Modified Extraction method and Characterization of sweet compounds from Stevia Rebaudiana

Jyoti Srivastava, Mahroz Khan, Padma S Vankar


Stevia is a natural sweetener obtained from the leaves of Stevia rebaudiana (Bertoni), belonging to the Asteraceae family. The objective of the present study was to develop a rapid and effective methodology for the extraction of stevioside and rebaudioside A from Stevia leaves and to compare the yields with different extraction techniques. Modified method of extraction was done with water finally the glycosides were precipitated by methanol-water solvent (0.25:1.0 v/v). The solvent from the extract was removed by lyophilization. Extracted product was analyzed by HPLC-PDA followed by mass-spectrometric (MS) detections. The decolorized extract was separated on an NH2 column using PDA detection with excitation at 210 nm. The mobile phase consisted of acetonitrile/water (80:20 v/v) with a flow rate of 1 ml/min. Evaluative criteria for stevioside and rebaudioside yield in this new method was high. An improved analytical method was developed which may be applied to quality control of stevioside content in dried leaves of Stevia before processing.


Stevia rebaudiana, Asteraceae, Lyophilization, HPLC-PDA, Stevioside, Rebaudioside a

Full Text:



Compadre CM, Hussain RA, Nanayakkara NP, Pezzuto

JM, Kinghorn AD. Mass spectral analysis of some

derivatives and in vitro metabolites of steviol, the

aglycone of the natural sweeteners, stevioside,

rebaudioside A and rubusoside. Biomed. Environ.

Mass Spectrom. 1998; 15(4): 211-222.

Medon PJ, Pezzuto JM, Havanec-Brown JM, Nanayakkara

NP, Soejarto DD, Kamath SK. Safety assessment of some

Stevia rebaudiana sweet principles. Fed Proc. 1982;

: 1568-1982.

Geuns JMC. Molecules of Interest-Stevioside.

Phytochemistry. 2003; 64(5): 913-921.

Hutapea AM, Toskulkao C, Buddhasukh D, Wilairat

P, Glinsukon T. Digestion of stevioside, a natural

sweetener, by various digestive enzymes. J Clin Biochem

Nutr. 1997; 23(3): 177-186.

Jakinovich W, Moon C, Choi YH, Kinghorn AD. Evaluation

of plant extracts for sweetness using the Mongolian

gerbil. J Nat Prod. 1990; 53(1): 190-195.

Kinghorn AD, Soejarto DD, Nanayakkara NPD, Compadre

CM, Makapugay HC, Hovanec-Brown JM, Medon, PJ,

Kamath S, Nanayakkara. A phytochemical screening

procedure for sweet ent-kaurene glycosides in the

genus Stevia. J. Nat. Prod. 1984; 47: 439- 444.

Haga T, Ise R, Kobayashi T. A method of purifying

stevioside (English abstr.) Jap. Patent 51-131900. 1976.

Ogawa T. Decolorization and purificationof stevia sweet

component (English abstr) Jap. Patent 55-111768.

Itagaki K, Ito T, Purification of Stevioside (English abstr)

Jap. Patent 54-041898. 1979.

Uneshi H, Ise R, Kobayashi T. Purification of stevia

sweetening agent (English abstr) Jap. Patent 54-


Matsushita K, Kitahara T. Separation of stevioside and

rebaudiosideA by crystallization (English abstr.) Jap.

Patent 56-121454. 1981.

Phillips KC. Stevia: steps in developing a new sweetner,

T.H.Grenby ed., Developments in sweeteners. Elsevier

Applied Science, London. 1989; 3: 41-43. .

Tan S, Ueki H. Methods of extracting and separating

sweet substances of Stevia rebaudiana Bertoni, (

English abstr.) Jap. Patent 06-007108. 1994.

Tanaka O. Steviol- glycosides: new natural sweetners.

Trends Anal.Chem.1982; 1: 246-248.

Fullas F, Kim J, Compadre CM, Kinghorn AD. Separation

of natural product sweetening agents using over

pressured layer chromatography. J. Chromatogr., 1989;

: 213-219.

Mizukami H, Shilba K, Ohashi H. Enzymatic

determination of stevioside in Stevia rebaudiana.

Phytochemistry 1982; 21: 1927-1930.

Liu J, Li SFY. Separation and determination of Stevia

sweeteners by capillary electrophoresis and high

performance liquid chromatography. J. Liq. Chromatogr.,

; 18: 1703-1719.

Shi R, Xu M, Shi Z, Fan Y, Guo X, Liu Y, Wang C, He B.

Synthesis of bifunctional polymeric adsorbent and its

application in purification of stevia glycosides. Reactive

and Functional Polymers 2002; 50(2): 107-116.

Crammer B, Ikan R. Sweet glycosides from the stevia

plant. Chem Br 1986; 22(10):915-917.

Wheeler A, Boileau AC, Winkler PC, Compton SC,

Prakash, J., Jiang, X. Pharmacokinetics of rebaudioside

A and stevioside after single oral doses in healthy men.

Food Chem Toxicol 2008; 46: S54-S60.

Pasquel A, Meireles MAA, Marques MOM, Petenate

A J. Extraction of stevia glycosides with CO2+water,

CO2+ethanol, and CO2+water+ethanol. Braz J Chem

Eng 2000; 17(3): 271-282.

Pol J, Hohnová B, Hyötyläinen T. Characterisation

of Stevia Rebaudiana by comprehensive twodimensional

liquid chromatography time-of-flight mass

spectrometry. Journal of Chromatography A 2007;

(1-2): 85-92.

Hupatea AM, Toskulkao C, Wilairat P, Buddhasukh D.

High performance liquid chromatographic separation

and quantitation of Stevioside and its metabolites,

Journal of Liquid Chromatgraphy. 1999; 22: 1161-1170.

Kolb N, Herrera JL, Ferreyra DJ, Uliana RF. Analysis of

sweet diterpene glycosides from Stevia rebaudiana:

improved HPLC method, Journal of Agri Food Chem,

; 49: 4538-4541.

Gardana C, Simonetti P, Canzi E, Zanchi R, Pietta P.

Metabolism of stevioside and rebaudioside A from

Stevia rebaudiana extracts by human microflora.

Journal of Agri Food Chem. 2003; 51: 6618 -6622.

Koyama E, Sakai N, Ohori Y, Kitazawa K, Izawa O,

Kakegawa K, Fujino A, U, M. Absorption and metabolism

of glycosidic sweeteners of stevia mixture and their

aglycone, steviol, in rats and humans. Food and

Chemical Toxicology. 2003; 41: 875-883.

Minne VJY, Compernolle C, Toppet S, Geuns JMC.

Steviol quantification at the picomole level by highperformance

liquid chromatography. Journal of Agri

Food Chem, 2004; 52: 2445-2449.

Wang LZ, Goh BC, Fan L, Lee HS. Sensitive highperformance

liquid chromatography/mass

spectrometry method for determination of steviol

in rat plasma, Rapid commun. Mass Spectrum. 2004;

: 83-86.


  • There are currently no refbacks.

Copyright (c) 2018 Journal of Drug Discovery and Development

Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.