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JOURNAL OF FOREST PRODUCTS & INDUSTRIES, 2015, 4(3), 107-113 ISSN:2325–4513(PRINT) ISSN 2325 - 453X (ONLINE)
Research Article 107
Histology, Photochemistry and Microbiology of
Spearmint [Mentha Spicata L. (Labiaceae)]
El Rasheed Ahmed Salim*1, Sakina Yagi
2 and Heba Mahoud M. Elyass
3
(1)*El Rasheed Ahmed Salim, Food Industry Dept., IRCC, Khartoum, Sudan.
Corresponding author E-mail: rasheedahmedsalim@hotmail.com.
Mobile: +249912204672 - +249122223676. (2)
Faculty of Science, University of Khartoum, Sudan. (3)
Faculty of Science University of Khartoum, Sudan
(Received: February 20, 2015; Accepted: July 21, 2015)
Abstract-This study was conducted to
investigate the histology, phytochemistry
and antimicrobial activities of spearmint
plant [Mentha spicata L. (Labiatae)]
obtained from Kuku area, Khartoum
Sudan.
Histology conducted by preparation,
fixation, dehydration, clearing, wax ebeding,
sectioning and mounting for microscopical
examination. Phytochemicls such as
moisture determined according to FAO
manuals, oil content determined according
to British pharmacopeia and physico-
chemicals determined according to BS2073.
The antimicrobiolcal activity test
investigated by preparation of extracts and
using four standard organisms;
staphylococcus aureus, Klepsella sp., E. coli
and Pseudomonas aeruginosa obtained from
Medicinal and Aromatic Plant Institute,
National Centre of Research, Sudan.
Histological features of spearmint result
showed upper epidermis (U.ep), lower
epidermis (L.ep), collenchyma (Coln), oil
gland (O.g), plastid cells (Plc), cuticle (cut)
and spongymesophyll (Sp). Phytochemical
of spearmint resulted showed the following
values; moisture content (75.5%), oil
content (0.83% (v/w)), refractive index
(1.4550), specific gravity (0.9375), odor and
taste (anise odor), evaporation (no traces
left), acid value (1.07) and ester value
(12.62). Microbilogical activities of
spearmint against staphylococcus aureus,
Klepsella sp., E. coli and Pseudomonas
aeruginosa resulted in high inhibition
against the four above microorganisms.
Index terms: Spearmint, Histology,
Antibacterial, Phytochemistry.
I. INTRODUCTION
Spearmint essential oil is very famous oil
through out the world. It was used oil in
medicines, perfumery, food and other
industries [1], [2]. The oil is also used in
carminative, pharmaceutical, antiseptic, tooth
paste, soap, insecticides, perfumery and food
industries [1], [2], [3]
Anatomical features of spearmint are avital
process for clear identification of the plant and
further investigations in plant anatomy,
phytochemistry and other uses, and plant
feature may differ to some extend externally or
internally from one location to another.
Phytochemical studies of spearmint oil is
important for identification of quality
assurance and other uses of essential for
medicinal and food purposes, because volatile
oil content and composition differ according to
climate and others [4] found that the amount of
oil contain to be 0.6 % of fresh leaves and
stems [5]. Other workers reported amounts of
0.75 – 2.00 ml / 100g dry weight [6], [7].
The aim of this study is to investigate
histology, phytochemistry and antimicrobial
activities of spearmint oil on staphylococcus
aureus, Klepsella sp., E. coli and Pseudomonas
aeruginosa using two concentrations (1:9 and
1:18).
II. MATERIALS AND METHODS
Source of the plant
Spearmint fresh herb was obtained from kuku
area, Khartoum north, Sudan. The plant was
kept in house-style refrigerator (5-1000C) for
further investigations.
Histology
Preparations
Soft tissue method was adopted by soaked
spearmint stem and leaves into running water
for two days and then run in calm water; water
temperature not exceed 600C.
JOURNAL OF FOREST PRODUCTS & INDUSTRIES, 2015, 4(3), 107-113 ISSN:2325–4513(PRINT) ISSN 2325 - 453X (ONLINE)
108
Fixation Leaves and stems were collected into
formaline: acetic acid: alcohol
(FAA)(5:5:90v/v). The fixative was changed
repeatedly till the solution looked transparent,
and calmed in an oven of 600C. The heating
liquefies oil contents of gland, so that to insure
complete leaching of the oil.
Dehydration
Leaves and stems were dehydrated through
passing it into a series of concentrations of
ethyl alcohol from 50 to 95%.
Clearing
The leaves and stems cleared through passing
them in a mixture of solutes; ethyl alcohol:
cedar oil (50:50%) and cedar oil: xylene
(50:50v/v) for at least 6 hours (incubation) and
the third one was pure xylene for over night
respectively in 60 600C.
Wax embedding
The above specimens were transferred from
xylene and parafine to pure wax to form
blocks.
Sectioning
The waxed specimens were sectioned using a
rotatory microtome (Letize 1512 – West
Germany).
Staining
Dewaxing of sectioned specimens by xylene
and repeated to insure dewaxing. Then
hydration of the dewaxed specimens by
decreasing dilution of alcohol from 95 to 50%
followed by hematoxylene for 60 minutes, then
finally in running water for 15 minutes. Then
again dehydration was conducted by passing
the specimens through ammonified water (4%)
for 30 seconds, followed by increasing
concentrations of ethyl alcohol and finally to
xylene.
Mounting
The prepared sections were mounted on
Canada balsam, and then the sections were
covered and placed immediately on hot
horizontal surface in the oven at 60 600C for
72 hours before microscopical examining.
Determination of moisture content Moisture content determined according to
FAO manuals [8].
Determination of oil content (v/w) Oil content determined according to British
pharmacopeia [9].
Physical and Chemical properties Physico-chemical determined according to
BS2073 [10], except odour and volatilization
according to British pharmacopeia (1980).
Antimicrobial activities
Preparation of extracts
The oil from fresh aerial plant parts was
extracted for 2 hours respectively. Dillution of
0.5 ml of spearmint essential oil in 4.5 ml
solvent (Methanol) (1:9) to make
Concentration A., 2.5 ml of concentration A
was further diluted with 2.5 ml solvent
(Methanol) (1:18) to makec Concentration B.
Source of microorganisms
Four standard organisms namely;
staphylococcus aureus, Klepsella sp., E. coli
and Pseudomonas aeruginosa were obtained
from Medicinal and Aromatic Plant Institute,
National Centre of Research, Sudan.
Test of extracts for antimicrobiolcal activity
To determine the effectiveness of spearmint oil
against the above four organisms’ cup-plate
agar diffusion method was adopted, with some
minor modification to assess the antibacterial
activity of the prepared extracts.
Two ml of the standard bacterial stock
suspension (10-10) colony forming units per
ml were thoroughly mixed with 200 ml of
sterile nutrient agar which was maintained at
450C 20 ml aliquots of the inoculated nutrient
agar distributed into sterile petridishes.
Alternate cups were filled with 0.1 sample of
each of the concentrations using standard
Pasteur pipette and allowed to diffuse at room
temperature for two hours. Two replicates
were carried out for each extract against each
of the test organisms. After incubation the
diameter of the resultant growth inhibition
zones were measured, averaged and the mean
value was tabulated.
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109
III. RESUTS AND DISCUSSION
Fig. 1. Transverse section through the leaf (X 100) of spearmint showing, upper epidermis (U.ep), lower
epidermis (L.ep), collenchyma (Coln), oil gland (O.g), plastid cells (Plc), cuticle (cut) and spongymesophyll.
Fig. 2. Transverse section through the leaf (X 200) of spearmint showing oil gland (O.g) embedded
between the epidermis cells (U.ep).
Histology Spearmint leaf histology with respect to oil
gland structure was shown in plate (1). Oil
gland was located embded between the
external epidermis of mint leaf, plate (2).
Focusing on plate (1) and (2) showed upper
epidermis (U.ep), lower epidermis (L.ep),
collenchyma (Coln), oil gland (O.g), plastid
cells (Plc), cuticle (cut) and spongmesophyll.
Oil gland was found to be born in the external
parts (both upper and lower epidermis) of the
leaves. This finding agreed with Abu-zeid who
said that oil secretory structure of mint plant
was found in an external structure known as
gland hairs [1]. Also the above results were
concide with Parry who illustrated the presence
of oil glands on both the upper and lower
epidermis of the leaves [11]. Spearmint
microanotamy of stem was shown in plate (3).
The feature of square shape of spearmint stem
and other anatomical features of the stem such
as endodermis (End), epidermis (Epd),
collenchyma (Coln), oil gland (O.g), cuticle
(Cut) , xylem (Xv) cortex (Cox) and Pith.
Figure (4) and (5) showed the presence of oil
gland on external structure of spearmint stem,
surrounded by thin layers of cells. This finding
agree with Abu-zeid [1] who indicated the
presence of oil secretory structure on the
external part of the stem and also to Parry who
illustrated the presence of oil glands on stem
exodermis of mint leave [11] (appendix 1).
Coln O.g Cut Plc
L.ep
Spg Coln
o.g
U.ep
U.ep
ep
O.g
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Fig. 3. Transverse section through the whole stem (X 100) of spearmint showing, endodermis (End), epidermis
(Epd), collenchyma (Coln), oil gland (O.g), cuticle (Cut) , xylem (Xv) and cortex (Cox) and Pith.
Fig. 4. Transverse section through the whole stem (X 200) of spearmint showing oil gland embedded in
the exodirmis.
Cut End Cox Epd
xy
O.g Pith Col
n
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111
Fig. 5. Transverse section through the whole stem (X 250) of spearmint showing oil gland
embedded in the endospirmis.
Phytochemistry
Moisture content, oil content, refractive index,
specific gravity, odor and taste, acid and ester
values of fresh herb of spearmint were shown
in table (1). Spearmint yielded higher amount
of oil content reach 0.83%, which exceeded the
range 0.4 – 0.6 % that mentioned by Abu-zeid
[1]. This finding may be due to the variety,
cultural practices, and soil or
microenironmental effect. Refractive index
observed was 1.4550 which was lower than
what mentioned by Guenther [12]. This result
may be due to inter and intra regional or/and
autogenetic differences under either direct or
indirect effect of environmental conditions. It
is well documented that genetic constituents
and environmental factors influence the yield
of volatile oils produced by medicinal plants
[13], [14], [15]. Spearmint odor was smooth
and near similar to fennel oil odor due to
presence of carvone which is the major
constituents of both oils with the exceptional
that spearmint carvone (-) rotary and fennul (+)
rotary. These findings agree with Guenther
[12] and Abu-zeid [1] they mentioned that
carvone is a major constituent in spearmint and
fennel. Spearmint oil evaporated through 24
hours without leaving grezy traces and concide
with evaporation test consept. Acid value was
1.07 which is very low compared to Abu-zeid
[1] (1.2 -1.7), while ester value yielded best
which was fell with in the range (12.0 - 15.5)
[1].
Table (1). Moisture content, oil content, physical and chemical properities of spearmint oil
Properties Values
Moisture content 75.5%
Oil content 0.83% (v/w)
Refractive index 1.4550
Specific gravity 0.9375
Odor and taste Anise odor
Evaporation No trace left
Acid value 1.07
Ester value 12.62
Microbiolgy
Microbilogical effect of spearmint using two
concentrations (1:9) and (1:18) was illustrated
in table (2). The results showed that the two
concentrations showed antimicrobial activities
against all the four organisms (staphylococcus
aureus, Klepsella sp., E. coli and Pseudomonas
aeruginosa). These results coincide with the
antimicrobial effect of Egyptain spearmint
essential oil against staphylococcus aureus and
E. coli. [16].
Oil
gland
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Table (2). Inhibition zones of bacterial using two diluted concentrations of spearmint oil
Micoorganisms Inhibition Zones (mm)
Concentration A (1:9) Concentration B (1:18)
Staphylococcus aureus 30 (H) 31.5 (H)
Klepsella sp. 25 (H) 20 (H)
Echerichia . coli 29.5 (H) 22.5 (H)
Pseudomonas aeruginosa 25 (H) 21.5 (H)
ACKNOWLEDGMENTS
Thanks are due to the staff of the Department
of Botany, Faculty of Science (U. of K.) for
generous helpfulness to conduct histological
investigation of this research. My thanks are
also extended to the staff of the Department of
Food Research Industries (IRCC) for their
generous helpfulness to conduct extraction and
phytochemical tests of this research. Thanks
are also due to the staff in the Department
Aromatic Plants Institute (NCR) for their
generous technical assistance to conduct the
microbiological investigations and friendly
attitudes and cooperation during the course of
this study. My thanks are also extended to
every body who contributed directly or
indirectly to perform this work.
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