Extraction and Characterization of Peppermint (Mentha piperita) Essential Oil and its Assessment as Antioxidant and Antibacterial

Abstract

The aim of this study is to extract and characterize peppermint essential oil and assess it as antioxidant and antibacterial activity. The extraction processes, chemical composition, total phenolic contents (TPC), total flavonoid contents (TFC), antioxidant and antibacterial activity were studied. The oil was extracted by three methods: steam distillation, solvent and soxhelet. The results indicated that the highest oil extraction was obtained by soxhelet method 1.5±0.12 and 1.2±0.12 ml/100 gm and the lowest result recorded by steam distillation 1.1±0.09 and 0.9±0.14 ml/100gm from fresh and dry samples, respectively. The chemical composition of fresh and dry sample were determined by using gas chromatography and the results showed that there were 26 components of volatile compounds were identified in the essential oil isolated from peppermint. The antibacterial potential of mint essential oils was evaluated by Muller Hinton agar well diffusion method against selected bacteria. The essential oils showed higher activity against Staphylococcus aureus and Salmonella 19±1.41 and 16.5±2.12 mm, respectively, for fresh sample and showed lower activity against Escherichia coli and Klebsilla pneumonia 8.5±0.70 mm and nil, respectively, for dry sample. The TPC of fresh and dry samples were 14.00±0.12 and 8.80±0.09 mg /kg, respectively. TFC of the mint essential oil was determined in comparison with rutin were 8.1±0.09 and 5.0±0.07mg/kg, for fresh and dry sample, respectively. The results showed decrease in peroxide value at 30, 60, 90, 120 and 150 min compared with blank sample without essential oil for wet and dry mint. DPPH inhibition percentages were recorded and inhibition concentrations at 50% activity (IC50) were 0.651±0.09, 0.683±0.6 and 0.161±0.07 mg/mL for wet, dry samples and standard respectively. It can be recommended that further study on sensory analysis of food products containing mint essential oil to evaluate its acceptability and shelf life.

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EDITORIAL
Gezira Journal of Engineering and Applied Sciences vol 13 (1)2018
vol 13 (1) 2016
Gezira Journal of Engineering and Applied Sciences vol 13 (1)2018
16) 201( 13vol
Extraction and Characterization of Peppermint (Mentha piperita) Essential Oil and its
Assessment as Antioxidant and Antibacterial
Azhari Siddeeg*, Zakaria A. Salih, Rabab M. E. Mukhtar & Ali O. Ali
Faculty of Engineering and Technology, University of Gezira, Wad Medani, Sudan, P.O Box,
20.
*Corresponding author: azhari_siddeeg@yahoo.com
ABSTRACT
The aim of this study is to extract and characterize peppermint essential oil and assess it as
antioxidant and antibacterial activity. The extraction processes, chemical composition, total
phenolic contents (TPC), total flavonoid contents (TFC), antioxidant and antibacterial activity
were studied. The oil was extracted by three methods: steam distillation, solvent and soxhelet. The
results indicated that the highest oil extraction was obtained by soxhelet method 1.5±0.12 and
1.2±0.12 ml/100 gm and the lowest result recorded by steam distillation 1.1±0.09 and 0.9±0.14
ml/100gm from fresh and dry samples, respectively. The chemical composition of fresh and dry
sample were determined by using gas chromatography and the results showed that there were 26
components of volatile compounds were identified in the essential oil isolated from peppermint.
The antibacterial potential of mint essential oils was evaluated by Muller Hinton agar well
diffusion method against selected bacteria. The essential oils showed higher activity against
Staphylococcus aureus and Salmonella 19±1.41 and 16.5±2.12 mm, respectively, for fresh sample
and showed lower activity against Escherichia coli and Klebsilla pneumonia 8.5±0.70 mm and nil,
respectively, for dry sample. The TPC of fresh and dry samples were 14.00±0.12 and 8.80±0.09
mg /kg, respectively. TFC of the mint essential oil was determined in comparison with rutin were
8.1±0.09 and 5.0±0.07mg/kg, for fresh and dry sample, respectively. The results showed decrease
in peroxide value at 30, 60, 90, 120 and 150 min compared with blank sample without essential
oil for wet and dry mint. DPPH inhibition percentages were recorded and inhibition concentrations
at 50% activity (IC50) were 0.651±0.09, 0.683±0.6 and 0.161±0.07 mg/mL for wet, dry samples
and standard respectively. It can be recommended that further study on sensory analysis of food
products containing mint essential oil to evaluate its acceptability and shelf life.
Keywords: Peppermint essential oil, antioxidant, antibacterial, ethanolic extract, GC.
INTRODUCTION
Peppermint (Mentha × piperita, also known as M. balsamea Willd is a hybrid mint, a cross
between water mint and spearmint. The plant, indigenous to Europe and the Middle East, is now
widespread in cultivation in many regions of the world ('Wang et al., 2008). It is herbaceous
rhizomatous perennial plant growing to 30–90 cm tall, with smooth stems, square in cross section.
The rhizomes are wide-spreading, fleshy, and bare fibrous roots. The leaves are from 4–9 cm long
and 1.5 – 4 cm broad, dark green with reddish veins, and with an acute apex and coarsely toothed
margins. The leaves and stems are usually slightly fuzzy. The flowers are purple, 6–8 mm long,
with a four-lobed corolla about 5 mm diameter; Peppermint is a fast-growing plant; once it sprouts,
and it spreads very quickly ('Zaidy and Dahiya, 2015). Humankind has used plants for healing for
many thousands of years, and it’s from this tradition of that the use of aromatic plant compounds
in medicine begun. Oils were used in the embalming process, in medicine and in purification
rituals. Essential oils are highly concentrated substances extracted from flowers, leaves, stems,
roots, seeds, barks, resins, or fruit rinds ('Yen et al., 2008). These oils are often used for their flavor

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and their therapeutic or odoriferous properties, in a wide selection of products such as foods,
medicines, and cosmetics. Extraction of essential oils is one of the most time- and effort consuming
processes. The increasing resistance of microorganisms to conventional chemicals and drugs has
prompted scientists to search for novel sources of biocides with broad spectrum activities. Since
ancient times, plants and their derivatives, such as essential oils, have been used in folk medicine
('Virendra, 2007). Mint oil has a number of uses it can be used to add a minty flavor to drinks, add
mint flavor to food such as chocolates and icing and used in a number of natural applications from
deterring ants to clearing up chest congestion. Making your own takes a few weeks, but is cheap
and easy to do ('Hayyan and Al-Taweil, 2014). Generally, Gram-negative bacteria are more
resistant to essential oil than Gram-positive bacteria. Before examining the effects of essential oil
on bacteria, should briefly consider the differing structures of the cell walls of Gram-positive and
Gram-negative bacteria. Approximately 90%–95% of the cell wall of Gram-positive bacteria
consists of peptidoglycan, to which other molecules, such asteicoic acid and proteins, are linked
('Felomina et al., 2013). Antioxidants are compounds found in virtually all plant foods. The
primary job of antioxidants is to protect cells against the oxidative stress caused by free radicals,
considered the primary cause of the aging process. Peppermint is an excellent source of
antioxidants like vitamin C and beta-carotene, which is converted to vitamin A in the body.
Peppermint is also a very good source of fiber, iron, calcium, magnesium, potassium, copper,
vitamin B2 and omega 3 fats. The objective of this study is to extract and characterize peppermint
essential oil and assess it as antioxidant and antibacterial.
MATERIAL AND METHODS
Fresh peppermint was collected from a local market in Wad Medani City, Gezira State, Sudan.
Then, raw materials and bacterial strains: Klebsiella pneumoniae, Staphylococcus aureus,
Salmonella typhimurium, Escherichia coli were transported to the Laboratory of food Analysis,
University of Gezira, Sudan. All other chemicals and reagents used of the highest grade
commercially available.
Steam distillation:
Steam distillation is one of the most popular ways will be used to extract essential oils from
plants, leaves and flowers. During steam distillation process, 100g of the plant raw material was
being placed in the chamber of the essential oil distillation still, and steam passed through the plant
matter. When the steam passed through the plant matter it picked up the oils and moved into
another chamber where it is cooled and condensed. Then, essential oil was separated from the
water and bottled for used (Hanbali et al., 2005).
Solvent extraction:
Sixty g of peppermint fresh and dry were mixed with 700 mL of ethyl alcohol using a shaker for
6 hour (70°C). The oil was then recovered by evaporating off the solvent using a rotary evaporator
and the solvent was removed under a laboratory fume hood for 30 min at 37°C stream and was
then stored in a refrigerator in dark bottle.
Soxhelet extraction:
100g of fresh and dry peppermint were weighted and put in soxhelet apparatus 300 mL of hexane
solvent were added. The solvent was heated to reflux then vapor travels up a distillation arm and
floods into the chamber housing the thimble of solid. The condenser ensures that any solvent vapor

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cools, and drips back down into the chamber housing the solid material. The chamber containing
the solid material slowly was filled with warm solvent. Some of the desired compound dissolves
in the warm solvent. When the Soxhlet chamber was almost full, the chamber was emptied by the
siphon. The solvent was returned to the distillation flask. The thimble ensures that the rapid motion
of the solvent does not transport any solid material to the still pot. This cycle was allowed to repeat
many times, over hours or days. During each cycle, a portion of the non-volatile compound
dissolves in the solvent. After many cycles, the desired compound was concentrated in the
distillation flask. The advantage of this system was that instead of many portions of warm solvent
being passed through the sample, just one batch of solvent was recycled. After extraction the
solvent was removed, typically by means of a rotary evaporator, yielding the extracted oil. The
non-soluble portion of the extracted solid remains in the thimble, and is usually discarded.
Chemical composition of essential oil:
The volatile compounds were sampled with a SPAM–fiber and separated with a GC/MS.
Volatile compounds were separated on a CP-Sil-8CB (Varian, Walnut Creek, CA, USA), fused
silica capillary column (30 m length, 0.25 mm, id, and 0.25 μm film thicknesses) in a Varian model
3800 gas chromatograph. Volatile compounds identification were carried out by matching the
compounds with the mass spectra of standard compounds found in the wily 130 K and national
institute of standards and technology (NIST) 98 library of MS spectra based on their retention
indices.
Determination of total phenolic (TPC) and flavonoid contents (TFC)
TPC was determined with the Folin-Ciocalteu method. Where, 100 mg oil equivalents·mL–1
were mixed with 0.5 ml of Folin-Ciocalteu reagent and 2 mL of methanol. The mixture was shaken
for 1 min, and then 1.5 ml of 15% Na2CO3 was added and the mixture was shaken again. Finally,
the solution was completed to 10 mL by adding distilled water. The mixture was incubated at 50°C
for 20 min and centrifuged at 3,000 rpm for 10 min. The absorbance was recorded at 750 nm. TPC
of the oil was calculated using Gallic acid as a standard (Liuand, Yao2007). The TFC of
peppermint oil was determined according to the method reported by (Bayat and Borice, 2014) with
slight modifications. Briefly, peppermint oil (2.5ml) was diluted with distilled water (10 ml),
followed by the addition of 0.75 ml of 5% NaNO2 solution. Then 10% AlCl3 solution (0.75 mL)
was added and the reaction mixture was allowed to stand at ambient temperature for 5 min. After
incubation, 5 ml of 1 M NaOH were added and the volume of the mixture was made to 25 mL with
distilled water. The mixture was shaken vigorously and the absorbance of the pink color developed
was measured at 510 nm using spectrophotometer.
Peroxide value:
200 mL of cotton seed oil were placed in flask and put in water path 60oC and added air. 0.50
ml and 1 mL peppermint oil added as antioxidant take recorded after 30 min, 1hour, 90 min, 2
hours and 150 min. About 1 mL of oil weighed into a clean dry boiling tube and 1g powder
potassium iodide was added while still liquid. 20 mL of acetic acid–chloroform solution (at
ratio2:1) were added. The tube was placed in the boiling water so that the liquid boils within 30

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seconds. The content was poured into flask containing 20 mL of potassium iodide solution (5%).
25ml of distilled water was immediately added by graduated cylinder, and was titrated with 0.002
M sodium thiosulfate solution using starch indicator. The starting deep red orange color of the
solution were the sign for titration with mixing slowly until the color lightness. When the blue gray
color disappears (upper layer) the titration was then stopped the mL of titrant used two decimal
places were accurately recorded.
DPPH radical scavenging activity:
The DPPH radical scavenging assay of peppermint oil was conducted using a method described
by Bradly, (1992), with a slight modification. Extract concentrations of 0.25, 0.50, 0.75 and 1.00
mg oil equivalents/ mL were mixed with 3.5 mL of DPPH solution with absorbance at 520 nm.
The mixtures were incubated for 30 min at 25°C. Then, the absorbance was recorded at 520 nm.
The DPPH radical scavenging activity was calculated by the formula: DPPH scavenging activity
% = (AC–AS /AC) ×100. Where: AS: absorbance of oil sample; AC: absorbance of control. The
percentage of scavenging activity was plotted against sample concentration to obtain the IC50,
defined as the concentration of the sample required to cause 50% inhibition. Ascorbic acid was
use as reference compounds.
Antibacterial activity:
The agar diffusion assay described by Smânia et al., (1999) was adopted. Briefly, bacterial
strains were grown in Mueller-Hinton agar and broth. The strains were incubated at 36oC for 18 h,
and were diluted to a final concentration of approximately 106 CFU/ml. Each bacterial suspension
was spread over the surface of Mueller-Hinton agar containing three wells of 7 mm diameter. The
wells were filled with 0.25, 0.50, 0.75 and 1 ml of extract dissolved in the medium. The plates
were incubated at 36oC for 20 h. An antibiotic was used as a control.
Statistical analysis:
All experiments were conducted at least in triplicate and statistical analyses were performed
using SPSS version 16.0 software for Windows (SPSS Inc., Chicago, IL, USA). One-way analysis
of variance (ANOVA) was used to determine significant differences between means and Tukey’s
test were used to perform multiple comparisons between means. The significance level was
defined as p<0.05.
RESULT AND DISCUSSION
Extraction process:
Peppermint leaves contains about 0.5 to 4 % volatile oil that is composed of 50-78 % free
menthol, monoterpene, menthofurane and traces of jasmine (Dew and Evans, 1984). Extraction of
peppermint by different methods gave change in volume of oil and was noticed that the oil
extracted from fresh peppermint more than from dry mint as shown in Table 1. Essential oil (EO)

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yield per plant on a dry weight basis was lower than that on a fresh weight basis for peppermint
herb. Rabak, (1917) suggested that a reduction in EO yield may occur if plant was dried before
extraction due to change that favor the formation of esters and the production of free acid in M.
piperita.
Table 1: Quantity of essential oil (mL/100g) of peppermint extracted by different methods.
Extraction method
Fresh mint oil (mL/100g)
Dry mint oil (mL/100g)
Soxhlet
1.5±0.12a
1.2±0.12a
Solvent
1.2±0.13a
1.0±0.16a
Steam distillation
1.1±0.09a
0.9±0.14a
Means ± standard deviation values having different superscript letter(s) in each row differ
significantly (p<0.005).
Chemical composition of peppermint essential oil:
The chemical composition of fresh and dry sample was determined by using gas
chromatography and the results were reported. As shown in Table 2 and 3, Twenty-six components
of volatile compounds were identified in the essential oil isolated from peppermint fresh and dry
sample. The results showed that α-Phellandrene acid, β-Bourbonene were the highest content
(8.176 and 6.14 respectively), followed by Trans-Carveol, Methyl caproate (C6) 5.884 and 4.541,
respectively in fresh sample. In Table 3, dry sample α-Humulene, Bornylacetate were the highest
content (149, 5.887) followed by Cis-Limonene oxide, 4, Methyloleate (C 18:1) (5.143, 8.184),
respectively.
Table 2: The chemical composition of fresh peppermint by GC.
No
Constituent
Retention time
Area
1
1,Hexane
4.281
107561924
2
2, Methylecaproate
4.404
11719
3
2, Methylecaproate (C6)
4.541
66683
4
α-Terpinene
4.681
5051
5
Trans-Sabinene hydrate
4.801
1255
6
Cis-Limonene oxide
4.983
2180
7
Trans-Limonene oxide
5.080
1097
8
Cis-p-Mentha-2.8-dien-1-ol
5.376
3977
9
Trans-p-Mentha-2.8-dien-1-ol
5.544
9397
10
Cis-Carveo
5.812
6717
11
Trans-Carveol
5.884
34050
12
Bornyl acetate
6.033
2884
13
β-Bourbonene
6.140
72946
14
α-Humulene
6.520
1256
15
Santene
6.670
1426

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Camphene
7.014
1898
17
Myrcene
7.087
3443
18
cis-3-Hexenyl acetate
7.730
9623
19
α-Phellandrene
8.176
259459
20
Linalool
8.613
4790
21
Nonanal
9.002
1410
22
trans-β-Caryophyllene
9.482
1114
23
Germacrene-D
10.582
2313
24
3, Methylepalmitoleate
10.898
6129
25
3, Methylepalmitoleate
14.568
1883
26
4, Methyleoleate (C 18:1)
15.721
10584
Table 3: The chemical composition of dry peppermint by gas chromatography
No.
Constituent
Retention time
Area
1
1,Hexane
4.276
109643237
2
2, Methylecaproate
4.431
47305
3
2, Methylecaproate (C6)
4.489
12282
4
3, Methylecaproate
4.538
19435
5
Trans-Sabinene hydrate
4.973
246820
6
Cis-Limonene oxide
5.143
390574
7
Trans-Limonene oxide
5.330
322749
8
Cis-p-Mentha-2.8-dien-1-ol
5.414
96728
9
Trans-p-Mentha-2.8-dien-1-ol
5.545
403518
10
Cis-Carveo
5.683
201447
11
Trans-Carveol
5.824
144494
12
Bornyl acetate
5.887
280072
13
β-Bourbonene
6.050
143714
14
α-Humulene
6.149
358481
15
Santene
6.296
66140
16
Camphene
6.380
154210
17
Myrcene
6.548
83188
18
Cis-3-Hexenyl acetate
6.684
141124
19
α-Phellandrene
7.015
10690
20
Linalool
7.293
4317
21
Nonanal
7.422
5616
22
Trans-β-Caryophyllene
7.623
7849
23
Germacrene-D
7.763
1982
24
3, Methylepalmitoleate
7.926
1519

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25
3, Methylepalmitoleate
8.093
23611
26
4, Methyleoleate (C 18:1)
8.184
64552
Total phenolic compounds and total flavonoid content:
As shown in Table 4, the TPC value of fresh sample was 14.00 ± 0.12 mg/kg while the TPC
values with dry samples 8.80 ± 0.09 mg/kg and all these values were significantly different (P<
0.05). The previous study of Zaidy and Dahiya, (2015) indicated that TPC of other kind of
Menthapiperita had the highest contents of total phenolic (12.63± 0.878). In this study, the total
phenolic content was determined in comparison with standard Gallic acid and the results expressed
in terms of mg GAE/g dry extract. Phenolic compounds are responsible for the antioxidant activity
of plant materials and they are highly effective radical scavengers (Pan et al., 2008). The
antioxidant activities of phenolics are due to their redox properties. The phenol moiety helps them
to work as reducing agents, hydrogen donors, and singlet oxygen quenchers (Chua et al., 2008).
Considering the heterogeneity of natural phenols and the possibility of interference from other
readily oxidized substances, several methods including Folin-Ciocalteu, permanganate titration,
colorimetric with iron salts, and ultraviolet absorbance have been used for total phenol
determination. However, in most direct comparisons, Folin-Ciocalteu method has been found
preferable and is being used by many researchers. In this method, phenols form a blue colored
phosphomolybdic-phosphotungstic-phenol complex in alkaline solution (Singleton et al., 1999).
Table 4: TPC and TFC of peppermint extract (mg/kg).
Samples
TPC (mg/kg)
TFC (mg/kg)
Fresh mint sample
14.00± 0.12a
8.10±0.09a
Dry mint sample
8.80± 0.09b
5.00±0.07b
Mean values ± standard deviation having different superscript letter(s) in each column differ
significantly (p<0.005).
TFC of the peppermint were determined in comparison with rutin as a standard and the results
are shown as mg /kg dry extract (Table 4). The TFC value of sample extracted by steam distillation
method was 8.1±0.09 mg/kg, 5.0±0.07 mg/kg for fresh and dry samples respectively. All these
values were significantly different (P<0.05) from traditional treatment value.
Peroxide value:
As illustrated in Table 5, the peroxide values of control were increased and the measure was.
for fresh and dry samples respectively. This is due to the formation of hydroperoxides of
unsaturated fatty acids that were obtained as a result of lipid oxidation (Choe and Min, 2006).
Detection of peroxide gives the initial evidence of rancidity in unsaturated fats and oils.
Autoxidation is a free radical reaction involving oxygen that leads to deterioration of fats and oil
which form off-flavor and off odors. Peroxide value concentration of peroxide in an oil and fat, is
useful for assessing the extent to which spoilage has advanced (Cheuibini et al .,1999). Auto-
oxidation, where peroxide is the main product that gives rise to objectionable flavor in food
products, proceeds through the free radical chain reaction, where it attacks on the double bond at
room temperatures. Photo-oxidation is a much faster reaction that involves attack at double bond
(Lawson, 1997). Rancidity of food items can be the result of auto and photo-oxidation, which are

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natural oxidation and chemical degradation processes of oils, where fatty acid esters of oils are
converted into free fatty acids giving a smell observed in many oils (Anwar et al., 2003). Oils that
are more unsaturated are oxidized more quickly than less unsaturated oils (Parker et al., 2003).
Table 5: Peroxide value of peppermint oil (meq O2·kg–1oil)
Dry peppermint oil sample (meq O2·kg–1oil).
Time (min)
Blank
0.5 mL
1.0 Ml
30
3.00
5.60
4.46
60
6.96
6.67
5.83
90
10.13
8.30
7.23
120
11.06
9.10
8.94
150
16.43
10.90
9.52
Fresh peppermint oil sample (meq O2·kg–1oil).
30
5.00
4.90
4.12
60
6.96
6.24
5.33
90
10.13
9.89
8.65
120
9.93
8.93
8.34
150
16.43
12.67
11.99
DPPH assay:
In the determination of antioxidant activity, the free- radical scavenging activity of
Menthapiperita essential oils was evaluated using the DPPH method is presented in Figure 1A and
1B for dry and fresh peppermint oil. The absorbance decreases because of a color change from
purple to yellow .DPPH assay is based on the reduction of DPPH radical in the presence of a
hydrogen donating antioxidant, and it has been extensively used for screening antioxidant activity
of natural compounds. It is sensitive enough to detect active ingredients at low concentrations and
can accommodate many samples in a short period of time (Chua et al., 2008). DPPH is a stable
free radical, which dissolves in ethanol or methanol.
The results indicated that the essential oils of Menthapiperita tested showed good antioxidant
capacities compared with vitamin C as standard antioxidant compound. The results of fresh
peppermint oil indicate that the radical scavenging activity inhibition of the essential oil was the
highest (62.41±0.12%) at the concentration of 1mL and (60.7±0.08%) as highest for dry sample at
same concentration 1 mL. It was noticed that the scavenging activities of the essential oils were
increased with the increase of the essential oils concentrations. All the tested samples showed
lower DPPH radical scavenging activity when compared with the standard. It is clear from the data
that the concentration of 1ml of Menthapiperita essential oil gave a percentage inhibition of DPPH
(62.41 and 60.7 %( for fresh and dry oil respectively while same concentration of 1 mL for vitamin
C was 77.3±1.09% inhibition. According to the results recorded, the DPPH radical scavenging
activity of fresh and dry peppermint oil of the IC50 were 0.651 and 0.681mg/mL respectively which
were higher than the control with an IC50 of 0.161 mg/mL. A previous study by (Hussain et al.,
(2011) indicated that the radical scavenging activity of the essential oil from Menthapiperita was
the highest (81.09±1.21%) at concentration of 150µg/mL. The result of the recent investigation
was comparable with previous study made by Tenore et al., (2011). De Oliveira et al., (2012)
reported that the DPPH radical scavenging activity of thymol rich essential oil from
Saturejamontana. The radical scavenging activity of its edible oil could be credited to the presence

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of its main total phenolic contents, especially thymol and their recognized impact on oil lipid
oxidation (Guimaraes et al., 2010).
Figure 1: DPPH radical scavenging activity (means ± standard deviation) of A: fresh sample and
B: dry sample with a reference (Ascorbic acid).
When an antioxidant scavenges the free radical by hydrogen donation, the purple color of DPPH
in assay solution turns to yellow, which can be monitored spectrophotometrically at 517 nm (Yen
et al., 2008). Antioxidants effect on DPPH radical scavenging was thought to be due to their
hydrogen-donating ability. Antioxidant activities of essential oils from aromatic plants are mainly
attributed to the active compounds present in them. This can be due to the high percentage of main
constituents, but also to the presence of other constituents in small quantities or to synergy among
them.
Antibacterial activity:
Peppermint extracts are bacteriostatic against Streptococcus pyrogens ,Streptococcus aureus,
Streptococcus pyrogens, Serratia marcescens, E. coli and Mycobacterium avium (Gotshall, et
al.,1949). For steam extraction of wet the results illustrated in Table 6, showed that the essential
oil had the highest antibacterial activity against Salmonella, Staphylococcus aureus with a mean
zone of inhibition of 18.5 and 17.5 mm respectively. The lowest mean zone of inhibition was as
16.5 and 11.5 mm obtained by E. coli, Klepsilla pneumonia, respectively, using a volume of
100µL, while 75µL gave 16.5 for Staph and Salmonella as highest zone and 11.5 and 10for E. coli
and Klepsilla pneumonia, respectively.
Table 6: Antibacterial activity of essential oil extracted by steam and ethanolic extraction .
Dry steam
Fresh steam
Volume
75 μL/disk
100μL/disk
75 μL/disk
100μL/dis
k
Diameter zone (mm)
Organism

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11±0.01b
11.5±0.70b
16.5±2.12a
17.5±0.70b
Staphylococcus aureus
12±1.41a
13±1.41a
16.5±0.70a
18.5±0.70a
Salmonella
0.0
0.0
10±0.70c
12.0±0.01d
Klebsilla pneumonia
8.5±0.70c
11±0.02c
11.5±070b
16.5±2.12c
E. coli
Antibacterial activity of essential oil extracted by ethanol extraction
12.0±1.41a
15±1.41a
17±1.41a
19±1.41a
Staphylococcus aureus
12.5±0.70a
13.5±2.10b
16 ±1.41ab
16.5±2.12b
Salmonella
8.5±0.70b
9.5±0.70c
10±0.70c
12.5±0.70c
Klepsilla pneumonia
11±1.414ab
15±1.41a
13.5±1.4b
16±1.41b
E.coli
Mean values ± standard deviation having different superscript letter(s) in each column differ
significantly (p<0.005).
The results showed that the essential oil of peppermint oil had antibacterial activity against all
the bacterial strains tested .Peppermint oil and menthol have moderate antibacterial effects against
both gram-positive and gram-negative bacteria Diaz et al., (1989). The dry steam extract gave
highest values of inhibition zone for Salmonella and Staph. as 13.5 and 11mm, respectively at
volume 100 µL, lowest inhibitionzone of this extract shown by E. coli and Klepsilla as11 and 0.0
mm respectively while 75 µL showed highest values as 12 and11 mm for salmonella and Staph
and in lowest 8.5 and 0 mm for E.coli and Klepsilla, respectively. For methanolic extraction of
fresh the result showed in Table 6, the peppermint oil had highest antibacterial activity against
Staphylococcus aureus and Salmonella with mean zone of inhibition19and 16.5mm respectively
while the lowest mean zone of inhibition was as 16.0 and 12.5 mm obtained by E.coli, Klepsilla
pneumonia, respectively using a volume of 100µL.while 75µL gave 17and 16 for Staph and
Salmonella as highest zone and 13.5 and 10for E.coli and Klepsilla pneumonia, respectively. The
dry methanol extract gave highest values of inhibition zone for Staph and Salmonella as 15 and
13.5 mm, respectively, at volume 100 µL, lowest inhibition zone of this extract shown by E. coli
and Klepsilla as 15 and 9.5mm while 75 µL showed highest values as 12.5 and 12 mm for
Salmonella and Staph and in lowest 11 and 8.5 for E. coli and Klepsilla, respectively. Zaidi and
Dahia, (2015),the results showed in Table 6, of methanolic extraction dry and wet samples of
Sudanese peppermint as15,16mm of dry and wet respectively higher as bacterial activity for E.
coli and Salmonella spp as 13.5 and 16.5, while, lowest value no inhibition zone for E. coli and
Salmonella. According to previous study also revealed the antibacterial activity of peppermint
against S.aureus, E.coli and Klebsilla spp (Jeyakumar et al., 2011; Sujana et al., 2013), however,
Sartoratto et al., (2004) reported that peppermint oil was found to be strongly effective against
Salmonella species. The results are in fair correlation with the studies in which peppermint oil of
antibacterial activities against Gram–ve and Gram+ve bacteria (Singh et al, 2011). Activity of
inhibition was expressed in terms of the diameter of the inhibition zone>15 mm: sensitive, 14-15
Intermediate, <14 mm: resistance.
CONCLUSION
The Results revealed that the pepper mint extracts can be described as primary antioxidants,
which suggests they can play beneficial role of preventing the initiation and propagation of free

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radical-mediated chain reactions and consequently, prevent the oxidative damage on skin and
avoid premature skin aging. The results also indicated that the mint essential oil can be used as a
potential source of natural antimicrobial compound and the presence of phenolic and alkaloids
possessing strong antioxidant potential. Hence it is essential to explore further by the identification
of biologically active compounds, characterization and purification of the crude extracts. Based on
the result obtained from the evaluation of the phenolic and flavonoid content and the DPPH values
of peppermint as medicinal herbs we conclude that it is important to educate consumer on the
benefit consumption, choosing those that have the highest antioxidant capacity in order to promote
healthy diet.
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Gezira Journal of Engineering and Applied Sciences vol 13 (1)2018
vol 13 (1) 2016
Gezira Journal of Engineering and Applied Sciences vol 13 (1)2018
16) 201( 13vol

Menthapiperita









    0.121.5 1.2 0.12100     
0.091.10.140.9100  

 Muller Hinton agar
               
Staphylococcus aureus

Salmonella
19±1.41 2.1216.5
Escherichia coli
8.5± 0.70
Klebsilla
pneumonia
 zero     Foline-Ciocaltue 
0.1214.000.098.8
rutin 0.098.10.075.0.
306090120150
DPPH 
      DPPH    500.09
50
IC0.651 
0.060.683  0.070.0161      

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