M00752

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_mart@yahoo.co.id.

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.

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, CaCO3, 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 optimization performed at different temperatures, at 50 and 90 0C, 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)

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

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 this case ethanol 95% (mol), Vm: molar volume of solution added (cm3/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

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.

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

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].

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

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.

6. REFERENCE

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Stevioside 0,35 mg/kg bw 3,69 x 103 71,54

Stevioside 2,50 mg/kg bw 1,48 x 104 13.85

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