Chemical composition of the essential oils of Stevia rebaudiana ...

Chemical composition of the essential oils of Stevia rebaudiana Bertoni leaves
/ Asian Journal of Traditional Medicines, 2010, 5 (2)
while in the United States it is used as a dietary
supplement and in Bangladesh as an antidiabetic
tea. Stevia rebaudiana Bertoni sweetener extracts
have been suggested to have a beneficial effect on
human health because of their antihypertensive [10-11] ,
antihyperglycemic [12] , anticariogenic [13] and antihuman
rotavirus activities [14] . These sweeteners are
also thought to influence glucose metabolism [15-16]
and renal function [17] . The increasing importance
of essential oils in various areas of human activity
(pharmacy, cosmetics as well as the food and drink
industries) has made this field very interesting
for chemical investigators. Current research into
Stevia rebaudiana Bertoni essential oil, is very
active. However, it should be remembered that the
sweetening effect is simply a pleasant by-product. The
primary reason that stevia is combined with other
herbs is to enhance the nutritive value of these other
herbs! Stevia is, after all, nutrient-rich, containing
substantial amounts of protein, calcium, phosphorous
and other important nutrients [18-19] .
Carrying the above considerations a step further,
there are many very legitimate reasons for using
stevia as a medicinal food. In spite of the prominence
stevia has attracted as a flavor enhancer, it contains
a variety of constituents besides the steviosides
and rebaudiosides, including the nutrients specified
above and lots of sterols, triterpenes, flavonoids, and
tannins, as well as being an extremely rich volatile
oil containing lots of aromatic agents, aldehydes,
monoterpenes and sesquiterpenes [20] . These and other,
as yet unidentified constituents, probably have a variety
of effects on human physiology and may help explain
some of the reported therapeutic uses of stevia.
Therefore, the aim of the present study is to
examine the chemical composition of the essential oils
isolated from the leaves of Stevia rebaudiana Bertoni
by GC–MS.
Material and methods
Plant material
The green leaves of Stevia rebaudiana Bertoni
were collected from the Bangladesh Sugarcane
Research Institute, Ishurdi, Pabna, Bangladesh. The
plants were harvested during the month of September,
2009. The leaves were collected at 2:00 pm-3:00 pm
on September 2, 2009 and packed in polyethylene
bags and stored at 4 °C until required. All chemicals
used were of research grade purity.
GC-MS analysis
The GC–MS analysis of the essential oil samples
of Stevia rebaudiana Bertoni was performed using a
Varian GC–MS instrument (Model Varian CP 3800,
USA) equipped with a VF-5 fused silica capillary
column (30 m×0.25 i. d. mm film thickness 0.25 µm,
Varian, USA). For GC–MS detection, an electron
ionization system with ionization energy of 70 eV
was used. Helium gas was used as a carrier gas at
a constant flow rate of 1 ml/min. The injector and
mass transfer line temperatures were set at 250 °C
and 300 °C, respectively. The oven temperature was
programmed from 50 °C to 200 °C at 8 °C/min, and
then kept isothermal for 20 min and finally raised to
300 °C at 10 °C/min. Diluted samples (1/100 v/v, in
methanol) of 0.2 µl were manually injected in splitless
mode. Identification of compounds of the essential oil
was based on GC retention times on the VF-5 capillary
column, with computer matching of mass spectra with
those of standards (Mainlab, Replib and Tutorial data
of GC–MS systems).
Extraction and isolation of essential oil
The air-dried leaves and stems (250 g each)
of Stevia rebaudiana Bertoni were subjected to
hydrodistillation for 3h using a Clevenger type
apparatus. The distillate was mixed with 1g NaCl and
20 ml dichloromethane (DCM) in a separatory funnel,
and shaking was continued by hand for 40 min and the
mixture was then allowed to stand for 15 min. Finally,
the DCM layer was collected in a round bottom flask
for rotary evaporation and evaporated at 30 °C until
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