MAUBIN UNIVERSITY

DEPARTMENT OF CHEMISTRY
A COMPARISON OF THE
ANTIOXIDANT ACTIVITY OF
SPEARMINT (MENTHA SPICATA L.)
LEAF BY DIFFERENT EXTRACTION
METHODS
By
Nyein Nyein Moe
(2 MSc Chem -1) 1
 MAUBIN UNIVERSITY
DEPARTMENT OF CHEMISTRY

Date of Seminar : 28.3.16

Title : A Comparison of the Antioxidant
Activity of Spearmint (Mentha
Spicata L.) Leaf by Different
Extraction Methods
Candidate : Nyein Nyein Moe
: (2 MSc Chem -1)
Supervisor : Dr Aung Kyaw Swar (Lecturer)
Department of Chemistry
Maubin University 2
 ABSTRACT
Many herbs and species, customarily used to add flavor to dishes,
are valued as commendable sources of natural antioxidant. The genus
Mentha, belonging to the family Lamiaceae, consists of more than 30
species. The plants of this family are a rich source of polyphenols and hence
could possess strong antioxidant properties. Mentha spicata L. is
characterized by its volitile oil of economical importance. Traditionally, M.
Spicata has been utilized in the foods as a flavoring agent and as an herbal
medicine in folk remedies.
In this research M. spicata will become a subject of scientific interest
in view of other potential uses of its extract, for the most part, as antioxidant
agents.
Firstly, the collected sample was screened the phytochemical
constituent of active compound by test tube method, in this case, alkaloids, a
amino acids, carbohydrates, flavonoids, glycosides, phenolic compounds,
reducing sugars, saponins, tannins, and terpenoids were present in the dry
M.spicata powder. But starch was not found in this species.
Secondly the dry and fresh samples were extracted with the different
extraction method such as cool and hot extracted with ethanol solvent,
2.52 % and 2.17 % of extracted yield were observed respectively.
3
 Final section contain two main parts, first, antimicrobial activity screening
and second is the determination of antioxidant activity of spearmint leaves.
Antimicrobial activities of extracts were examined by Agar Well Diffusion
Method at the Pharmaceutical and Food Research Department, Yangon. The
five bacterial strains and the one fungal strain were used in the present
study. For all the tested microorganism not significantly different between
the cool and hot extract. The maximum inhibition zone diameter was
obtained in E-coli has more potent with the diameter of 15 mm in cool
extract. Similarly in the hot extract showed maximum inhibition zone with
the diameter of 13 mm in B-pumilus, Candida and E-coli.
DPPH (1,1-diphenyl-2-picryl-hydrazyl) free radical scavenging assay was
chosen to assess the antioxidant activity of plant materials. The IC 50 values
were found to be 1.27 μg / mL for hot extract of spearmint, 1.01 μg / mL for
cool extract in 70% EtOH. Since the lower the IC 50, the higher the free
radical scavenging activity, cool extract has the highest free scavenging
activity followed by hot extract. Therefore, it can be deduced that the
antioxidant activity was found in the decreasing order of cool and hot
extract. All of these extracts were found to possess the lower antioxidant
activity than standard ascorbic acid (IC50 = 0.78 μg / mL ).

Keywords : Mentha spicata L., phytochemical screening,

different extraction, antimicrobial, antioxidant 4
 AIM AND OBJECTRIVE
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSIONS
 CONCLUSIONs
 REFERENCES

5
 To observe the effect of extraction on the estimation of
antioxidant activity of crude extract from Spearmint
(Mentha spicata L.) leaves

To collect the samples

To examine the moisture content of dry and fresh samples
To perform the phytochemical investigation on selected samples
To extract the sample with different extraction methods
 To test and comparison the antioxidant activity of different crude
extract by DPPH assay

6
 Scientific Classification
Kingdom : Plantae
Family : Lamiaceae
Genus : Mentha
(a)
Species : spicata
Binomial name : Mentha spicata
Botanical name : Mentha spicata L.
Myanmar name : Pudina
Part used : Leaves
(b)

7
Figure 1: (a) Fresh and (b) dry of Spearmint (Mentha spicata L.) leave
 CHEMICAL COMPOSITION OF
SPEARMINT
O

carvone beta pinene

(A) (C)
HO

limonene dihydrocarveol

(B) (D)

Figure 2 2D and 3D structure of main chemical constituent of

spearmint leaf (a) carbone (b) limonine (c) bita pinene (d) 8
dihydrocarveol
Materials
Test for alkaloids
and Methods
Sample + 1 % Boiled (3 filtrate 1) Dragendorff’s 1) Orange ppt
(3g) HCl min) filtered reagent 2) White ppt
(15 mL) 2) Mayer’s reagent 3) Reddish
3) Wagner’s reagent brown ppt
Test for a amino acids

Sample+ distilled D, (10 min), filtered, Spray with ninhydrin

(3g) Purple spot
water filtrate reagent
(25 mL) transfer to filter paper dry in oven (100 C)

Test for carbohydrates (Molisch’s test)

Sample+ distilled D, (20 min), filtered, Added conc: H2SO4 (1mL) Red ring
(3g) (between
water filtrate inclined at 45 two
(10 mL) added 10 % a-naphthol
layer)

9
Test for flavonoids
Sample + 95 % Boiled (3 filtrate1) Added HCl (2 mL)
(0.5g) ethanol min) filtered Pink colour
2) Added Mg turning
(25 mL)
added sodium Added dilute
hydroxide Yellow colourless
colour acid
solution
Test for glycosides
Sample + ethanol D, (10 min), filtered,
filtrate white ppt
(3g) (25 mL)
Added 10 % lead
acetate solution
Test for phenolic compounds
Sample+ ethanol D, (20 min), filtered, green
(3g) (10 mL) filtrate colour
added 5 % FeCl3
solution
Test for starch

Sample + distilled D, (20 min), filtered, filtrate No deep blue

(3g) water added I2 solution
10
(10 mL)
Test for reducing sugars
1) Boil (3 min) 1) D
Sample+ distill 2) Filter 2) Added Fehling’s Red ppt
(0.5g) water 3) Hydrolyzed with 1 M HCl solution A & B
4) Neutralized with alkali
Benedict’s Orange ppt
reagent
Test for saponins
shaked a few minutes
Sample + distilled frothing
(1g) water

Test for Tannins

Sample + distilled D, (20 min), filtered, white ppt
(2g) water filtrate
(20 mL) added 2 % galatin solution

Test for Terpenoids

+ Chloroform Soaked (6 hrs), filtered

Sample Pink colour ppt
(50 mL) added a few dps of
(3g)
acetic anhydride
11
conc H2SO4
Extraction
Choice of solvent
Good solvent
low toxicity
ease of evaporation at low heat
rapid physiologic absorption of the extract
preservative action

COOL EXTRACTION HOT EXTRACTION

Fresh sample
Grinded in a blender
Added solvent (95 % ethanol)
Shaken vigorously for 5 to 10 min
Left for 1 week
Filter
filtrate
Dry under reduced
pressure
Crude extract (calculate the
yield % and continuing )
dry sample
Grinded in a blender
Placed in the soxhlet assembly
Added solvent (95 % ethanol)
Heat the solvent for 6 hr
Filter
filtrate
Dry under reduced pressure
Crude extract (calculate the yield
12
% and continuing)
Antimicrobial Activity
Drugs that destroy microbes, prevent their multiplication or growth, or
prevent their pathogenic action

Agar well method

one fungal strain
The five bacterial strains
Candida albicans.
Bacillus subtilis,
Staphylococcus aureus,
Pseudomonas aeruginosa,
bacillus pumilus and
Escherichia coli.

13
Antioxidant Activity

Method
DPPH assay
Used instrument - Cary 60 (UV, spectrophotometry)

14
Table 1. Phytochemical Constituents in Spearmint (Mentha spicata L.)
No. Types of Extract Test reagent Observation Re
compound ma
rk
1. Alkaloids Ether, 1) Dragendorff’s reagent Orange ppt +
1% 2) Mayer’s reagents White ppt +
HCl 3) Wagner’s reagents Reddish brown ppt +
2. a – amino acids H2O Ninhydrin Purple spot +
3. Carbohydrates H2O Alcoholic a naphthol, conc Red ring +
H2SO4
4. Flavonoids EtOH 1) conc: HCl & Mg turning Pink colour +
2) NaOH sol, dilute acid Colourless after +
yellow
5. Glycosides EtOH 10 % lead acetate sol White ppt +
6. Phenolic EtOH 5 % FeCl3 Green colour +
compounds
7. Reducing H2O 1) dil HCl & neutralized with Red ppt +
sugars alkali, Fehling A & B
2) Benedict’ test Orange red ppt +
8. Saponins H2O Shaked vigorously frothing +
9. Starch H2O 10 % ethanol, I2 sol no deep blue -
10. Tannins H2O 1) gelatin sol White ppt +
2) 10 % FeCl3 Bluish black +
11. Terpenoids CHCl3 Acetic anhydride and conc Pink colour +
H2SO4
15
+ = present, - = absent
 (a) (c) (a) (c)
(a) (c)

(b) (b)
(b)

(1) (2) (3)

(a) (c) (a) (c)

(a) (c)

(b) (b)
(b)

(4) (5) (6)

Figure 3. Antimicrobial activity (zone of inhibition, mm) of various plant
extracts Spearmint, Mentha Spicata L. against clinical pathogens
(a) cool extract (b) hot extract (c) control (EtOH)
1) Bacillus subtilis 2) staphylococcus aureus 3) Pseudomonas aeruginosa 16
4) Bacillus pumilus 5) Candida albican 6) E - coli
Table 2. Antimicrobial activity (zone of inhibition, mm) of various
plant extracts Spearmint Mentha Spicata L. against clinical
pathogens

Organism
Extraction Solvent B-sub S-aureus Pseudomo B-pumilus Candida E-coli
types nas
Cool EtOH 13 mm 12 mm 11 mm 12 mm 12 mm 15 mm
extract (+) (+) (+) (+) (+) (++)
Hot extract EtOH 12 mm 12 mm 11 mm 13 mm 13 mm 14 mm
(+) (+) (+) (+) (+) (+)

Agar well = 10 mm + = 10 mm -14 mm

++ = 15 mm -19 mm
+++ = 20 mm above

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Table 3 Mean % Oxidative Inhibition of Speamint Extracts and
Ascorbic acid in Different Concentrations and Their IC50
Values

Samples Mean % Inhibition in Different Concentration (mg/mL)

0.625 1.250 2.500 5.000 10.000
Ascorbic acid 39.92 80.08 84.88 87.90 98.30
Hot extract 49.83 49.91 57.13 69.98 72.17
Cool extract 31.08 62.08 76.29 77.24 77.73

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 Ascorbic acid Hot extract Cool extract
100.00
Oxidation inhibition (%)

80.00
60.00
40.00
20.00
0.00
0.625 1.250 2.500 5.000 10.000
Concentration (m g/mL)

Figure 4 A plot of % oxidative inhibition Vs concentrations of

hot, cool extract of spearmint and standard ascorbic acid

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Table 4 Comparison of IC50 Values of Hot and Cool Extract of
Spearmint and Standard Ascorbic Acid
Samples IC50 (μg/mL)
Hot extract 1.27
Cool extract 1.01
Ascorbic acid 0.78

1.40
1.20
1.00
IC50 (m g /m L )

0.80
0.60
0.40
0.20
0.00
Hot extract Cool extract Ascobic acid
Samples

Figure 5 Comparison of IC50 values of hot and cool extract of

20
spearmint and standard ascorbic acid
 The screening of phytochemical constituent of the M.
spicata L. was also investigated. In this case, active
phytocompounds such as alkaloids, a- amino acids, flavonoids,
glycosides, phenolic compounds, saponins, tannins and
terpenoids were present in this leaves, so these compound will
active therapeutic action of some drugs, such as antimicrobial,
anthelmintic, antidirrhoeal activities.
Starch is absent in the M.spicata L. according to the
phytochemical screening, if too much starch is present, it
contributes calories and have the potential of rising the blood
sugar levels. The antimicrobial potential of both extract
was evaluated according to their zone of inhibition against
various pathogens and the results (zone of inhibition)
were compared with the activity of the standard.
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 For all the tested microorganism not significantly different between the
cool and hot extract. The maximum inhibition zone diameter was obtained in
E-coli has more potent with the diameter of 15 mm in cool extract. Similarly
in the hot extract showed maximum inhibition zone with the diameter of 13
mm in B-pumilus, Candida and E-coli.
Hot and cool ethanol extracts of spearmint, Mentha Spicata L. leaves
were subjected to screening of radical scavenging activity by DPPH assay
method. Five kinds of concentrations: 0.625, 1.25, 2.5, 5.0 and 10 μg / mL
were prepared by dilution with ethanol. Ascorbic acid was used as a
standard. The IC50 values were found to be 1.27 μg / mL for hot extract of
spearmint, 1.01 μg / mL for cool extract in 70% EtOH and all of these
extracts were found to possess the lower antioxidant activity than standard
ascorbic acid (IC50 = 0.78 μg / mL ). The high radical scavenging activity of
cool extract may be probably due to extraction method.
Thus proved the cultivation of M. spicata L. is not only adding flavor to dishes
22
but also valuable sources of natural herbal medicine.
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