Water-Based Crystallization and Formulation of Stevioside

from Stevia rebaudiana (Bert.) As Natural Sweetener With

Antidiabetic Activity

Yohanes Martono, Fandi Ade Darmawan, November Ratuaminu, Dewi K.A.K.H

Chemistry Department, Faculty of Science and Mathematics
Satya Wacana Christian University, Salatiga, Indonesia
E-mail: yohanes.martono@staff.uksw.edu

ABSTRACT

Stevioside is compound that can be extracted from the Stevia rebaudiana (Bert.). The steviol
glycosides of stevoside have potential, functions, and characteristics as natural sweetener. The
purposes of this experiment were to optimize the crystallization method of stevioside, to indentify
stevioside in crystall, to determine the content of stevioside by using High Performance Liquid
Chromatography (HPLC), and to determine organoleptic assay by hedonic test from crystall
formulated. Based on research obtained the percent yield of each sample was 3,27%, 1,23% (w/w) in
stevioside crystall form, then 3,24%, 4,48%, 6,99% (w/w) in stevioside isolate form. The content of
stevioside in sample 1, 2, 3, 4, 5 were 45,99%, 93,17%, 0,76%, 75,39%, 6,65% (w/w), respectively.
The method developed can be used for stevioside crystallization. Stevioside crystals can be
formulated with maltodextrin as a natural sweetener. Organoleptic tests showed that the sweetness of
stevioside crystal was more than 100 times sucrose. In vivo assay using glucose tolerance test
showed that lowering blood glucose levels activity of stevioside crystal in low dosage (0.35 mg / kg
bw) was higher (71.54%) compared with 1.8 mg aspartame / kg bw (36.15% ).

Keywords: crystallization, stevioside, natural sweetener, antidiabetic

1. INTRODUCTION
There is kind of low-calorie natural sweetener that does not have a negative impact
on the health of the body is expected by the public. Among the wide variety of sweeteners,
there is a glycoside compound which can be extracted from the herb Stevia rebaudiana
(Bert.). Steviolnya glycoside compounds have potential, functions and characteristics of
sweeteners that are larger than other types of sweeteners [11]. Stevioside and rebaudioside
A are the component glycosides of principal interest for their sweetening property. Associated
glycosides include rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside F,
dulcoside A, rubusoside and steviolbioside. Stevioside is a diterpenoid glycoside, comprising
an aglycone(steviol) and three molecules of glucose. Structure of Stevioside and
Rebaudioside A are shown in figure 1 [3].
Stevioside and extracts of Stevia rebaudiana leaves are commercially available and
used in many countries including Japan and several South American countries as sweetener
for a variety of food and beverages [6]. Stevioside also has pharmacological effect as
antidiabetic agent since this compound has antihyperglycemic activity [3].
From the results of previous studies suggest that the natural glycosides S.
rebaudiana (Bert.) can be crystallized. But the yield of crystallization is still in low quantity and
has small solubility in water [8]. The aims of this research were to optimize the crystallization
method, to determine stevioside content in crystall obtained by High Performance Liquid
 Chromatography (HPLC), to create a natural sweetener formulations of the resulting crystals
and to assay hypoglycemic activity of crystall.

Figure 1. Chemical structures of stevioside and rebaudioside A

2. METHODS
Sample, Materials and Equipments
The samples used were the leaves of Stevia rebaudiana (Bert.) obtained from Bandungan,
Central Java. Chemicals used were hexane, distilled water, citric acid, CaCO 3, ethanol,
H2SO4 (MERCK, Germany), Anthrone reagent (MERCK, Germany), acetonitrile (LC, JT
Baker), methanol (LC, MERCK, Germany), standard steviosida (Wako, Japan, with a purity of
99.2%).
Equipments used were drying cabinet, technical grinder, Soxhlet, filter paper, analytical
balance (Mettler H80), water bath (Memmert LK1714), pH-meter (Hanna, Romania), rotary
evaporator (Buchi R114), spectrophotometer (Shimadzu, UVmini 1240), High Performance
Liquid Chromatography (HPLC) (Smart Line, Knauer Advanced Scientific Instruments), and
glassware.

Method
Sample Preparation
Samples were cleaned of soil, then dried with a drying cabinet for 24 hours and the
leaves were grinded by using grinder technical. A total of 150 g sample was deffated with 1 L
hexane using soxhlet for 17.5 hours.
Sample Extraction
100g samples were macerated with 1.5 L distilled water. In this step, maceration
0
optimization performed at different temperatures, at 50 and 90 C, for 60 minutes. The
solution was filtered and twice repeated maceration with 1 L distilled water, each for 30
minutes.
Optimization of Crystallization Samples
The solution was filtered and the filtrate added 50% citric acid to pH 4. The filtrate
was added CaCO3 to pH 10. The solution was filtered and added 50% citric acid to pH 6.4.
The solution was evaporated with a rotary evaporator. Concentrated solution was added by
30 mL of ethanol. Solution was added 10 g bentonite / charcoal and then filtered, these steps
were repeated three times. The filtrate was evaporated with a rotary evaporator and
recrystallized from ethanol.
Analysis Steviosida Extract with High Performance Liquid Chromatography (HPLC)
Identification steviosida was done using HPLC RP C18 stationary phase. Elution
isocratic mobile phase was done using a solvent (A) water: methanol (70:20 v / v). 76% and
(B) 24% acetonitrile. The flow rate used was 1.5 mL/min. Injected sample volume was 20 mL.
Detection separation was done using Smart Line Knauer UV detector at a wavelength of 217
nm.
 Analysis of the extract spectra Steviosida in Spectroscopy
Stevioside crystall was dissolved in solvent: H3PO4 : acetonitrile (1:1 v / v). Spectra
was recorded at wavelength of 200-400 nm. Stevioside standard was used as reference to
compare spectra pattern.

Formulation stevioside crystall as natural sweetener and Organoleptic Assay

Stevioside crystal was formulated with maltodextrin DE 35-40 in various formulations
as shown in Table 1. Hedonic test was used to organoleptic assay of sweetness on various
stevioside sweetener formulated of 25 panelists. Tests was carried out with 6 parameters
hedonic, ratings at 5 = totally unsweet, 4 = unsweet, 3 = litle bit sweet, 2 = sweet, 1 = very
sweet. Each mixture formulated was dissolved in 100 ml of water. In the current study used a
comparison of 5% sucrose solution [10].

Glucose Tolerance Test [13]

Animal testing rats were grouped into 5 treatment groups. Test subjects were fasted
(12-18 hours) to be offered a drink ad libitum, before treatment. Distribution groups as follows:
negative controll were given distilled water, positive controll was given a low-calorie sugar
solution brand "X". Treatment 1, 2, and 3 respectively - were given a solution of stevioside
formulation with 0.35, 2.50, and 4.90 mg / kg bw. All groups received a glucose load of
glucose 50%, 5 ml / kg bw at 30 min after administration of the treatment. After administering
glucose load, blood samples were taken from the lateral tail vein as many rats from 0.1 to 0.2
ml at minute -45; 0; 45; 90, and 135. Blood was then added to a solution of anti-coagulation
(NaEDTA 5%) and then centrifuged for 5 minutes at 3000 rpm. Clear solution (plasma) was
taken for measurement of blood glucose levels.

Blood Glucose Levels Assay [12]

Measurement of blood glucose levels determined by the enzymatic method using glucose
oxidase reagent (GOD-PAP). 10 mL sample / standard glucose added to 1.0 ml of glucose
oxidase reagent and then incubated for 10 min at 37 0C. Absorbance measurements were
performed every 10 minutes in 60-minute intervals at a wavelength of 546 nm. Calculation of
blood glucose levels using the formula:
[ ]( ) ) (1)

Table 1. Formulation of stevioside crystall and maltodextrin

No Stevioside Maltodextrin DE 35-40 (g)
crystall (g)
1 0,0 0,8
2 0,05 0,75
3 0,1 0,7
4 0,3 0,5
5 0,5 0,3

3. RESULT AND DISCUSSION

Optimization of Water-Based Stevioside Crystallization
Optimization-based research focused on the water based crystallization of stevioside.
The method was expected to be optimized more efficiently and effectively in the formation of
crystals. Some basic extraction-crystallization are the adjustment of pH of the solution
developed more efficiently and easily obtained, purification solution by clarification using
activated charcoal and bentonite, as well as the attainment of a state of the supersaturated
solution to form stevioside crystals. The use of water at 50 ⁰ C was found more effective to
extract stevioside. This results agree with [1]. Crystallization can be achieved by changes in
the pH of the solution in the extreme conditions [4]. Therefore, in this optimization, the change
in pH from 3 to 10 was maintained by using economical ingredients such as calcium
carbonate and citric acid. Crystal formation is strongly influenced by the achievement of a
super saturated solution, after which the super saturated solution is reached then when added
to a solvent that does not dissolve the crystals will accelerate the formation of crystals, but
this addition is redundant if it will re-dissolve crystals [5]. Therefore, the addition of ethanol to
the super saturated solution can lead to the formation of crystals. Based on this optimization,
the percent of maximum yield obtained was 6.25%.
In classical modeling based on the Arrhenius equation below, assuming that the
critical point (nucleation) will be formed, after the formation of the crystal starts to grow at the
rate of optimum growth nucleation.

Ra = R Vm dt (2)

Description: Ra: average molar optimum solution (dR / dt), R: molarity addition of reactants in
3
this case ethanol 95% (mol), Vm: molar volume of solution added (cm /mol)
When the reactants are added constantly, will form new nucleation. Furthermore, the
nucleation rate will decrease as the number of points increases the crystal nucleation and
eventually be completely replaced by the crystal growth only up to a maximum of crystal
growth [7]. So when the value of R is too large (the addition of ethanol is too much) or not in
accordance with the balance of the formation of nucleation, the nucleation will decrease
steviol glycosides (Vm decreases) and Ra did not reach the optimum. In addition, if the value
of Vm in this case is the molarity of a solution of steviol glycosides in supersaturated
conditions are not in the right balance of nucleation and increasing volumes of ethanol were
added less precise, the crystals will not form steviol glycosides.

Identification and Content Determination of Stevioside in Crystals By HPLC and

Spectroscopy
Steviosida crystallization results are analyzed qualitatively and quantitatively by
HPLC. The results of this analysis showed the percentage of crystalline glycoside obtained for
each sample are shown in Table 2.

Table 2. Data Steviosid2 Content % (w / w) of Crystals Each Repetition of Crystallization

Sampel Stevioside content Keterangan
Sample 1 45,99 Cryatal
Sample 2 93,17 Cryatal
Sample 3 0,766 Isolate
Sample 4 75,39 Isolate
Sample 5 6,65 Isolate

Qualitative analysis by HPLC steviosida known by comparing the value of time retention (tR)
of stevoside standard with tR value of each sample (Figure 2). On the results of the
chromatogram shows that in each of the highest peak is visible steviosida that have almost
the same tR stevioside standards. When compared with previous studies by [9], the levels
obtained steviosida have differences. This is because the different samples used in different
seasons so that the composition of the leaves of S. rebaudiana (Bert.) is also different.
Besides differences in the methods used greatly affect the results obtained, both when the
removal of the compounds are impurities, and the stage of crystallization.
 [A] [B]

[C] [D]

[F]
[E]

Figure 2. Crystallization Stevioside Chromatogram Results: Standard stevioside (tR = 12.117) [A], Sample 1 (tR =
11.783) [B], Sample 2 (tR = 12.117) [C]. Sample 3 (tR = 13.833) [D], Sample 4 (tR = 12.267) [E]. Sample 5 (tR =
13.233) [F], with tR is the retention time

These results were confirmed by identification using spectroscopy. The spectra can be seen
in Figure 3. Based on the identification of the pattern looks similar to a standard sample
spectra. This indicates that the sample contains steviosida.

[A] [B]
 Figure 3. Spectra of [A] stevioside standard and [B] stevioside crystall obtained

Formulation of Stevioside Crystall with Maltodextrin

To determine the level of sweetness stevioside crystall, organoleptic tests was
performed. In the current study, the sweetness test was used a comparison of 5% sucrose
solution. Organoleptic test results can be views and in Table 3. The results showed that the
sweetness level of stevioside crystall formulated with 0.05 g 0.075 g of maltodextrin has
similar of sweetness with 5% sucrose solution. It means that the stevioside crystal have a
level 100 times the sweetness of sucrose. This result agree with [11] that stevioside has
sweetness 100-300 time sucrose.

Table 3. Organoleptic Assay Results of Stevioside crystall formulated with maltodextrine

Averages of sweetness ± SE
Sample G E D C B F A
Sweetness 4,67±0,111 4,13±0,150 3,40±0,183 2,63±0,162 2,60±0,123 2,37±0,169 1,07±0,046

(a) (a) (b) (c) (c) (c) (d)

Description: Figures followed by different small letters indicate significantly different between treatments
Samples: A (stevioside: maldex = 0,0:0,8), B (5% sucrose), C (0,05:0,75), D (0,1:0,7), E (0, 15:0,65), F (sample
Tawangmangu = 0,05:0,75), G (0,2:06)
Level of sweetness: (1 = very not sweet, 2 = not sweet 3 = less sweet, 4 = sweet, 5 = very sweet)

Effect of Stevioside Crystall on Lowering Blood Glucose in Wistar rats

In the determination of the effect of stevioside crystall on lowering blood glucose
levels, stevioside crystals was used formulation at a dose of 0.35, 2.50, and 4.90 mg / kg bw.
Adjusted dose used ADI (Acceptable Daily Intake), where the maximum demand of stevioside
per day was 5 mg / kg bw [2]. Steviosida in low doses has been able to lower blood glucose
levels. These results can be seen from Table 4 and Figure 4.

Table 3. Blood Glucose Levels of Wistar Rat on various groups and period
Periods Blood glucose levels (mg/dL)
C (-) C (+) (Aspartame P1 (Stevioside P2 (Stevioside P3 (Stevioside
1,8 mg/kg bb) 0,3 mg/kgbb) 2,50 mg/kgbb) 4,90 mg/kgbb)
-45 137,0787 35,9551 39,3258 62,9213 111,2360
0 166,2921 25,8427 5,6180 79,7753 55,0562
45 20,2247 53,9326 12,3596 71,9101 48,3146
90 21,3483 50,5618 33,7079 76,4045 24,7191
135 23,5955 74,1573 21,3483 137,0787 31,4607

At a dose of 0.35 mg / kg bw, stevioside gave the effect of lowering the mean blood
glucose levels (71.54%) compared with 1.8 mg aspartame / kg bw (36.15%) (Table 4). This
dose (0.35 mg / kg bw) when converted for human consumption is by 0.28 mg / kg bw. This
means that in addition can be used as a low-calorie natural sweetener, safe for consumption
steviosida crystals even by diabetics because it does not cause a rise in blood glucose levels
but lowers blood glucose levels. This dose does not exceed the ADI established so it is safe
for health. Stevioside can increase the amount and sensitivity of insuline from pancreas. This
hormon can induce glucose absorbtion in blood and lowering its level [3].
 C-
225,0000 Aspartam 1,8 mg/kg bb
Kadar Glukosa Darah (mg/dl) 200,0000 0,35 mg/kg bb
175,0000 2,5 mg/kg bb
150,0000 4,9 mg/kg bb
125,0000
100,0000
75,0000
50,0000
25,0000
0,0000
-45 0 45 90 135
Waktu (menit)

Figure 3. Profile Curve Blood Glucose Levels (mg / kg bw) vs. Time (min) at Various Doses
Treatment

Table 4. Area Under Curve (AUC) and Lowering Blood Glucose Activity (LBGA, %) of various
treatments
Treatments AUC % LBGA
4 -
Negatife control 1,30 x 10
3
positif control (aspartam1,8 mg/kg bw) 8,34 x 10 36,15
3
Stevioside 0,35 mg/kg bw 3,69 x 10 71,54
4
Stevioside 2,50 mg/kg bw 1,48 x 10 13.85
3
Stevioside 4,90 mg/kg bw 8,97 x 10 31,00

4. CONCLUSION
The method developed can be used for stevioside crystallization. Based on research obtained
the percent yield of each sample was 3,27%, 1,23% (w/w) in stevioside crystall form, then
3,24%, 4,48%, 6,99% (w/w) in stevioside isolate form. The content of stevioside in sample 1,
2, 3, 4, 5 were 45,99%, 93,17%, 0,76%, 75,39%, 6,65% (w/w), respectively. Stevioside
crystals can be formulated with maltodextrin as a natural sweetener. Organoleptic tests
showed that the sweetness of stevioside crystal was 100 times sucrose. In vivo assay using
glucose tolerance test showed that lowering blood glucose levels activity of stevioside crystal
in low dosage (0.35 mg / kg bw) was higher (71.54%) compared with 1.8 mg aspartame / kg
bw (36.15% ).

5. ACKNWOLEDGMENT
We would thank to DP2M DIKTI of Minister of Education Indonesia for funding this research
on Hibah Bersaing program 2012.

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