antioxidant activities

Indian J Pharmacol. 2011 Sep-Oct; 43(5): 557–562.doi: 10.4103/0253-7613.84972

PMCID: PMC3195127

Antioxidant activities of bark extract from mangroves, Bruguiera cylindrica (L.)Blume and Ceriops decandra PerrM. Krishnamoorthy, J. M. Sasikumar, R. Shamna, C. Pandiarajan, P. Sofia, and B. Nagarajan

Division of Plant Biotechnology, Institute of Forest Genetic and Tree Breeding (IFGTB), Coimbatore 641 021, IndiaDepartment of Biotechnology, School of Life Sciences, Karpagam University, Coimbatore 641 021, India

Correspondence to: Dr. J. M. Sasikumar, E-mail: [email protected]

Received May 3, 2010; Revised September 24, 2010; Accepted July 1, 2011.

Copyright : © Indian Journal of Pharmacology

This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unported,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Objectives:

The antioxidant activities of two Indian mangrove plants, Bruguiera cylindrica and Ceriops decandra,were investigated.

Materials and Methods:

Total phenolics and total flavonoid contents of the mangroves were determined using folin-ciocalteureagent method and aluminium chloride method, respectively. Antioxidant capacity was assessed bythe following methods: 1,1-diphenyl-2-picryl hydroxyl (DPPH.) quenching assay; 2,2’- azinobis-3-ethylbenzothiozoline-6-sulfonic acid (ABTS. ) cation decolorization test; scavenging capacitytowards hydroxyl ion radicals (.OH); reductive capacity; and antihemolytic activity.

Results:

The mangroves yielded 233.3 ± 0.062 and 283.31 ± 0.04 mg gallic acid equivalent/g phenoliccontents and 11.6 ± 0.12 and 15.1 ± 0.02 mg quercetin equivalent/g flavonoid contents. The methanolextracts of both mangroves exhibited high antiradical activity against DPPH., ABTS. , and .OHradicals. The reductive capacity of the extracts increased with increasing concentration of samples.The extracts also inhibited H O induced hemolysis in cow blood erythrocytes. The antioxidantactivities were found stronger than that of the reference standard, butylated hydroxy toluene (BHT).The antioxidant activity of mangrove plants was correlated with total phenolics and flavonoidcontents.

Conclusion:

Both plants can be considered as good sources of natural antioxidants for medicinal uses. Furtherstudies are necessary to isolate active principles responsible for the overall antioxidant activity of theextracts.

Keywords: ABTS assay, Bruguiera cylindrica, Ceriops decandra, DPPH., H induced mangroves,red blood cell hemolysis, total flavonoids, total phenolics

Introduction

Free radicals, such as reactive oxygen species (ROS) and reactive nitrogen species (RNS), induceoxidative damage to biomolecules, such as deoxyribonucleic acid (DNA), lipids, proteins, and

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Antioxidant activities of bark extract from mangroves, Bruguiera cylindr... http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3195127/?report=printable

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carbohydrates. They have been implicated with variety of pathological processes including cancer,diabetes, atherosclerosis, neurodegenerative disorders and arthritis. Antioxidants of plant origin arebelieved to help protect the cells from free radical damage. Therefore, several studies in recent yearsshowed that polyphenols in plants scavenge active oxygen species and effectively prevent oxidativecell damage.[1] Hence, considerable attention has already been focused on the isolation,characterization, and utilization of natural antioxidants as potential disease preventing agents.However, little is known about the antioxidant potentials of Indian mangroves.

Bruguiera cylindrica (L.) Blume, (family: Rhizhophoraceae) is a rare tree mangrove found along thewestern coast of India. The bark of B.cylindrica is used to stop hemorrhage and applied to malignantulcers.[2] Pentacyclic triterpenoid esters from the fruits[3] and brugine alkaloid from stem andbark[4] have been isolated. Ceriops decandra (Griff.) Ding Hou (family: Rhizhophoraceae) isreported to cure hepatitis.[5] Earlier, antiviral activity of B. cylindrica and C. decandra leaves wasstudied.[6] A study was carried out to evaluate phenolic content, DPPH (1,1 - diphenyl - 2-picrylhydroxyl) reduction activity and reducing power of leaves on B. cylindrica and C. decandra leaves.[7]Leaf extract of C. decandra exhibited antidiabetic activity in alloxan induced diabetic rats.[8] Leaf ofC. decandra was found to have radical scavenging activity against superoxide anion.[9] However,there has been no detailed in vitro studies on antioxidant properties of stem bark of these mangrovemedicinal plants from Pichavaram forest, Tamil Nadu, India. Hence, in the present study, we aimedto evaluate the total phenolic content and total flavonoid content and to examine the antioxidantactivities. In the present study, the antioxidant effects of B. cylindrica and C. decandra wereevaluated using in vitro assays including DPPH radical quenching test, ABTS radical decolorizationtest, ˙ OH scavenging assay, reducing power capacity, and antihemolytic activity.

Materials and Methods

General

The ultraviolet (UV) spectra were recorded on Elico SL 177, India UV-visible (UV-VIS)spectrophotometer. All chemicals used including the solvents were of analytical grade; 1, 1-diphenyl-2-picryl hydrazyl (DPPH), 2, 2’- azinobis-3-ethylbenzothiozoline-6-sulfonic acid (ABTS), quercetin,butylated hydroxy toluene (BHT) were purchased from Merck Chemical Co., Mumbai, India. Gallicwas procured from Riedel-de-Hahn, Germany.

Plant Material

The stem barks of B. cylindrica and C. decandra were collected from Pichavaram mangrove forest(area 1400 ha), Tamil Nadu, India in June 2008. The taxonomic identification was done in BotanicalSurvey of India (BSI), Southern circle, Coimbatore. A voucher specimen was deposited in theDepartment of Plant Biotechnology, IFGTB, Coimbatore, India.

Preparation of Extract

The stem barks of B. cylindrica and C. decandra were washed in tap water, shade dried, andpowdered. The powder (100 gm) was exhaustively extracted with methanol in the ratio of 1:7 (w/v) for24 h by using soxhlet apparatus. The extract was evaporated to dryness using rotary flash evaporator(Buchi type, Switzerland). Different concentrations of extracts were prepared from the resultant crudemethanol to determine in vitro antioxidant assays and polyphenolic contents.

Total Phenolic Content

The content of phenolics was determined UV spectrophotometrically using folin-ciocalteumethod.[10] Aliquots (0.1 ml) of the extracts were mixed with 0.5 ml of folin-ciocalteu reagent andmade up to 3 ml with distilled water. After 3 min, 2 ml of sodium carbonate (20%) was added andmixed thoroughly. The sample was then incubated for 5 min at 50°C and cooled. The absorbance wasmeasured at 650 nm against the reagent blank. The total phenolic content was expressed as mg gallicacid equivalent per gm of extract. The coefficient of determination was r = 0.9968.

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Total Flavonoid Content

The determination of total flavonoid content in B. cylindrica and C. decandra was based onaluminium chlroride method.[11] A volume of 0.5 ml of AlCl ethanol solution (2 %) was added to 0.5ml of sample solution. After 1 h incubation at room temperature, the absorbance was measured at 420nm. Extract samples were evaluated at a final concentration of 0.1 mg/ml. The total flavonoid contentwas calculated as mg quercetin equivalent per gm of extract. The coefficient of determination was r =0.9965.

DPPH Quenching Activity

DPPH radical scavenging activity was determined using the method described previously[12] withminor changes. Briefly, 5 ml of methanol solution, DPPH (0.1 mM) was added to 1 ml of the samplesolution at different concentrations (5-1000 μ/ml) and vortexed. The mixtures were still stood for 20min at room temperature. After incubation, the absorbance was spectrophotometrically measured at517 nm against a blank. BHT was used as reference compound. The inhibition percentage wascalculated using the following formula:

Inhibition of DPPH (%) = 1 – Sample /Control × 100, where, Sample was theabsorbance of the sample and Control was the absorbance of the control. The results wereexpressed as EC , which means the concentration at which DPPH radicals were quenched by 50%.

2, 2’- Azinobis (3-Ethylbenzothiazoline Sulphonic Acid) ABTS Cation Decolorization Test

ABTS cation decolorization activity of the mangrove extracts was determined by the methoddescribed previously.[13] The test was based on the relative activity of antioxidants to quench theradical cation ABTS . The stock solution included ABTS solution (7 mM) and potassium persulfatesolution (2.4 mM). The working solution was then prepared by mixing the two stock solutions inequal quantities and allowing them to react for 12 h at room temperature in the dark. The solutionwas then diluted by mixing 1 ml ABTS solution with 60 ml methanol to obtain an absorbance of0.706 ± 0.001 units at 734 nm using the spectrophotometer. Bark extracts and BHT of variousconcentrations (5-1000 mg/ml) were allowed to react with 1 ml of the ABTS solution and theabsorbance was taken at 734 nm after 7 min using the spectrophotometer. The ABTS scavengingcapacity of the extract was compared with that of BHT. Percentage inhibition was calculated asABTS radical scavenging activity (%) = 1 – Sample /Control × 100, where, Samplewas the absorbance of the sample and Control was the absorbance of the control.

Hydroxyl Radical Scavenging Activity

The hydroxyl radical (˙ OH) scavenging activity of the B. cylindrica and C. decandra extracts wasmeasured based on the method described previously.[14] Various concentrations of extracts (5-1000μg/ml) were added with 1.0 ml of iron-ethylenediaminetetraacetic acid (EDTA) solution (0.13%ferrous ammonium sulfate and 0.26 % EDTA), 0.5 ml of EDTA solution (0.018%), and 1.0 ml ofdimethyl sulfoxide (DMSO) (0.85% v/v in 0.1 M phosphate buffer, pH 7.4). The reaction was initiatedby adding 0.5 ml of ascorbic acid (0.22%) and incubated at 80-90°C for 15 min in a water bath. Afterincubation, the reaction was terminated by the addition of 1.0 ml of ice-cold tricholoro acetic acid(TCA) (17.5% w/v). Three ml of Nash reagent (75.0 g of ammonium acetate, 3.0 ml of glacial aceticacid, and 2 ml of acetyl acetone were mixed and raised to 1 l with distilled water) was added and leftat room temperature for 15 min. The reaction mixture without sample was used as control. Theintensity of the color formed was measured spectrophotometrically at 412 nm against reagent blank.BHT was used as positive control. The ˙ OH scavenging activity was calculated by the followingformula: ˙ OH scavenging activity = 1 – (difference in absorbance of sample/difference in absorbanceof blank) ×100.

Reducing Capacity

The reducing power of the extracts of B. cylindrica and C. decandra was measured using thepotassium ferricyanide reduction method.[15] Various concentrations of the extracts and standards

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(50, 100, 250, 500, 750, 1000 mg/ml) were added to 2.5 ml of (0.2 M) sodium phosphate buffer (pH6.6) and 2.5 ml of potassium ferricyanide [K Fe (CN) ] (1%) solution and vortexed. After incubationat 50°C for 20 min, 2.5 ml of TCA (10%, w/v) was added to all the tubes and centrifuged at ×3000 gfor 10 min. Afterwards, upper layer of the solution (5 ml) was mixed with deionized water (5 ml). Tothis, 1 ml of FeCl (1%) was added to each test tube and incubated at 35°C for 10 min. The formationof Perl's Prussian color was measured at 700 nm in a UV-VIS spectrophotometer. Increasedabsorbance of the reaction mixture indicated increasing reducing power. Here, the EC value is theeffective concentration at which absorbance was 0.5 % for the reducing capacity. BHT was used forcomparison.

Antihemolytic Activity

Inhibition of H 0 induced red blood cell (RBC) hemolysis of methanolic extract was examined by thein vitro method described previously.[16] The erythrocytes from cow blood were separated bycentrifugation and washed with saline or isotonic sodium phosphate buffer (pH 7.4) until thesupernatant was colorless. The erythrocytes were then diluted with saline or phosphate buffer to givea 4% suspension. Varying amounts of sample (200-1000 μg/ml) with saline or buffer were added to 2ml of the suspension of erythrocytes and the volume was made up to 3.5 ml with saline or buffer. Thismixture was preincubated for 5 min and then 0.5 ml H 0 solutions of appropriate concentration insaline or buffer were added. The concentration of H 0 in the reaction mixture was adjusted so as tobring 90% hemolysis of blood cells after 120 min incubation. About 80-90% hemolysis of raterythrocytes was obtained after 4-6 h. Incubation was concluded after these time intervals bycentrifugation during 5 min at ×l000 g and the extent of hemolysis was determined by measurementof the absorbance at 540 nm corresponding to hemoglobin liberation. Antihemolytic activity wasexpressed as the inhibition percentage and was calculated using the following formula: Antihemolyticactivity (%) = 1 – Sample /Control × 100, where, Sample was the absorbance of thesample and Control was the absorbance of the control.

Statistical Analysis

The experimental results concerning the study were mean ± standard deviation of three parallelmeasurements. Linear regression analysis was used to calculate the EC values. One-way analysis ofvariance (ANOVA) and Duncan's Multiple Range Test (DMRT) were carried out to determinesignificant differences between the parameters by Statistical Package for the Social Sciences (SPSS)(version 10 for Windows 98, SPSS Inc.).

Results

Total Phenolic and Flavonoid Contents

The phenolic and flavonoid contents of the bark of B. cylindrica and C. decandra are reported inTable 1. The amount of phenolic contents was 233.3 and 258.33 mg of gallic acid equivalents/g dryplant material in B. cylindrica and C. decandra, respectively. The flavonoid content was 11.6 and 15mg of quercetin equivalents (QE)/g of dry material in B. cylindrica and C. decandra.

DPPH Quenching Capacity

The extracts of B. cylindrica and C. decandra exhibited a concentration dependent antiradical activity[Figure 1a] by quenching DPPH radical with values EC of 5.5 ± 0.58 and 2.1 ± 0.28, respectively.The DPPH scavenging activity of the extracts is higher than that of the BHT (EC 26 ± 0.005) (P <0.05) [Table 2].

ABTS Scavenging Capacity

Stable free radical ABTS was scavenged by bark extracts of both the mangrove species produced bythe oxidation of 2, 2-azinobis-3-ethylbenzothiazoline-6-sulphate is illustrated in [Figure 1b]. Themethanol extracts of the selected mangroves mopped up more than percentage of radicals in vitro.ABTS scavenging activity of the B. cylindrica bark extract was higher than that of C. decandra and

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comparable to that of BHT (P < 0.05) [Table 2].

OH Scavenging Capacity

Figure 1c depicts a concentration-dependent inhibition by the extracts of B.cylindrica and C.decandra against ˙ OH as evident by low EC value 41 ± 0.002 mg/ml [Table 2]. The extractsshowed significantly better scavenging activity than BHT, the reference standard [Table 2].

Reducing Capacity

The reduction of ferrous ion (Fe ) to ferric ion (Fe ) is measured by the intensity of the resultantblue-green solution which absorbs at 700 nm. The methanol extracts exhibited a concentrationdependent increase in reducing power [Figure 1d]. They caused significant elevation of reducingpower potential with optical density (OD) value of 1.56 ± 0.021 in B. cylindrica and 1.927 ± 0.56 in C.decandra at 1000 mg/ml.

Antihemolytic Capacity

The methanolic extracts of B. cylindrica and C. decandra bark inhibited the hemolysis of RBC byH 0 induced peroxy radicals in a concentration dependent manner [Figure 1e] with the values ofEC 11.5 ± 0.24 and EC 24.4 ± 0.04 mg/ml, respectively [Table 2].

Correlation Between Phytoconstituents and Antioxidant Assays

In this present study, the association between antioxidant activities and total phenolic content andtotal flavonoid content was observed based on regression analysis. A positive linear correlation wasestablished between total phenolic content and corresponding inhibitory efficacy on DPPH radical bythe extract (r = 0.833) of B. cylindrica. Positive correlations were observed between total phenoliccontent and ABTS cation scavenging (r = 0.788) and reducing power capacity (r = 0.8). Lowercorrelations were found between total phenolic content and hydroxyl ion radical scavenging (r =0.524) and antihemolytic activity (r = 0.461). In the methanol extract of C. decandra, positivecorrelations were established between total phenolic content and DPPH (r = 0.96), hydroxyl ion (r= 0.93), ABTS cation (r = 0.733) radical scavenging and antihemolytic activity (r = 0.646).

Discussion

Recent years have seen an exponential increase in research on the antioxidant properties of medicinalplants. The identification and investigation on antioxidants from medicinal plants is a fast expandingfield of research and several antioxidants have been investigated such as flavonoids and otherphenolic compounds. In this study, we examined total phenolics and flavonoids and evaluated theantioxidant effects of crude methanolic extracts of B. cylindrica and C. decandra bark. Methanolicextracts of both the plants exhibited promising antioxidant activity in all the experimental modelsadopted.

In this study, the barks of the mangroves, B. cylindrica and C. decandra, collected from Pichavaram,South India were found to contain appreciable content of polyphenolics. The total phenolic content inC. decandra is higher when compared to the species (94.4 mg/g) found in Sundarbans, India.[17]Mangroves in Sundarbans, North India were found to contain high content of polyphenolics liketannins.[18] Previous reports revealed that mangroves are rich in polyphenols and tannins.[19,20]Phenolics widely encountered in the plants tested as the most active radical scavengers. They arepresent in a variety of plants utilized as important components of both human and animal diets.[21,22] There is a strong evidence on the preventive effects of phenolics on age related chronicdiseases.[23]

The DPPH assay constitutes a quick and low cost method which has frequently been used for theevaluation of the antioxidative potential of various natural products.[24] In this study, the extracts ofB. cylindrica and C. decandra exhibited a concentration dependent antiradical activity. The DPPHscavenging activity of the extracts are higher than that of mangrove studied from Sundarbans, India(IC = 0.65 mg/ml).[17] It was evident that the bark extracts of B. cylindrica and C. decandra

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hydrogen donating ability to act as antioxidants.[25] In this study, C. decandra showed higherpotential among the mangroves hitherto investigated.

The decolorization of ABTS cation reflects the capacity of an antioxidant species to donate electronsor hydrogen atoms to deactivate these radical species.[26] In the present study, the methanol extractsof the investigated mangrove plants showed strong decolorizing effect towards ABTS cation in adose dependent manner. It is noteworthy that ABTS scavenging activity of the B. cylindrica barkextract was higher than that of C. decandra and comparable to that of BHT.

Hydroxyl radical (OH ), the most reactive free radical, has the capacity to conjugate with nucleotidesin DNA, cause strand breakage, and lead to carcinogenesis, mutagenesis and cytotoxicity.[27] The OHion scavenging results indicate that extracts scavenged OH radicals and percentage inhibition wasproportional to the concentration of the extract. Earlier, Babu et al.,[28] observed a strong OHradical scavenging activity of a mangrove, Acanthus ilicifolius leaf extract. In our study, thescavenging effect of methanolic bark extracts of B. cylindrica and C. decandra was found strongerthan that of A. ilicifoilius.

The methanol extracts caused significant elevation of reducing power. The reducing powers of theextracts were higher when compared with BHT (1.022 at 1000 mg/ml). The reduction of ferrous ion(Fe ) to ferric ion (Fe ) is measured by the intensity of the resultant blue-green solution whichabsorbs at 700 nm. The reducing properties are generally associated with the presence of reductones.Gordan[29] reported that the antioxidant action of reductones is based on the breaking of the freeradical chain by donating a hydrogen atom. The result presented here indicates that the markedantioxidant activity of bark extracts of B. cylindrica and C. decandra seems to be due to the presenceof polyphenols which may act as reductones to convert free radicals into more stable products andterminate free radical chain reaction.

H O mediated lipid oxidation in cow blood erythrocyte membrane induce membrane damage andhemolysis. In the present study, the extracts of the mangroves were reported to possess dosedependent inhibitory effects towards hemolysis of cow blood erythrocyte. The antihemolytic effect ofthe C. decandra was found stronger than that of B. cylindrica and BHT (EC 22 ± 0.48 mg/ml).

In this study, a linear correlation was found between phenolics contents and antioxidant activity.Many supportive reports emphasize the positive correlation between phenolic content and antioxidantefficacy.[30–33] A positive correlation between antioxidant activity and polyphenol content wasfound suggesting that the antioxidant capacity of the plant extracts is due to a great extent to theirpolyphenols.[34,35]

In summary, the results indicate the potential of bark extracts as dietary supplement and in thetreatment of chronic diseases caused by overproduction of free radicals. Additional work is thereforenecessary to fractionate the extract further to elicit a better understanding of how each chemicalfraction contributes to the overall antioxidant activity and to determine whether the unique mixtureof plant phenolics contributes to a synergistic antioxidant activity.

Acknowledgments

The authors are grateful to the management of Karpagam Educational Institutions for its generoussupport and encouragement.

FootnotesSource of Support: Nil,

Conflict of Interest: None declared.

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Figures and Tables


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Table 1

Total phenolic and flavonoid contents of B. cylindrica and C. decandra


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Figure 1a

DPPH quenching activity of methanol extracts of B. cylindrica (BC), C. decandra (CD) and butylated hydroxytoluene (BHT). All the values are presented as mean ± S.E.M (n = 3)


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Table 2

EC values for the methanol extracts of B. cylindrica and C. decandra and standards in various freeradical scavenging assay models

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Figure 1b

ABTS. scavenging activity of methanol extracts of B. cylindrica (BC), C. decandra (CD) and butylated hydroxytoluene (BHT). All the values are presented as mean ± S.E.M (n = 3)

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Figure 1c

(OH˙) scavenging activity of methanol extracts of B. cylindrica (BC), C. decandra (CD) and butylated hydroxytoluene (BHT). All the values are presented as mean ± S.E.M (n = 3)


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Figure 1d

Reducing capacity of methanol extracts of B. cylindrica (BC), C. decandra (CD) and butylated hydroxy toluene(BHT). All the values are presented as mean ± S.E.M (n = 3)


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Figure 1e

Antihemolytic of methanol extracts of B. cylindrica (BC), C. decandra (CD) and butylated hydroxy toluene (BHT).All the values are presented as mean ± S.E.M (n = 3)

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