research article 107 histology, photochemistry and...

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: [email protected].

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.

mailto:[email protected]


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.


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


110

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


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


112

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.

REFERENCES

[1] Abu-zeid, E. N. (1992). Aromatic Plants

and their Agricultural and Pharmaceutical

Products. 1st edition. Al Dar Al Arabia for

Printing and Distribution. Cairo, Egypt. 473

p. (Arabic).

[2] Ahmed, D. G.; Elsayed, A. A.; Badwi M.

E. and Atman, A. M. (1993). Medicinal and

Aromatic Plants. Faculty of Pharmacy

Printing Press, Cairo University. Cairo,

Egypt. Pp:162-169. (Arabic).

[3] Murry, M. J.; Fass, W. and Phillips M.

(1972). Chemical composition of Mentha

arvensis var. piperascens and four hybrids

with Mentha crips harvested at different

times in India. Crop Science, volume 12.

[4] Salim, E. A.; Hassan, G. M. and Khalid,

H. E. (2013). Effect of Cultivar and

Seasonal Variation on Growth

Parameters, Yield and Oil Content of

Spearmint (Metha spicata L.) and

Japanesemint (Metha arvensis L.). Sudan

Academy of Science Journal, vol. 4 No. 2.;

Sudan Academy of Sciences, Khartoum,

Sudan.

[5] Schooley, J. (2003). Mint. Food and Rural

Affairs. Minstry of Agriculture. Ontario,

Canada.

[6] Hussein, F. T. G. (1987). The Medicinal

Plants: Cultivation and Composition.

Kymfeto Press Company, Sudan Street, Jeza,

Cairo, Egypt. (Arabic).

[7] Telci, I.; Sahbaz, N.; Yilmaz, G., and

Tugay, M. E. (2004). Agronomical and

chemical characterization of spearmint

(Mentha spicata L.) originating in Turkey.

Economic Botany, 58: 721–728.

[8] FAO (1986). Food analysis. FAO manuals,

14/7 Food and Agriculture Organization of

United Nations, Rome. Pp.205 , 227, 228,

232, 258.

[9] British Pharmacopoeia. (1968).

Anonynous. pp. 727 – 728. Pharmaceutical

Press. London, U. K.

[10] BSI (1976). Methods of test for essential

oils. BSI: 2073 British standard Institute,

London, UK.

[11] Parry, W. J. (1969). Spices morphology,

histology and chemistry. Vol. II. Pp. 96 – 99,

108 -111, 120 – 123, 136 – 137. Chemical

publishing company, Inc., New York, USA.

[12] Guenther, E. (1975). The essential oils.

5th

printing, D. Van. Nostard Company, Inc.

(Princeton, New Jersey) vol. I, 50-77, 77 -

83, Vol.III, 676 - 683.

[13] Aflatuni, A. (2005). The Yield and

Essential Oil Content of Mint (Mentha ssp.)

in Northern Ostrobothnia. Ph. D. Thesis

(Science), Faculty of science, University of

Oulu, Finland.British Pharmacopoeia.

(1968). Anonynous. pp. 727 – 728.

Pharmaceutical Press. London, UK.

[14] Omidbaigi R., Kazemi F., and Sefidkon,

F. (2004). Influence of drying methods on

the essential oil content and composition of

Roman chamomile. Flavour and Fragrance

Journal, 19(3):196-198.

[15] Ramezani, S.; Ramezani, F.; Rasouli,

F.; Ghasemi, M. and Fookian, M. H.

(2009). Diurnal variation of essential oil of

four medicinal plants species in central

region of Iran. Research Journal of

Biological Science, 4 (1):103-106.

[16] Ela, M.A.A.; El-Shaer, N. S. and

Ghanem. N. B. (1996). Antimicrobial

evaluation and chromatographic analysis of

some essential and fixed oils. Department of

Pharmacognosy, Faculty of pharm.

University of Alexandaria, Pharmazic.

Pp.993 -994; 7 ref.

http://www.itis.gov/servlet/SingleRpt/SingleRpt?search_topic=all&search_value=Mentha+Spicata&search_kingdom=every&search_span=exactly_for&categories=All&source=html&search_credRating=All

http://www.bioone.org/perlserv/?request=get-toc&issn=0013-0001&volume=58&issue=4


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Appendices:

Appendix 1. Cross section through leaf midribs, stem and oil gland mint (Parry (1969))

research article 107 histology, photochemistry and...

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