acid (TCA), thiobarbituric acid (TBA), ethylenediaminetetraacetic acid (EDTA), sodium hydroxide, H2O2, butylated hydroxy anisole, deoxyribose, Folin-Ciocalteu's phenol reagent, and carbon tetrachloride (CCl4) were purchased from Sisco Research Laboratories Pvt. Ltd., Mumbai, India. All other chemicals were used in high analytical grade.

Plant materials

The bark of H.Salicifolia was collected from the upper hilly region of Eastern Himalayan, Sikkim, India. Authenticated air dried whole bark (400 g) was powdered in a mechanical grinder, and the powdered materials were extracted successively by petroleum ether, chloroform, and methanol using Soxhlet extraction apparatus. The solvents were completely removed from the methanol extract of H.salicifolia (MEHS) (14.3% w/w, yield) under reduced pressure in a rotary vacuum evaporator (Buchi R-210). The concentrated extracts were stored in vacuum desiccators for further use.

Animals

Male Swiss Albino mice (20–25 g) were taken from Rita Ghosh and Co. Kolkata, India. The mice were grouped and housed in polyacrylic cages (38 cm × 23 cm × 10 cm) with not more than six animals per cage. The animals were maintained under standard laboratory conditions (temperature 25–30°C and 55–60% relative humidity with dark/light cycle 12/12 h) and were allowed free access to standard dry pellet diet (Hindustan Lever, Kolkata, India) and water adlibitum. The mice were acclimatized to laboratory conditions for 7 days before commencement of the experiment. All the procedures described were reviewed and approved by the University Animal Ethical Committee.

Acute toxicity

MEHS was administered orally to male Swiss Albino mice to evaluate the acute toxicity as per the reported method.[11]

In vitro free radical scavenging activity

Different in vitro assay were used to measure the scavenging of ROS and RNS. Determination of in vitro DPPH, superoxide, nitric oxide, hydroxyl radical, H2O2 free radical scavenging activities of MEHS was measured, and percentage inhibition was calculated according to the previously used standard methods.[10],[12] The 50% inhibitory concentrations (IC50) of the extracts were calculated from the graph as concentration versus percentage inhibition. The experiments were performed in triplicate.

In vitro antioxidant effect on macrophages

Isolation of mouse peritoneal macrophages cells Mouse peritoneal macrophages cells were lavaged aseptically using ice-cold phosphate buffer saline (0.02 M, pH-7.4). After centrifugation (3000 rpm × 10 min) of macrophages cells at 4°C the pellet was resuspended in phosphate-buffered saline (PBS), and cell viability was confirmed by trypan blue exclusion method.[13],[14]

Inhibition of hydrogen peroxide-induced DNA damage in macrophages cells Isolated mouse peritoneal macrophages were pre incubated with different concentrations (5, 10, 25, 50, and 100 µg/ml) of MEHS for 1 h and further incubated with H2O2 (10 mM) for 2 h. After that DNA was isolated by TCA precipitation methods and oxidative DNA damage was estimated by standard diphenylamine reaction.[15],[16]

In vitro effect on attenuation of nitrite-induced lysis of murine erythrocytes Blood was collected from the mice by cardiac puncture in an EDTA-containing tube. Then the blood containing cells were immediately centrifuged at 2000 rpm for 5 min and subsequently washed with PBS (0.02 M, pH-7,4) for three times to remove excess plasma. Red blood cell (RBC) was lysed by adding 20 volumes of phosphate buffer (20 mM, pH-7.4). In 1.5 ml freshly prepared hemolysate, 1.0 ml of different concentrations of MEHS (10–100 μg/ml) were added each time concomitantly with 0.1 ml sodium nitrite (6.0 mM) and the formation of methemoglobin (MetHb) was monitored spectrophotometrically (631 nm) at 10, 25 and 50 min interval.[17],[18]

In vivo antioxidant activity in mouse peritoneal macrophages The animals were divided into four groups (n = 12):

Group I: Normal vehicle control: Received liquid paraffin (1 ml/kg i.p.) as vehicle control for 2 days (Day 1 and 2).

Group II: CCl4 control: Received CCl4 (1 ml/kg i.p.) in liquid paraffin (1:1 v/v) for 2 days (Day 1 and 2).

Group III: N-acetyl cysteine (NAC) control: Received CCl4 (1 ml/kg i.p.) in liquid paraffin (1:1 v/v) for 2 days (Day 1 and 2). NAC (150 mg/kg i. p.) treatment was started before 24 h of the CCl4 treatment and continued for Day 1 and Day 2.

Group IV and V: MEHS control: Received CCl4 (1 ml/kg i.p.) in liquid paraffin (1:1 v/v) for 2 days (Day 1 and 2). MEHS (50 and 100 mg/kg i. p.) treatment was started before 24 h of CCl4 treatment and continued for Day 1 and 2.

At the day third, the six mice were sacrificed under ether anesthesia, and peritoneal macrophages were lavaged aseptically using ice cold PBS (0.02 M, pH-7.4). Both Superoxide anion production in macrophages cells and SOD in the cell lysate were estimated by NBT methods. Oxidative damage to the DNA was estimated according to the standard protocol. The lipid peroxidation was measured by TBA-reactive substances (TBARS) assay. The nonenzymatic antioxidant reduced GSH in the cells lysate was estimated by 5,5'-dithiobis-(2-nitrobenzoic acid methods.[19]

Data analysis

Statistical analysis was performed using GraphPad Prism (version 5.0, San Diego, California, USA) Software. All data were expressed as mean ± standard error of mean (SEM). The data were statistically analyzed using one-way analysis of variance followed by Dunnett's post hoc test. Results were considered statistically significant when P < 0.05.


   Results Top


The H.salicifolia extract shows a remarkable capacity to scavenge both ROS and RNS as shown by the IC50 values (mean ± SEM) for three individual experiments taken at the μg/ml level. The extract also reduces the oxidative DNA damage in mouse peritoneal macrophage and also reduced the formation of MetHb from murine erythrocyte in vitro. The extract also significantly reduced the generation of free radical and increased the level of endogenous antioxidants in vivo.

In acute toxicity study, the extract was safe up to the dose of 800 mg/kg b.w. p.o. for mice and 50 and 100 mg/kg b.w. doses were used in the present study.

DPPH is a potent stable nitrogen compound which is used for the assessment of free radical scavenging activity. Free radical scavenging potential of the extract at different concentrations was tested by DPPH method [Figure 1]. The results showed that the MEHS reduced the free radical in a concentration-dependent manner. The IC50 values [Figure 1] were successively found to be 5.80 ± 0.05 μg/ml for MEHS and 11.4 ± 0.32 μg/ml for ascorbic acid. The results thus demonstrated good free radical scavenging activity of the MEHS. Superoxide radicals generated from dissolved oxygen by PMS-NADH coupling can be measured by their ability to reduce NBT. The IC50 values [Figure 1] of MEHS and ascorbic acid (96.7 ± 0.26 μg/ml and 22.7 ± 0.23 μg/ml) indicate that the extract has good superoxide radical scavenging activity as compared to standard ascorbic acid. MEHS effectively reduced the generation of nitric oxide radicals from sodium nitroprusside solution in a concentration-dependent manner as compared to standard ascorbic acid. The IC50 values were 76 ± 0.32 μg/ml and 18.7 ± 0.17 μg/ml for MEHS and standard ascorbic acid, respectively [Figure 1]. The extract showed potent hydroxyl radical scavenging activity as compared to standard ascorbic acid. The IC50 values of the extract and standard in this assay were found to be 106 ± 0.19 μg/ml and 21.9 ± 0.31 μg/ml, respectively [Figure 1]. The extract showed potent H2O2 radical scavenging activity as compared to standard ascorbic acid. The IC50 values of the extract and the standard in this assay were respectively found to be 61.4 ± 0.35 µg/ml and 50.1 ± 0.55 µg/ml [Figure 1].
Figure 1: Inhibitory concentrations values. The inhibitory concentrations values of the H. salicifolia and standard ascorbic acid for 1, 1-diphenyl-2-picryl-hydrazyl, superoxide, nitric oxide, hydrogen peroxide, hydroxyl radical scavenging activity are 5.80 ± 0.05 μg/ml and 11. 4 ± 0.32 μg/ml, 96.7 ± 0.26 μg/ml and 22.7 ± 0.23 μg/ml, 76 ± 0.32 μg/ml and 18.7 ± 0.17 μg/ml, 61.4 ± 0.35 μg/ml and 50.1 ± 0.55 μg/ml, 106 ± 0.19 μg/ml and 21.9 ± 0.31 μg/ml. All the values are mean ± standard error of mean (n = 3)

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H.salicifolia attenuates the oxidative DNA damage (P < 0.05) to the macrophage cells in a concentration-dependent manner as compared to the respective standards (deoxyribose for in vitro, NAC for in vivo) and normal control both in vitro and in vivo [Table 1]. To assess the effect of MEHS on oxidative DNA damage induced by H2O2 in the mouse macrophages cells, we measured its effect on the cells at different concentrations and subsequently the isolated DNA was estimated by diphenylamine reagent. H2O2 (10 mM) significantly (P < 0.01) caused DNA damage in mouse macrophages that was attenuated by MEHS in a concentration-dependent manner [Table 1]. To highlight the mechanism of action, we also checked the in vitro H2O2 scavenging activity of MEHS. We found that MEHS scavenged the H2O2 in a concentration depended on manner as discussed above.
Table 1: MEHS attenuates the oxidative DNA damage on mouse peritoneal macrophages in vitro and in vivo. Different concentrations of MEHS (in vitro 10-50 μg/mL, in vivo low and high dose) decreased the oxidative DNA damage in concentration dependent manner

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H.salicifolia extract showed a concentration dependent inhibition of the RBC hemolysis as compared to control [Table 2]. Several free radical species are generated during the course of nitrite-induced oxidation of Hb. Hb is the primary oxygen-transport protein in vertebrates; it can be converted to MetHb by multiple pharmacological and chemical insults, including the nitrites, with consequent loss of its oxygen-carrying capacity which may predispose to hypoxia. Moreover, several free radical species are generated during the course of nitrite-induced oxidation of Hb; the elevated ROS formation in the vascular wall is a key feature of cardiovascular diseases and contributes to endothelial dysfunction and vascular inflammation.[14] Different concentrations of MEHS showed good antioxidant activity against the nitrite-induced damage to RBC in a concentration depended manner.
Table 2: Effect of MEHS in nitrate induces lysis of murine erythrocytes: Different concentrations of MEHS (10-100 μg/mL) decreased the lysis of RBC concentration dependent manner in response to time

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Generation of free radical by CCl4 inside the cells occur by at least two mechanisms. The first of which is the classical example of the biotransformation of CCl4 to a free radical species. Metabolism of CCl4 to the trichloromethyl radical by the hepatic mixed-function oxides system results in the initiation of lipid peroxidation, protein-lipid cross linkages, and altercation of DNA. The second mechanism of free radicals generation involves reduction of CCl4 to less stable free radical intermediates, which are oxidized by molecular oxygen to give superoxide.[20] The generation of superoxide anion reduces the SOD level in mouse peritoneal macrophages.[21]

Intraperitoneal administration of CCl4 induces the production of superoxide anions in mouse macrophages and also in the peripheral blood lymphocytes which lead a subsequent lipid peroxidation and protein oxidation.[14] The lipid peroxidative degradation of the cell membrane was evident from the elevation of TBARS which is due to increased level of malondialdehyde (MDA) and also from the decrease in activity of free radical scavenging enzyme SOD in the CCl4 treated animals. SOD is the key enzyme in scavenging the superoxide radicals, and this antioxidant enzyme plays an important role in the body defense mechanism against the harmful effects of the free radicals in biological systems.[14] Different concentrations of MEHS was able to mitigate the toxic effects of CCl4 in a concentration depended manner. CCl4 in mouse peritoneal macrophages resulted in a significant rise in intracellular superoxide anions when compared to normal mouse macrophages. MEHS significantly reduced the superoxide anion level [Figure 2]. The activity of SOD was measured for further investigation of the effect of CCl4 on the regulation of intracellular superoxide anions level. There was a significant reduction of SOD level in CCl4 treated mouse cells which were significantly enhanced by NAC and MEHS treatment, respectively, due to their free radicals scavenging property [Figure 3].
Figure 2: Effect of methanol extract of Hippophae salicifolia on carbon tetrachloride induces Superoxide anion in mouse peripheral blood macrophage. All data were expressed as mean ± standard error of mean (n = 6). Where **P< 0.001 (with respect to normal) and *P< 0.01 (with respect to carbon tetrachloride control) are considered to be highly significant and significant, respectively

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Figure 3: Effect of methanol extract of Hippophae salicifolia on carbon tetrachloride induces Superoxide Dismutase in mouse peritoneal macrophages. All data were expressed as mean ± standard error of mean (n = 6). Where **P< 0.001 (with respect to normal) and *P< 0.01 (with respect to carbon tetrachloride control) are considered to be highly significant and significant, respectively

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Lipid peroxidation results in the formation of ROS species and subsequently elevates the level of MDA. In the present study, the MDA level was significantly increased in CCl4 control animals when compared to normal control animals. Interestingly, treatment with MEHS and NAC significantly reduced the MDA levels as compared to the CCl4 control group [Figure 4]. The level of reduced GSH was significantly decreased in CCl4 control group when compared to normal control group. Administration of MEHS in a dose dependent manner significantly raised the reduced GSH level as compared to CCl4 control animals [Figure 5].
Figure 4: Effect of methanol extract of Hippophae salicifolia on carbon tetrachloride induces lipid peroxidation in mouse peritoneal macrophages. All data were expressed as mean ± standard error of mean (n = 6). Where **P< 0.001 (with respect to normal) and *P< 0.01 (with respect to carbon tetrachloride control) are considered to be highly significant and significant, respectively

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Figure 5: Effect of methanol extract of Hippophae salicifolia on carbon tetrachloride induces lipid peroxidation in mouse peritoneal macrophages. All data were expressed as mean ± standard error of mean (n = 6). Where **P< 0.001 (with respect to normal) and *P< 0.01 (with respect to carbon tetrachloride control) are considered to be highly significant and significant, respectively

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   Discussion Top


Typical antioxidants are usually proton donors, ex-ascorbic acid, tocopherol, GSH or L-cysteine are all known to be reductive antioxidants that scavenge superoxide, hydroxyl radical or hydrogen peroxy radical. Many researchers examine the capability of proton donative antioxidants as free radical scavengers. DPPH in methanol or chloroform solution shows strong absorption at around 520 nm, which gives deep violet color to the solution. This compound can accept an electron or a hydrogen radical to become a stable irreversible product.[22]

Oxidative DNA damage causes mutations and consequently genomic instability thereby leading to various diseases including cancer and Alzheimer's disease. Endogenous DNA damage is generally triggered by intermediates of oxygen metabolism that either attack the DNA bases or the deoxyribosyl backbone of DNA.[13] Alternatively, oxyradicals can attack other cellular components such as lipids to generate reactives that couple to DNA bases. Hence, in the present experiment, studying of the effects of various free radicals such as OH , H2O2, or nitric oxide and their subsequent mitigation by the plant extract provide supportive justification to the ameliorative role of the extract against DNA damage.[15] In our experiment, H2O2 was used to induce oxidative DNA damage. This reactive intermediate produced in the body via metabolism may interact with various metals especially Fe 2+ present in the vicinity of DNA strands thereby generating the reactive hydroxyl radical as is implicated in the Fenton reaction:

Fe 2+ + H2O2 ----------> Fe 3+ + OH + OH

The highly reactive hydroxyl radical is a major player in oxidative DNA damage that adds to DNA bases or abstracts hydrogen atoms to form various adducts. Hence, H2O2 is a precursor of this oxidant that insults DNA and hence quenching of H2O2 is imperative to protect DNA.[23] To assess the effect of MEHS on oxidative DNA damage induced by H2O2 in the mouse macrophages cells, we measured its effect on the cells at different concentrations and subsequently the isolated DNA was estimated by diphenylamine reagent. H2O2 (10 mM) significantly (P < 0.01) caused DNA damage in mouse macrophages that was attenuated by MEHS in a concentration-dependent manner [Table 1]. To highlight the mechanism of action, we also checked the in vitro H2O2 scavenging activity of MEHS. We found that MEHS scavenged the H2O2 in a concentration depended manner as discussed above.

The free radicals generated can also cause damage to RBC by bringing about its hemolysis. Several free radical species are generated during the course of nitrite-induced oxidation of Hb. Hb is the primary oxygen-transport protein in vertebrates; it can be converted to MetHb by multiple pharmacological and chemical insults, including the nitrites, with consequent loss of its oxygen-carrying capacity which may predispose to hypoxia. Moreover, several free radical species are generated during the course of nitrite-induced oxidation of Hb; the elevated ROS formation in the vascular wall is a key feature of cardiovascular diseases and contributes to endothelial dysfunction and vascular inflammation.[5] The peroxynitrate (ONO2) is coupling product of nitric oxide and is also implicated in DNA damage. The ONO2 is an extremely reactive oxidant capable of oxidizing DNA. Hence, quenching of this radical along with nitric oxide is important in protecting DNA. H.salicifolia extract showed a concentration dependent inhibition of the RBC hemolysis as compared to control [Table 2]. Different concentrations of MEHS showed good antioxidant activity against the nitrite-induced damage to RBC in a concentration depended manner.[16]

Generation of free radical by CCl4 inside the cells occur by at least two mechanisms. The first of which is the classical example of the biotransformation of CCl4 to a free radical species. Metabolism of CCl4 to the trichloromethyl radical by the hepatic mixed-function oxides system results in the initiation of lipid peroxidation, protein-lipid cross linkages and altercation of DNA.[12] The second mechanism of free radicals generation involves reduction of CCl4 to less stable free radical intermediates which are oxidized by molecular oxygen to give superoxide, a strongly reactive substance that too has its share in DNA damage.[20] This superoxide radical is scavenged in the body by the endogenous enzyme SOD. When the burden of superoxide anion increases considerably in the body, SOD level dramatically reduces in the body.[21]

Intraperitoneal administration of CCl4 induces the production of superoxide anions in mouse macrophages and also in the peripheral blood lymphocytes which subsequently leads to lipid peroxidation and protein oxidation.[14] The lipid peroxidative degradation of the cell membrane was evident from the elevation of TBARS, which is due to increased level of MDA.[14] Different concentrations of MEHS was able to mitigate the toxic effects of CCl4 in a concentration depended manner. CCl4 in mouse peritoneal macrophages resulted in a significant rise in intracellular superoxide anions when compared to normal mouse macrophages. MEHS significantly reduced the superoxide anion level [Figure 2]. Activity of SOD was measured for further investigation of the effect of CCl4 on the regulation of intracellular superoxide anions level. There was a significant reduction of SOD level in CCl4 treated mouse cells which was significantly enhanced by NAC and MEHS treatment, respectively, due to their free radicals scavenging property [Figure 3].

Lipid peroxidation results in the formation of ROS species and subsequently elevates the level of MDA. The MDA has a role to play in DNA damage. It reacts with DNA to form adducts such as dG, dA, and dC, which consequently leads to mutation of the DNA.[5] In the present study, the MDA level was significantly increased in CCl4 control animals when compared to normal control animals. Interestingly, treatment with MEHS and NAC significantly reduced the MDA levels as compared to the CCl4 control group [Figure 4]. The level of reduced GSH was significantly decreased in the CCl4 control group when compared to normal control group. Administration of MEHS in a dose-dependent manner significantly raised the reduced GSH level as compared to CCl4 control animals [Figure 5].


   Conclusion Top


The ability of MEHS to neutralize the oxidative stress both in vitro and in vivo may be due to the presence of high flavonoid content. Flavonoids are hydroxyl group containing compounds. These hydroxyl groups may be responsible in donating a proton (H +) or in forming hydrogen bonds with reactive species thereby neutralizing their toxic effects. The accentuation of the activities of SOD and GSH by the MEHS further buttresses the antioxidant potential of the plant. The significant reduction in the levels of MDA, DNA damage, MetHb formation by the extract (MEHS) in this study suggests a possible preventive role from oxidative stress which may be because of diminization of the free radicals or activation of the endogenous antioxidant system or may be due to presence of reductants in the extract. Hence, it can be concluded that in vitro study including hydroxyl radical, H2O2 and reducing the power of the extract bear a good correlation with the in vivo model in establishing the free radical scavenging activity of MESH. Subsequently the H + donating ability of the MEHS implies that it can have a protective role against DNA damage by decreasing the free radical insults to DNA.

Financial support and sponsorship

Department of Science and Technology (DST), Govt. of India is highly acknowledged for financial support.

Conflicts of interest

There are no conflicts of interest.

 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2]



 

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