15 free radical scavenging (dpph) potential in nine mentha species

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DOI: 10.1177/0748233711407238 2012 28: 83 originally published online 6 June 2011Toxicol Ind Health

Nisar Ahmad, Hina Fazal, Iftikhar Ahmad and Bilal Haider AbbasiFree radical scavenging (DPPH) potential in nine Mentha species

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Free radical scavenging (DPPH)potential in nine Mentha species

Nisar Ahmad1,2, Hina Fazal3,4, Iftikhar Ahmad2, andBilal Haider Abbasi1

AbstractMentha species are used in every day life in various food items. These species produce valuable secondarymetabolites that scavenge toxic free radicals. Toxic free radicals can cause different diseases in the humanbody. In the present study free radical scavenging potential (1,1-diphenyl-2-picrylhydrazyl scavenging activity)in nine Mentha species were investigated to evaluate and explore new potential sources for natural antioxi-dants. The activity was performed after different time intervals with incubation period of 30 minutes. Themethanolic extracts revealed that significantly higher activity (82%) was observed in Mentha suaveolens, fol-lowed byMentha longifolia (79%),Mentha officinalis (76%) andMentha piperita,Mentha pulegium,Mentha royleana(75%), respectively. Significantly same activity was observed inMentha arvensis andMentha spicata. Lower activ-ity was observed in Mentha citrata (64%). The present study revealed that these species can be used as naturalantioxidants.

KeywordsFree radical scavenging activity, antioxidant, Mentha species, DPPH

Introduction

The active ingredients of a medicinal plant are mainly

its secondary metabolites, among which is the

phenolic compound that is an important antioxidant

(Huda-Faujan et al., 2009; Khanavi et al., 2009). These

ingredients are naturally produced during a plants

growth metabolic process, the active substances with

antioxidant function such as scavenging reactive

oxygen species, free radicals (hydroxyl radicals, OHand superoxide anion radicals, O2) or non-free radi-cal reactive oxygen species (peroxide, H2O2) produc-

tion from body metabolism (Ramarathnam et al.,

1995). Natural antioxidants are known to exhibit a wide

range of biological effects, including antibacterial,

antiviral, anti-inflammatory, anti-allergic, antithrombo-

tic and vasodilatory activity (Cook and Samman, 1996;

Liyana-Pathirana andShahidi, 2006). In recent develop-

ments, it has been recorded that free radicals are

involved in causing many diseases (Ames et al., 1993).

For instance unsaturated fatty acids in the biomembranes

are attackedby free radicals causing lipid peroxidation in

membranes, a decrease in membrane fluidity, loss of

enzymes and receptor activity and damage to mem-

brane proteins leading to cell inactivation (Dean and

Davies, 1993). Free radicals also attack DNA and

cause mutation leading to cancer. For these reasons

antioxidants are of interest for the treatment of many

kinds of cellular degeneration (Tutour, 1990).

Restriction on the use of synthetic antioxidants has

been imposed, because of their carcinogenicity

(Bronen, 1975). Thus, the interest in natural antioxi-

dants has been increased considerably. As compared

to other antioxidants much attention has been paid to

plants (Couladis et al., 2003).Especially, the antioxidants

present in edible plants have recently been considered

1Department of Biotechnology, Faculty of Biological Sciences,Quaid-i-Azam University, Islamabad, Pakistan2Nuclear Institute for Food and Agriculture (NIFA), Peshawar,Pakistan3Department of Plant Sciences, Faculty of Biological Sciences,Quaid-i-Azam University, Islamabad, Pakistan4Pakistan Council of Scientific and Industrial Research (PCSIR)Laboratories Complex, Peshawar, Pakistan

Corresponding author:Nisar Ahmad, Department of Biotechnology, Faculty of BiologicalSciences, Quaid-i-Azam University, Islamabad 45320, PakistanEmail: [email protected]

Toxicology and Industrial Health28(1) 8389 The Author(s) 2012Reprints and permission:sagepub.co.uk/journalsPermissions.navDOI: 10.1177/0748233711407238tih.sagepub.com


as food additives (Fukuda et al., 1990). Several

methods have been developed to evaluate the total

antioxidant activity of fruits or other plants and animal

tissues. One of the methods is the 1,1-diphenyl-2-

picrylhydrazyl (DPPH) assay, which can accommodate

a large number of samples in a short periodof time and is

sensitive enough to detect natural compounds at low

concentrations so it was used in the present study for the

primary screening of antioxidants (Benzie and Strain,

1996; Cao and Prior, 1999; Evelson et al., 2001;

Ou et al., 2001; Van den Berg et al., 1999).

The objective of the current study is to evaluate the

free radical scavenging activity (FRSA) (DPPH activ-

ity) of nine Mentha species to find new potential

sources of natural antioxidants, which were subjected

to extraction using methanol as a solvent and to com-

pare their highest antioxidant potential in each specie.

Materials and methods

Plant materials

Nine of the Mentha species of family Lamiaceae, that

is, Mentha arvensis, Mentha citrata, Mentha longifo-

lia,Mentha officinalis,Mentha piperita,Mentha pule-

gium, Mentha royleana, Mentha spicata and Mentha

suaveolens were collected from different localities

(Swat, Bunir, Khanpur, Haripur, Abottabad, Islamabad

and Peshawar) of Pakistan from 2008 to 2009. These

plants were identified by the experts of the Department

of Plant Sciences, Quaid-i-AzamUniversity, Islamabad

and the Medicinal Botanical Centre PCSIR Complex,

Peshawar, Pakistan.

Preparation of the extract

Dried plant materials were ground and sieved to a fine

powder from which the extracts were prepared.

Methanolic extract of the plant was obtained by tak-

ing 10 g of powdered material in a separate container.

With this 50 ml of methanol was added and kept for

1 week with periodic shaking (the soaked material

was stirred every 18 h using a sterilized glass rod),

filtered and the filtrate was collected. This procedure

was repeated three times with fresh volume of metha-

nol. The filtrates were pooled. The final extracts were

passed throughWhatman filter paper No. 1 (Whatman

Ltd., England). The pooled methanol extracts were

concentrated separately by rotary vacuum evaporator

at 40C and evaporated to dryness and stored at 4C inan air tight bottle (Ahmad et al., 2010). The extracts

obtained from each part were dissolved in methanol

independently to get stock solutions. The stock

solution was prepared by dissolving 5 mg of pure

extract in 20 ml of methanol independently.

DPPH free radical scavenging activity

The FRSA of methanolic extracts of nine Mentha

species was measured in terms of hydrogen donating

or radical scavenging ability using the stable radical

DPPH. The test extracts were prepared in methanol

therefore the DPPH was also prepared in methanol.

A total of 3.96 mg of DPPH was dissolved in 20 ml

of methanol to get stock solution. A 0.5 ml of sample

solution was added to 1 ml of DPPH solution sepa-

rately. These solution mixtures were kept in dark for

30 minutes (incubation period) at room temperature.

Thirty minutes later, the absorbance was measured

at 517 nm. Lower absorbance of the reaction mixture

indicated higher FRSA. All tests were carried out in

triplicate. Finally the radical scavenging activity was

calculated as percentage of DPPH discoloration using

the equation;

% scavenging DPPH free radical 100 1 AE=AD

Where AE is absorbance of the solution, when

extract has been added at a particular level and AD

is the absorbance of the DPPH solution with nothing

added (blank, without extract).

Results

Antioxidant constituents of plant origin are vital

substances which protect the body from coronary

diseases and from damage caused by free radical-

induced oxidative stress. The overall objective of the

current study was to evaluate the antioxidant activity

in nine Mentha species. % FRSA in each of nineMentha extracts was performed in time dependent

manner. The data was recorded in triplicates and the

mean values are presented in Figures 19.

Antioxidant activity or FRSA of nine Mentha spe-

cies of which M. suaveolens had shown significantly

higher activity to detoxify DPPH free radicals than

other species (Figure 9). According to our results

M. suaveolens have higher antioxidant activity

(82%) as compared to other species of Mentha. Thedata was collected after 10, 20 and 30 minutes to com-

pare highest antioxidant activity. From the comparison

it was concluded that at 30 minutes the antioxidant

activity was highest. M. arvensis detoxify free radical

after 10, 20 and 30 minutes and showed best

84 Toxicology and Industrial Health 28(1)


activity (67%, 68% and 70%) after incubation period of30 minutes in the dark. In the present experiment

M. citrata have 56%, 62% and 64% activity as shownin Figure 2.

The antioxidant activity recorded for M. longifolia

ranges from 67%, 75% and 79% (Figure 3), respec-tively. Lamaison et al. (1991) also observed the

antioxidant activity (LC50 29.0 mcg/ml) in aqueous-

alcoholic extracts of the shade-dried inflorescence

of M. longifolia. From the experiment it was con-

cluded that the plant secondary metabolites scavenge

more free radicals after 30 minutes than 10 minutes,

as the time interval and incubation period increases

the plant extract scavenge more free radicals. Several

studies suggested that the phenolic compounds con-

tributed significantly to the antioxidant capacity of

the 112 wild grown Chinese herbs (Cai et al., 2004).

The present data are consistent with the findings of

67.0 67.5 68.0 68.5 69.0 69.5 70.0

67.0 67.5 68.0 68.5 69.0 69.5 70.0

Tim

e in

terv

als

(min

)

c

b

10

20

Free radical scavenging activity (%)

30a

Figure 1. Time dependent free radical scavenging activityin Mentha arvensis. Values are means of three replicates.Columns with common letters are not significantly differentat p < 0.05.

56 58 60 62 64

56 58 60 62 64

30

20

c

b

Tim

e in

terv

als

(min

)

Radical scavenging activity (%)

a

10

Figure 2. Time dependent free radical scavenging activityin Mentha citrata. Values are means of three replicates.Columns with common letters are not significantly differentat p < 0.05.

66 68 70 72 74 76 78 80

66 68 70 72 74 76 78 80

Tim

e in

terv

als

(min

)

10

20

30

c

b


a

Figure 3. Time dependent free radical scavenging activityin Mentha longifolia. Values are means of three replicates.Columns with common letters are not significantly differentat p < 0.05.

69 70 71 72 73 74 75 76 77

69 70 71 72 73 74 75 76 77T

ime

inte

rval

s (m

in)

10

20

30

c

b


a

Figure 4. Time dependent free radical scavenging activityin Mentha officinalis. Values are means of three replicates.Columns with common letters are not significantly differentat p < 0.05.

Ahmad et al. 85


many research groups who reported such positive cor-

relation between total phenolic content and antioxi-

dant activity (Ahmad et al, 2010; Cai et al., 2004;

Zheng and Wang, 2001).

The methanolic extract of M. officinalis shows

70, 72 and 76% FRSA, respectively (Figure 4), whileM. piperita shows 67, 74 and 75% activity,respectively (Figure 5). Mimica-Dukic in 2004, also

detemined the antioxidant activity of M. piperita.

Studies conducted on free radical scavenging or anti-

oxidant activities of medicinally important plant

reveal that the efficiency of each plant species differs

depending on the particular assay methodology,

reflecting the complexity of the mechanisms

involved in total antioxidant capacity (Matkowski and

Piotrowska, 2006). It is suggesting a great complexity

of the mechanisms involved that can vary even among

the related species. M. pulegium showed activity in

66 68 70 72 74 76

66 68 70 72 74 76

Tim

e in

terv

als

(min

)

c

b

a

10

20

30


Figure 5. Time dependent free radical scavenging activityin Mentha piperita. Values are means of three replicates.Columns with common letters are not significantly differentat p < 0.05.

71.5 72.0 72.5 73.0 73.5 74.0 74.5 75.0 75.5

71.5 72.0 72.5 73.0 73.5 74.0 74.5 75.0 75.5

Tim

e in

terv

als

(min

)

c

b

a

10

20

30


Figure 6. Time dependent free radical scavenging activityin Mentha pulegium. Values are means of three replicates.Columns with common letters are not significantly differentat p < 0.05.

62 64 66 68 70 72 74 76

62 64 66 68 70 72 74 76

Tim

e in

terv

als

(min

)

c

b

a

10

20

30


Figure 7. Time dependent free radical scavengingactivity in M. royleana. Values are means of three replicates.Columns with common letters are not significantly differentat p < 0.05.

60 62 64 66 68 70 72

60 62 64 66 68 70 72T

ime

inte

rval

s (m

in)

c

b

a

10

20

30


Figure 8. Time dependent free radical scavenging activityin Mentha spicata. Values are means of three replicates.Columns with common letters are not significantly differentat p < 0.05.



order of 72%, 73% and 75% as shown in Figure 6. M.pulegium antioxidant activity was also determined by

Souria et al. (2004). In this study it was observed that

M. royleana have 63%, 68% and 75% activity. Thedata on antioxidant activity recorded for M. spicata

was 61%, 64% and 71%, respectively (Figure 8;M. spicata extract has been found to have antioxidant

and antiperoxidant properties due to the presence of

eugenol, caffeic acid, rosmarinic acid and a-tocopheroland it could enhance error-free repair for DNA damage

and hence could be antimutagenic. (AI-Sereiti et al.,

1999; Vokovic-Gacis and Simic, 1993).M. suaveolens

one the potential specie for natural antioxidant, it

showed 79%, 81% and 82% activity in the presentstudy. M. suaveolens showed appreciable antioxidant

activity only in the polar fractions and its decoction

was also very effective in the inhibition of AChE and

as a scavenger of radicals (Ferreira et al., 2006).

Natural antioxidant substances are considered to be

safe since they occur in plant foods, and are seen as

more desirable than their synthetic counterparts. From

the current data it was summarized that these Mentha

species contain a large variety of metabolites that

possess antioxidant activity.

Discussion and conclusion

Many species of the Mentha family have been

experimentally studied and the efficiency of some tra-

ditional applications was confirmed by many workers.

According to the literature cited different Mentha

species are used for rheumatism, dysentery, dyspepsia,

skin allergies, stimulant, chills, jaundice, throat infec-

tions, diaphoretic, diuretic, reconstituent, stomach

tonic, constipation, spasm, bladder stone, gall stone,

jaundice, diarrhoea, toothache, stomachache, anti-

infection, dyspnea, flatulence, gastrodynia, sedative,

insect repellent, cholagogue antimycobacterial, anti-

fungal, anti-allergic, virucidal, radioprotective,

cyclooxygenase inhibitor, anti-inflammatory and

haemostatic (Naghibi et al., 2005).

The aerial parts of Mentha species are commonly

used in commercial spice mixtures for many

processed foods as well as in herbal teas, flavour

liqueurs, breads, salads, soups and cheese, as well as

an ingredient of cosmetics (Kofidis et al., 2006;

Moreno et al., 2002; Yadegarinia et al., 2006). In

addition these have been utilized traditionally for

the treatment of many digestive tract diseases due to its

carminative, antiemetic spasmodic, analgesic and anti-

inflammatory attributes (Gulluce et al., 2007; Moreno

et al., 2002). The essential oils of some Mentha species

viz: M. arvensis, M. piperita, M. longifolia and

M. spicata are potential candidates for exhibiting anti-

microbial, antioxidant and radical scavenging activities

(Dorman et al., 2003; Gulluce et al., 2007; Kaur and

Kapoor, 2002; Pandey et al., 2003), which are mainly

attributed to the presence of phenolic substances

(Hosseinimehr et al., 2007).

Nine Mentha dry materials were subjected to

extraction with solvent methanol. Highest antioxidant

potential was found in the extract of M. suaveolens

followed by other eight species. From the experiment

it was observed that these plants have certain impor-

tant constituents such as essential oil, phenolics, and

so on which is responsible for radical scavenging

activity. Mentha species prevent cell damage through

its strong antioxidant activity, by scavenging free

radicals and neutralizing toxic invaders. It also pro-

motes the release of superoxide dismutase, a powerful

antioxidant especially potent in destroying free radi-

cals caused by imbalanced oxidation. Radical scaven-

ging activity was observed when discoloration

occurred. M. suaveolens were observed to have high

discoloration followed by M. longifolia, M. officina-

lis,M. piperita,M. pulegium,M. royleana,M. spicata,

M. arvensis and M. citrate, respectively.

When the difference in the results was high

between the DPPH solution and sample, the percent

free radical activity is high or the sample had high

potential to scavenge the free radical of DPPH. This

study reveals that the tested plant materials have

79.0 79.5 80.0 80.5 81.0 81.5 82.0 82.5

79.0 79.5 80.0 80.5 81.0 81.5 82.0 82.5

Tim

e in

terv

als

(min

)

c

b

a

10

20

30


Figure 9. Time dependent free radical scavenging activityin Mentha suaveolens. Values are means of three replicates.Columns with common letters are not significantly differentat p < 0.05.

Ahmad et al. 87


significant FRSA. The result of the present study sug-

gests that these plant materials especially M. suaveo-

lens and M. longifolia can be used as natural source

of antioxidants for different diseases.

Funding

This research received no specific grant from any funding

agency in the public, commercial, or not-for-profit sectors.

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15 free radical scavenging (dpph) potential in nine mentha species

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