in vitro free radical scavenging activity of ethanolic extract of the whole plant of evolvulus...
TRANSCRIPT
• 1 •Chin J Integr Med
Plant-derived bioactive compounds are attractive candidates for drug development.(1) It constitutes an important source of active natural products which differ widely in terms of structures, biological properties and mechanisms of actions. Living cells may generate free radicals and other reactive oxygen species by products as a result of physiological and biochemical processes. An antioxidant is a molecule capable of slowing or preventing the oxidation of other molecules which can produce free radicals to initiate chain reactions that damage cells.(2) An antioxidant terminates the chain reaction by removing the free radical intermediate and inhibits other oxidation reactions by being oxidized itself. Some antioxidants are produced in the body, while others must be sequestered from the diet or supplemented. Plants are endowed with free radical scavenging molecules, such as vitamins, terpenoids, phenolic acids, lignins, stilbenes, tannins, flavonoids, quinones, coumarins, alkaloids, amines, betalains, and other metabolites, which are rich in antioxidant activity.(3) These effects are mainly attributed to their antioxidant activities in scavenging free radicals, inhibiting peroxidation and chelating transition metals. In generally, polyphenols share the same chemical patterns, one or more phenolic groups for which they react as hydrogen donors and in that way neutralize free radicals.(4)
Studies have shown that many of these
antioxidant compounds possess anti-inflammatory, anti-atherosclerotic, anti-tumor, anti-mutagenic, anti-carcinogenic, anti-bacterial, and anti-viral activities.(5) The ingestion of natural antioxidants has been associated with reduced risks of cancer, cardiovascular disease, diabetes, and other diseases associated with ageing.(6) And in recent years, there has been a worldwide trend towards the use of the natural phytochemical present in berry crops, teas, herbs, oilseeds, beans, fruits and vegetables.(7) Currently available synthetic antioxidants like butylated hydroxyl anisole (BHA), butylated hydroxy toluene (BHT), tertiary butylated hydroquinon and gallic acid esters have been suspected to cause or prompt negative health effects. Hence, strong restrictions have been placed on their application and there is a trend to substitute them with naturally occurring antioxidants. Recently there has been an upsurge of interest in the therapeutic potentials of medicinal plants as antioxidants in reducing such free-radical-induced tissue injury. Many plant extracts and phytochemicals have shown to have free radical
ORIGINAL ARTICLE
In Vitro Free Radical Scavenging Activity of Ethanolic Extract of the Whole Plant of Evolvulus Alsinoides (L.) L.
Duraisamy Gomathi, Ganesan Ravikumar, Manokaran Kalaiselvi, Balasubramaniam Vidya, and Chandrasekar Uma
©The Chinese Journal of Integrated Traditional and Western Medicine Press and Springer-Verlag Berlin Heidelberg 2014Department of Biochemistry Karpagam University, Coimbatore (641021), IndiaCorrespondence to: Dr. Chandrasekar Uma, Tel: 91-422-2611146, Fax: 91-422-2611043, E-mail: [email protected]: 10.1007/s11655-014-1763-0
ABSTRACTABSTRACT ObjectiveObjective: To identify the free radical scavenging activity of ethanolic extract of : To identify the free radical scavenging activity of ethanolic extract of Evolvulus Evolvulus
alsinoidesalsinoides. . MethodsMethods: The free radical scavenging activity was evaluated by : The free radical scavenging activity was evaluated by in vitroin vitro methods like reducing methods like reducing
power assay, total antioxidant activity, 2,2-diphenyl-1-picrylhydrazyl (DPPH) reduction, superoxide radical power assay, total antioxidant activity, 2,2-diphenyl-1-picrylhydrazyl (DPPH) reduction, superoxide radical
scavenging activity, 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTSscavenging activity, 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS+) scavenging activity, ) scavenging activity,
hydroxyl radical scavenging assay, and nitric oxide radical scavenging assay, which were studied by using hydroxyl radical scavenging assay, and nitric oxide radical scavenging assay, which were studied by using
ascorbic acid as standard. ascorbic acid as standard. ResultsResults: The extract showed signifi cant activities in all antioxidant assays compared : The extract showed signifi cant activities in all antioxidant assays compared
with the reference antioxidant ascorbic acid. The total antioxidant activity as well as the reducing power with the reference antioxidant ascorbic acid. The total antioxidant activity as well as the reducing power
was also found to increase in a dose-dependent manner. was also found to increase in a dose-dependent manner. ConclusionConclusion: : Evolvulus alsinoidesEvolvulus alsinoides may act as a may act as a
chemopreventive agent, providing antioxidant properties and offering effective protection from free radicals. chemopreventive agent, providing antioxidant properties and offering effective protection from free radicals.
KEYWORDSKEYWORDS Evolvulus alsinoidesEvolvulus alsinoides, ethanolic extract, total antioxidant, reducing power, ascorbic acid, , ethanolic extract, total antioxidant, reducing power, ascorbic acid,
convolvulaceaeconvolvulaceae
• 2 • Chin J Integr Med
scavenging properties,(8) but generally there is still a demand to find more information concerning the antioxidant potential of plant species.(9)
Evolvulus alsinoides (L.) L. (Convolvulaceae), commonly known as "vishnukranti" in India, Africa, and the Philippines, is an important medicinal plant employed for different ailments in India traditionally. It grows in open and grassy places throughout almost all of India and subtropical countries of the world. This plant is used to cure fever, cough, cold, venereal diseases, azoospermia, adenitis and dementia. It has a known nootropic and anti-infl ammatory activity.(10,11) The present study was taken to explore the in vitro antioxidant status of ethanolic extract of Evolvulus alsinoides (L.) L. using various in vitro scavenging assays.
METHODS
Collection of Plant MaterialThe whole plant of Evolvulus alsinoides (L.) L.
used for the investigation was obtained from Coimbatore District, Tamilnadu, India. The plant was authenticated by Dr. P. Satyanarayana, Botanical Survey of India, TNAU Campus, Coimbatore, India. The voucher number is BSI/SRC/5/23/2011-12/Tech-514. Fresh plant material was washed under running tap water, air dried and powdered.
Preparation of Plant ExtractA total of 100 g of dried powdered plant
material were mixed with 500 mL of ethanol by soxhlet apparatus at room temperature. The solvent was completely removed by rotary evaporator and stored at 0–4 ℃ in an air tight container. This crude extract was used for further investigation for potential antioxidant properties. Two milligram of crude extract was dissolved in 1 mL of ethanol and which was used for the study. Ascorbic acid was used as a reference standard and was dissolved in distilled water to make the stock solution with the same concentration (5 mg/mL). Control sample was prepared containing the same volume without any extract or reference standard ascorbic acid.
Determination of In Vitro Antioxidant ActivityThe reduc ing power o f the whole p lant
extract was quantified according to the method of Oyaizu,(12) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity was adopted from those previously described with slight modifi cations.(13) The
total antioxidant potential of sample was determined using ferric reducing antioxidant power (FRAP) by the method of Benzie and Strain.(14) The hydroxyl radical scavenging activity was measured according to the method of Klein, et al.(15) Measurement of superoxide radical scavenging activity was done using the standard method of Liu, et al.(16) Nitric oxide (NO) scavenging activity of the extract was determined by the method of Green, et al(17) and 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS+) radical activity was estimated by the method of Re, et al.(18) The antioxidant activity was evaluated by the phosphomolybdenum method according to the procedure described by Prieto, et al.(19)
Statistical AnalysisFor all the experiments three samples were
analyzed and all the assays were carried out in triplicate. The results were expressed as mean±standard deviation (x–±s). Statistical analysis was performed with SPSS 10.0 software. Differences between groups were evaluated using t-test, and P<0.05 was considered signifi cantly.
RESULTS
Reducing Power AssayThe reducing capacity of a compound may serve
as a significant indicator of its potential antioxidant activity. Standard curves of ascorbic acid as well as the ethanolic extract of Evolvulus alsinoides are shown in Figure 1. The Evolvulus alsinoides ethanolic extract ability increases with the increasing concentration (200–1000 μg/mL) like the antioxidant activity of standard curve.
Figure 1. Reducing Power Assay of Evolvulus Alsinoides
Notes: P<0.05, compared with 200 μg/mL concentration; and the same below
Concentration (μg/mL)
0 200
Opt
ical
den
sity
at 7
00 n
m
1.00.90.80.70.60.50.40.30.20.1
0400 600 800 1000
Ascorbic acid
Evolvulus alsinoides
Total Antioxidant ActivityTotal antioxidant capacity of the Evolvulus
alsinoides ethanolic extract, expressed as the number
• 3 •Chin J Integr Med
Figure 2. Total Antioxidant Activity of Evolvulus Alsinoides
DPPH Radical Scavenging ActivityThe scavenging of DPPH by the extract was
increased in dose-dependent manner. Figure 3 illustrates a signifi cant decrease in the DPPH radical due to the scavenging ability of extracts and ascorbic acid. The ethanol extract showed maximum activity of 75% at 5 mg/mL, where as ascorbic acid at the same concentration exhibited 85% inhibition. The half maximal inhibitory concentration (IC50) values were found to be 560 μg/mL for ethanolic extract and 510 μg/mL for ascorbic acid, respectively.
Figure 3. DPPH Radical Scavenging Activity of Evolvulus Alsinoides
Figure 4. Hydroxyl Radical Scavenging Activity of Evolvulus Alsinoides
Figure 5. Superoxide Radical Scavenging Activity of Evolvulus Alsinoides
high radical scavenging activity at 87% and ethanolic extract exhibited stronger scavenging effect of 78% at the high dose of 1000 μg/mL.
Inhi
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Concentration (μg/mL)
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Ascorbic acid
Evolvulus alsinoides
Inhi
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Evolvulus alsinoides
Concentration (μg/mL)
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Evolvulus alsinoides
Concentration (μg/mL)
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1.8
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1.4
1.2
1.0
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0.6
0.4
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0400 600 800 1000
Evolvulus alsinoides
Superoxide Radical Scavenging ActivityFigure 5 shows the superoxide scavenging
activity of Evolvulus alsinoides in which the plant extract exhibited inhibitory concentration of 80% and ascorbic acid showed the inhibitory activity of 83%.
ABTS+ Scavenging ActivityThe principle behind the ABTS assay technique
involves the reaction between ABTS and potassium persulphate to produce the ABTS radical cation (ABTS+) a blue green chromogen. In the presence of antioxidant reductant, the coloured radical is converted back to colourless ABTS, the absorbance of which is measured at 734 nm (Figure 6). The ethanolic extract were found to exhibit an inhibition of ABTS+ radical with an IC50 value of 420 μg /mL when compared with reference standard ascorbic acid (IC50 : 660 μg /mL).
NO Scavenging ActivityIn vitro inhibition of NO radical is a measure of
antioxidant activity of plant drugs. The NO radical scavenging activity of Evolvulus alsinoides plant
of gram equivalents of ascorbic acid, is shown in Figure 2. The phosphomolybdenum method was based on the reduction of Mo (Ⅵ) to Mo (Ⅴ) by the antioxidant compound and the formation of a green phosphate/Mo (Ⅴ) complex with a maximal absorption at 695 nm. Total antioxidant activity was found to increase in a dose-dependent manner.
Hydroxyl Radical Scavenging ActivityThe results of hydroxyl radical-scavenging
powers of the plant extract and ascorbic acid are given in Figure 4. The sample as well as standard exhibited obvious scavenging activity in a dose-dependent manner. The standard ascorbic acid showed valuable
• 4 • Chin J Integr Med
as inflammation, cataract, cirrhosis and reperfusion of injury. Herbal drugs containing radical scavengers are gaining importance in the prevention and treatment of such diseases.(20) In this present study, the antioxidant activity of the ethanolic extract of the whole plant Evolvulus alsinoides was investigated by using different scavenging methods like reducing power assay, total antioxidant, DPPH, ABTS, etc. All the methods have proven the effectiveness of the ethanolic extract compared with the reference standard antioxidant ascorbic acid.
The reducing capacity of a compound may serve as a significant indicator of its potential antioxidant activity. However, the activity of antioxidants has been assigned to various mechanisms such as prevention of chain initiation, binding of transition metal ion catalysts, decomposition of peroxides, prevention of continued hydrogen abstraction, reductive capacity and radical scavenging.(21) The reducing power activity of Evolvulus alsinoides extract increased with the increasing dosage from 200–1000 μg/mL. This result showed that it consists of hydrophilic compound that causes reducing power.(22)
The antioxidative effect is mainly due to phenolic components, such as phenolic acids and phenolic diterpenes. The antioxidant activity of phenolic compounds is mainly due to their redox properties, which can play an important role in absorbing and neutralizing free radicals, quenching singlet and triplet oxygen, or decomposing peroxides.(23) Total antioxidant capacity mainly concentrates on the thermodynamic conversion and measures the number of electrons or radicals donated or quenched by a given antioxidant molecule, and measures the capacity of biological samples under defined conditions. This study revealed that the antioxidant activity of the extract exhibited increasing trend with the increasing concentration of the plant extract. Thus, the extract demonstrated electron donating capacity, may act as radical chain terminators, transformating reactive free radical species into stable non-reactive products.(24,25) In this assay extract was found to have higher activity when compared with the standard (ascorbic acid).
DPPH is used for the evaluation of antioxidant capacity in a short time and frequently applied for testing food products. The result of DPPH scavenging
Figure 7. NO Scavenging Activity of Evolvulus Alsinoides
extract were studied and compared with ascorbic acid (IC50 : 560 μg /mL). Concentration of plant extract required for IC50 was found to 580 μg /mL (Figure 7).
Inhi
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Ascorbic acid
Evolvulus alsinoides
Concentration (μg/mL)
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Figure 6. ABTS+ Scavenging Activity of Evolvulus Alsinoides
Concentration (μg/mL)
0 200
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)70
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Ascorbic acid
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DISCUSSION
Damage to cells caused by free radicals is believed to play a central role in the aging process and in disease progression. Many aromatic, medicinal and spice plants contain compounds that possess confirmed strong antioxidative components. The medicinal value of plants has assumed an important dimension in the past few decades owing largely due to the discovery as a rich source of antioxidants that combat oxidative stress through their redox active secondary metabolites and the rising concerns about the side effects of synthetic drugs. These factors have inspired the widespread screening of plants for possible antioxidant properties. Scientifi c interests in medicinal plants are emerging as plants are invaluable sources of new drugs and plant based antioxidants are preferred to the synthetic ones because of safety concerns.
Recently, much attention has been focused on the reactive oxygen species and free radicals, which play in the genesis of various diseases such
• 5 •Chin J Integr Med
activity assay in this study indicated that the plant was potently active. This suggests that the plant extract contains compounds that are capable of donating hydrogen to a free radical in order to remove odd electron which is responsible for radical's reactivity. DPPH free radical scavenging assay can be helpful for primary screening and fi nding of novel antioxidants.(26)
The hydroxyl radical in the cells can easily cross cell membranes at specific sites, react with most biomolecules and furthermore cause tissue damage and cell death. Thus, removing hydroxyl radical is very important for the protection of living systems.(27) Superoxide anion radical is known as an initial radical and plays an important role in the formation of other reactive oxygen-species, such as hydrogen peroxide, or singlet oxygen in the living systems.(28) Superoxide anion radical is one of the strongest reactive oxygen species among the free radicals that are generated. The scavenging activity of this radical by the plant extract compared favourably with the standard reagents such as gallic acid suggesting that the plant is also a potent scavenger of superoxide radical.(5)
ABTS+ radical assay, can be used in both organic and aqueous solvent systems,(29) therefore, is often used in evaluating total antioxidant power of single compounds and complex mixtures of various plants.(30) The free radical scavenging activity by this method of ethanolic extract of Evolvulus alsinoides suggests for the antioxidant activity. NO is a free radical which plays an important role in the pathogenesis of pain, inflammation, neural signal transmission, immune response, and control of blood pressure, etc.(31) Results obtained in the present study indicated that ethanolic extract of Evolvulus alsinoides showed a free radical scavenging activity which was signifi cantly compared with the free radical scavenging activity of ascorbic acid.
The results obtained in present study indicate that ethanolic extract of whole plant Evolvulus alsinoides inhibits free radical scavenging activity. The overall antioxidant activity of these extracts might be attributed to its flavonoids, phenolic and other phytochemical constituents. These could be a source of natural antioxidant that could have greater importance as therapeutic agent in preventing or slowing oxidative stress related degenerative diseases.
Confl ict of InterestNone.
AcknowledgementThe authors are thankful to the Chancellor, Advisor, Vice
Chancellor and Registrar of Karpagam University for providing
facilities and encouragement.
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(Received March 18, 2012)Edited by Yu Ming-zhu