Cation Exchange Properties of a Terpolymer: Synthesis and Characterization

Determination of Stevioside and Rebaudioside A in Stevia Rebaudiana Leaves via preparative High Performance Liquid Chromatography (prep-HPLC)

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:

Tel: +6094592476

Fax: +6095492766

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Determination of Stevioside and Rebaudioside A in Stevia Rebaudiana Leaves via preparative High Performance Liquid Chromatography (prep-HPLC)

Abstract

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Determination of Stevioside and Rebaudioside A in Stevia Rebaudiana Leaves via preparative High Performance Liquid Chromatography (prep-HPLC)

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

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Determination of Stevioside and Rebaudioside A in Stevia Rebaudiana Leaves via preparative High Performance Liquid Chromatography (prep-HPLC)

1.  Introduction

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Determination of Stevioside and Rebaudioside A in Stevia Rebaudiana Leaves via preparative High Performance Liquid Chromatography (prep-HPLC)

Stevia rebaudiana leaves as an herbaceous perennial plant native of Paraguay and Brazil. This wondrous herb is also known as “Sweet Weed”, “Honey-Leaf”, “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 (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.  Materials and Methods

Dried stevia leaves material which come from a zero calorie plant known for its sweetness was used about 39.5 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.

Table I. Differences mass against area percentage of analyte

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 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

Figure 3. Chromatogram of rebaudioside A standard solution for 1 mg/L concentration

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 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.

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.

REFERENCES

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