June 2012, MURJPR-332
International Journal of Chemical and Environmental Engineering
Determination of Stevioside and Rebaudioside A in
Stevia Rebaudiana Leaves via preparative High
Performance Liquid Chromatography (prep-HPLC)
1
N. A. Samaha, A. D. A. Hisham a and S. A. Rahima
a
Department of Industrial Chemistry
Faculty of Industrial Sciences & Technology
Universiti Malaysia Pahang
Lebuhraya Tun Razak
26300 Kuantan
Pahang Darul Makmur
E-mail: nurlin_abusamah@yahoo.com
Tel: +6094592476
Fax: +6095492766
Abstract
Stevia rebaudiana leaves known as sweet leaf and it contain with non-caloric sweeteners (steviol-glycosides) whose
consumption could exert beneficial effects on human health. In Malaysia, the applicable of steviol-glycosides was very limited.
The sweetness of steviol-glycosides was about 300 times more than sugar produce from sugar cane. Nowadays, sugarcane has
critical value due to its high consumptions in community. The objective of this study is to determine stevioside and rebaudioside
A in Stevia Rebaudiana leaves qualitatively by using soxhlet extraction method analyzed via preparative HPLC. A series of
standard solution for both stevioside and rebaudioside A were analyzed. Samples were sequentially extracted using methanol as
the solvent and the column had been used in this study was Waters XBridge C18 column (150 mm x 4.6 mm I.D., 5µm). The
mobile phase for C18 column performed in isocratic mode elution consisting of acetonitrile:water (80:20,v/v). Stevioside were
the most abundant steviol-glycosides (Rt = 4.23 min) found in stevia rebaudiana leaves samples followed by rebaudioside A (Rt
= 4.28 min). The objective of analysis was achieved and this leads to the suggestion of using the Capcell Pak C18 – MGII
Column (250 mm x 4.60 mm ID, 5 µm) as the replacement to the C18 column that was used in this study. Also, it is a good
practice if the sample was extracted using solid phase extraction method compare to the conventional practice using sohxlet
extraction method.
Keywords: stevia-rebaudiana, steviol-glycosides, stevioside, rebaudioside A, prep-HPLC
1. Introduction
Stevia rebaudiana leaves as an herbaceous
perennial plant native of Paraguay and Brazil. This
wondrous herb is also known as “Sweet Weed”, “HoneyLeaf”, “Sweet-Leaf” and “Sweet-Herb” and which is
estimated to be 300 times sweeter than sugar cane
(Chalapathi, M.V. et al., 1997). However, Stevia
rebaudiana leaf is the only species to have the natural
sweetness to replace artificial sweeteners.
Stevia rebaudiana leaves which are extracts can
be sold as a dietary supplement. The extracts have been
used for sweetening soft drinks such as diet coke, soy
sauce, dried seafood, candies, ice-cream, chewing gum,
yoghurt, and as well as in toothpaste and mouthwash in
Japan, Korea, and Brazil (Erkucuk, A. et al., 2009).
Stevia rebaudiana plant is one of 154 members
of the genus Stevia and one of two species that produce
sweet steviol glycosides (Madan, S. et al., 2010). It
consist about nine active components of steviol
glycosides (SG) which are they, stevioside (SV),
rebaudioside A (RbA), rebaudioside B (RbB),
rebaudioside D (RbD), rebaudioside F (RbF),
steviolbioside (Stb), rubusoside (Rub), rebaudioside C
Determination of Stevioside and Rebaudioside A in Stevia Rebaudiana Leaves via preparative High Performance Liquid
Chromatography (prep-HPLC)
(RbC) and dulcoside A (DuA). Each of them contributes
their own percentage of sweet flavor to the Stevia leaf.
While, according to the research of determining
the two active compounds (SV and RbA) of Stevia leaves,
one of the best ways is by extracting among the several
types of extraction method such as soxhlet extraction,
hydro distillation, ultrasonic, and others.
The soxhlet extraction is the most conventional
of all methods and consists of a simple distillation process
repeated a number of times. Soxhlet extraction method is
a straight forward method. The sample phase is always in
contact with fresh solvent, thereby enhancing the
displacement of target compound from the matrix and the
compound are not discomposed due to the moderate
extraction condition.
In this study, the experimental procedure for this
research in Stevia rebaudiana leaves focus on one main
instrument method which is preparative High
Performance Liquid Chromatography (HPLC) for study
the qualitative analysis of the SV and RbA compounds
content in Stevia leaves. The preparative HPLC Waters
AutoPurificationTM System with the UV/Vis detector is
used in this research. It is well known as versatile
purification and good isolation solution among other
Waters HPLC models.
While for the most important component role
play in the HPLC, is the HPLC column itself. So for this
research, using the analytical column named Waters
XBridge C18 column, with features; 4.6 mm ID x 150
mm, 5µm. This column has own unique itself which is it
gives maximizing column efficiency. This XBridge
packing incorporate the use of well-characterized, state of
the art, proprietary procedures for bonding and
endcapping, that make it show very little retention loss
and exhibit a column lifetime equivalent to that sterically
hindered C18 silica bonded phase.
Currently, Stevia rebaudiana leaves have very
high demand and prices in the world market for its
popular uses in medicine and as a sweeter of drinks.
However, there is limited research on Stevia leaves
extraction in the world. Stevia leaf is also used in
production of pharmaceutical products widely.
Currently, a new crop of Stevia rebaudiana plants
produced in Malaysia. Estimate about 50-60 hectare
mostly it grown in Pahang, Selangor and Perak.
Because of the climate and rainfall in Malaysia are
suitable for Stevia to be grown commercially as
profitable ventures.
The objective of this preliminary study is to
determine the alternative method of analysis for
extraction of the stevioside and rebaudioside A
compounds using sohxlet extraction method via
preparative HPLC qualitatively.
2.
grams in this study. These leaves were purchased from
Agro Sweet Pak Long under commerce with NADIMAS
at Sungai Besi, Kuala Lumpur. The chemical reagent,
Methanol (QRec, HPLC Grade, 99.9%, M.wt 32.04
g/mol,) was used on 1.2 liter as the main solvent in this
research.
2.1 Equipments
The equipments are soxhlet extractor apparatus and
High Performance Liquid Chromatography (Preparative
HPLC Waters AutoPurificationTM System).
2.2 Sample preparation
The dried Stevia leaves are grinded into powder form
using dry blender. Then the dried powder was divided
into five samples with different quantities, from 1.0 g to
10.0 g as shown on the table of data list samples. The first
sample of dried Stevia leave was put into extraction
thimble and place sample into soxhlet extractor. The
soxhlet apparatus using 200 mL of methanol was
assembled. The thimbles are inserted and heat the reflux
within period three hours or got two cycles solutions.
Heating mantle was set at 70 ºC. The completed
extraction, when got two cycles extraction solutions, and
removed the flask. The other four samples were repeated
same steps until got two cycle’s extraction.
2.3 Standard solution preparation
The 2.5 mg of each two standards solution were
prepared by diluting with 80% methanol in water into
volumetric flask repectively in prior to sonicate them for
15 minutes.
2.4
Preparative High Performance Liquid
Chromatography (prep-HPLC)
To optimize the separation of the steviol-glycosides
which are steviol and rebaudioside A, the following
HPLC operating conditions were used. Isocratic mode
elution.
Acetonitrile:water
(80:20,v/v).
Flowrate,
1mL/min. UV detector range, 205-215 nm. Temperature,
25oC. Waters XBridge HPLC C18 Column analytical
(150mm X 4.6 ID, 5µ). The compounds in each sample
were identified by comparing their retention times with
those of the standards reference solution.
3.
Results and Discussion
The five sample solutions of dried stevia leaves
powders are extracted linear in the range mass amount of
(1.0 – 10.0 g) by through soxhlet extraction method for
first run analysis, prior to further run analysis via
preparative HPLC. Thus, mass of sample 1.0 g is
selective for the further analysis with stevioside and
rebaudioside A standard solutions. This is due to the
optimum mass of 1.0 g (37.3939 %) compare to other
four samples at the UV wavelength detection signals, 210
nm as show in Table I.
Materials and Methods
Dried stevia leaves material which come from a zero
calorie plant known for its sweetness was used about 39.5
8
Determination of Stevioside and Rebaudioside A in Stevia Rebaudiana Leaves via preparative High Performance Liquid
Chromatography (prep-HPLC)
Table I. Differences mass against area percentage of analyte
No.
Mass Sample
Area Percentage
Retention Times
(g)
(%)
(Mins.)
1
1.0
37.3939
26.971
2
2.5
25.3640
26.998
3
3.0
23. 1498
26.997
4
5.0
18.5475
27.026
5
10.0
16.1723
27.073
Figure 3. Chromatogram of rebaudioside A standard solution for 1
mg/L concentration
By reviewing the data result above (Fig. 1), the data
shows inversely proportional of these mass samples to
their area percentages. As the smaller mass of samples,
the larger area percentages of samples can be observed
which have high potential for stevioside and rebaudioside
A compounds to be detected for further analysis.
Figure 4. Chromatogram of stevioside and rebaudioside A in sample
Determination of stevioside compound in the
Stevia sample was determined as the figures above
showed. This is due to the signal UV wavelength is in the
range 205 – 215 nm and the retention time for the
stevioside compound in the sample is 4.26 minutes close
to the retention time of stevioside standard solution, 4.28
minutes. While referring the Vanek, T. et al., (2001)
research about the determination of stevioside in Stevia
plant material and fruit teas, which used same type
Waters HPLC instrument method and C18 column, was
determined the stevioside compound at the retention time,
15 minutes with UV signal at 205 nm.
Rebaudioside A compound (Fig. 3 and Fig. 4),
also was identified in the Stevia leaves sample at the same
retention time of stevioside compound, 4.26 minutes.
Although the retention time of both compounds in their
standard solutions respectively, were different in retention
time about 5 minutes. This can be said that, after through
a few references journal, they reported that the
percentages extracted between them are similar in the
extracted stevia sample.
This statement above, was approved by taken
one of a few previous researches, that the 63rd JECFA
reported, where the commercially available extracts of
Stevia leaves are stevioside and rebaudioside A, in
Figure 1. Graph plotted for different mass sample versus area
percentage from the chromatogram
3.1
Qualitative determination of Stevioside
and Rebaudioside A
Figure 2. Chromatogram of stevioside standard solution for 1 mg/L
concentration
9
Determination of Stevioside and Rebaudioside A in Stevia Rebaudiana Leaves via preparative High Performance Liquid
Chromatography (prep-HPLC)
various amounts ranging from about 10-70% stevioside
and 20-70% rebaudioside A. On the other hand, in the
journal of Cacciola, F. et al., (2010) and Pol, J. et al.,
(2007) also reported that the stevioside compound most
abundant steviol glycoside (4-13% w/w) in plant leaves
followed by rebaudioside A (2-4% w/w) and other seven
minor active compounds of steviol glycoside.
The chromatogram for the separation stevioside
and rebaudioside A in the Stevia sample were showed
that, their peaks are broadening, give poor result of
resolution. The soxhlet extraction method applies in this
research produces large quantities of stevioside and
rebaudioside A.
The mass of dried Stevia leaves sample
extracted is 1 gram. Due to the Pol, J. et al., (2007),
Woelwer-Rieck, U. et al., and Gardana, C. et al., (2010)
researches currently, used the amount of mass dried
Stevia leaves less than 1 gram in the range 0.25 – 0.8
grams.
[7]
[8]
[9]
[10]
[11]
ACKNOWLEDGEMENT
The authors thank Universiti Malaysia Pahang for the
equipment services from Central Laboratory. We also
thank Professor Dr. Mashitah Mohd Yusoff for her kindly
supporting us in giving ideas for this labwork.
[12]
REFERENCES
[1]
[2]
[3]
[4]
[5]
[6]
[13]
Cacciola, F., Delmonte, P., Jaworska, K., Dugo,
P., Mondello, L. and Rader, J. I. 2010.
Employing
ultra
high
pressure
liquid
chromatography as the second dimension in a
comprehensive two-dimensional system for
analysis of Stevia rebaudiana extracts. Journal of
Chromatography A.
Chalapathi, M.V. and S. Thimmegowda, 1997.
Natural non-calorie sweetener stevia (Stevia
rebaudiana Bertoni): A future crop of India.
Crop Research Hisar, 14 (2): 347-350.
A., Akgun, I.H. and Yesil-Celiktas, O. 2009.
Supercritical CO2 extraction of glycosides from
Stevia rebaudiana leaves: Identification and
optimization. Journal of Supercritical Fluids.
51: 29-35.
European Stevia Association. 2006. Steviol
glycosides as food additive. Document 3, 2007.
Gardana, C., Scaglianti, M. and Simonetti, P.
2010. Evaluation of steviol and its glycosides in
Stevia rebaudiana leaves and commercial
sweetener by ultra-high-performance liquid
chromatography-mass spectrometry. Journal of
Chromatography A. 1217:1463-1470.
Geuns, J.M.C. 2003. Molecules of interest
stevioside. Journal of Phytochemistry. 64:913.
[14]
[15]
[16]
10
Joint Expert Committee on Food Addictive.
1956. Steviol glycosides, Chemical and
Technical Assessment. Document 3, 2007.
Jumpatong, K., Phutdhawong, W. and
Buddhasukh, D. 2006. Dechlorophyllation by
electrocoagualation. Journal of Molecules. 11:
156-162
Liu, J. and Li, S.F.Y. 1995. Separation and
determination of stevia sweeteners by capillary
electrophoresis and high performance liquid
chromatography.
Journal
of
Liquid
Chromatographic. 18: 1703-1719.
Madan, S., Ahmad, S., Singh, G.N., Kohli, K.,
Kumar, Y., Singh, R. and Garg, M. 2010. Stevia
rebaudiana (Bert.)Bertoni – A Review. Journal
of Indian Natural Products and Resources. 1(3):
267-286.
Pieri, V., Belancic, A., Morales, S. and Stuppner,
H. 2011. Identification and quantification of
major steviol glycosides in Stevia rebaudiana
purified extracts by 1H NMR Spectroscopy.
Journal of Agricultural and Food Chemistry.
59:4378-4384.
Pol, J., Hohnova, B. and Hyotylainen, T. 2007.
Characterisation of Stevia Rebaudiana by
comprehensive
two-dimensional
liquid
chromatography
time-of-light
mass
spectrometry. Journal of Chromatography A.
1150: 85-92.
Torabi, K., Karami, A., Balke, S. T. and Schunk,
T.C. 2001. Quantitative FTIR detection in sizeexclusion chromatography.
Journal of
Chromatography A. 910:19-30.
Sivaram, L. and Mukundan, U. 2003. In vitro
culture studies on stevia rebaudiana, in vitro
cellular and development biology. Journal of
Plant. 39:520-523.
Vanek, T., Nepovim, A. and Valicek, P. 2001.
Determination of stevioside in plant material and
fruit teas. Journal of Food Composition and
Analysis.14: 383-388.
Woelwer-Rieck, U., Lankes, A., Wawrzum, A.
and Wiist, M. 2010. Improved HPLC method for
the evaluation of the major steviol glycosides in
leaves of Stevia rebaudiana. Journal of Eur Food
Res Technol. 231: 581-588.