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Stevia (Stevia rebaudiana Bertoni): Futuristic View of the Sweeter Side of Life

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 46
Stevia (Stevia rebaudiana Bertoni): Futuristic
View of the Sweeter Side of Life
M. Angela A. Meireles1* • Gui-Min Wang2 • Zai-Bin Hao2 • Kasumi Shima3 •

Jaime A. Teixeira da Silva4**

1 LASEFI - DEA / FEA (College of Food Engineering) – UNICAMP (State University of Campinas). Rua Monteiro Lobato, 80; 13083-862 Campinas, SP, Brasil
2 College of Life Science, Northeast Agricultural University, Harbin 150030, China
3 Stevia+ Miki cho Post Office, Kagawa ken, Kita gun, Miki cho, Ikenobe 3011-2, P.O. Box 7, 761-0799, Japan
4 Department of Horticulture, Faculty of Agriculture, Kagawa University, Ikenobe, 761-0795, Japan
Corresponding author: * Meireles@fea.unicamp.br ** jaimetex@yahoo.com

Keywords: glycoside, rebaudioside, stevia, stevioside, steviol

ABSTRACT

This review summarizes several characteristics of Stevia rebaudiana (Bertoni). The plant, whose extract is commonly used as a natural
sweetener, does not as yet have any ornamental application, despite its showy leaves and petite, attractive flowers. But through this chapter we
hope to expose the importance of Stevia, not only to human health, but also its potential for ornamental greenhouse or field cultivation as a foliar
spray for pest control and in stress studies. This possibility lends itself from the particularly wide-ranging characteristics of its major sweetening
compound, stevioside. Aspects including the tissue culture, bioreactors, chemistry and genetics will reveal that Stevia may be more than just a
greener view to healthy living, but may actually become an important model plant, along with the ranks of Arabidopsis, tobacco and tomato. We
demonstrate, too how this importance may often be masked by the numbers of patents which heavily outweighs the numbers of original
publications. An open exposure of the current status of this plant through this chapter may reverse this trend, allowing R&D to find solutions to
the presently major challenge facing Stevia today: how to increase the endogenous levels of both stevioside and rebaudioside-A.

1. INTRODUCTION

Stevia rebaudiana (Bertoni) is a plant with a long history of medicinal and pharmaceutical use, with potential as an ornamental garden or pot
plant. Throughout this chapter we wish to highlight some of the aspects underlying its importance, and perhaps provide the reader with a means
to explore new biotechnological avenues to further the research field of this plant.

1.1. The plant: botany and historical importance

A B
Stevia is a herbaceous perennial plant of the Asteraceae.
Records of stevia can be found in mid-19th C. Spanish
publications. From 1899-1905, a Paraguayan botanist
Moises S. Bertoni classified stevia as Stevia rebaudiana
Bertoni after having observed that native Indian populations
consumed the herb as an infusion. It is a sweet herb
indigenous to the north-eastern corner of Paraguay
(Kinghorn et al. 1984) and grows naturally in sandy soils Fig. 1 Stevia in vegetative stage (A) and in flower (B).
near streams 25-26ºS latitude (Katayama et al. 1976),
while Valio and Rocha (1976) and Monteiro and Gifford (1988) claimed it was a critical short-day length plant. The best temperature for S.
rebaudiana growth is 20-28ºC during the day and 13-20ºC at night at 80% relative humidity. Stevia can grow up to about 65 cm tall, is sessile,
and has oppositely arranged lanceolate oblancoelate leaves to oblancoelate leaves from the middle of the plant upwards (Fig. 1A). Trichome-
like structures on the leaf surface are of two distinct sizes, one large (4-5 μm), one small (2.5 μm) (Shaffert and Chetobar 1994). The flowers are
small (7-15 mm), white and arranged in an irregular cyme (Fig. 1B). The seed is an achene with a feathery pappus (Robinson 1930) whose
embryo development conforms to the Asterad type (Shen et al. 1989). Plants can initiate flowering after 4 true leaves have been produced
(Carneiro et al. 1997) and showed from a complete diallel cross that Stevia was a self-incompatible plant (Brandle et al. 1998, Brandle 1999).

Abbreviations: R-A, rebaudioside-A; ST, stevioside

Meireles et al. 417                 Stevia

1.2. Sweet shock: compounds conferring Stevia’s sweetness

Stevia is composed of several natural, heat-stable ent-kaurene glycosides (steviol glycosides) whose intensities of sweetness and flavour
profiles differ from each other and vary according to concentration and environment (Geuns 2003). Collectively, they give stevia 100-300 times
the sweetness of sucrose (0.4% solution). The potency can vary depending on the purity and the proportion of stevioside and rebaudioside found
in the extract. Unlike sucrose and other conventional sweeteners, whose energy value is high, that of Stevia is low, hence its medicinal
importance.
Ten leaf diterpenic glycosides have been identified as having a sweet taste, the two main ones being stevioside (ST), traditionally 5-10% of
the dry weight of leaves, and Rebaudioside A (R-A), being 2-4%, i.e. a 1:2 ratio (ST:R-A), these being the sweetest compounds, and respectively
tasting about 300 and 450 times sweeter than sucrose, respectively (reviewed in Tanaka 1982, Geuns 2003, Dacome et al. 2005 and references
therein). The R-A tastes very similar to sucrose and Soejarto et al. (1982) suggested that the bitter taste, common to many Stevia species, is
probably due to sesquiterpene lactones especially due to the dominance of ST. In crossing experiments it was shown that R-A is controlled by a
single gene, which is in turn controlled by an additive multiallelic locus. S. rebaudiana is one of 154 members of the genus Stevia and only one
of two that produces sweet steviol glycosides (Robinson 1930, Soejarto et al. 1982).
The most valuable extracts, however are those that have R-A as the main component due to its organoleptic and physiochemical features,
i.e. the best taste profile from all the glycosides and the greatest solubility in water (Crammer and Ikan 1986) allowing for a wider application and
greater use in formulations.

1.3. Uses

ST has a long history as a sweetener in China, the EU, Japan, South Korea and having been used as a dietary supplement by the United States
since 1995 (Brandle et al. 1998, Kinghorn 2002, Mizushina et al. 2005).
The steviol glycoside industry is divided into 3 parts: upstream, midstream, and downstream. Upstream industry includes genetic breeding,
cultivation, plant physiology; study, improvement, and evaluation of germplasm resources; breeding, renewal, and generalization of new
varieties; prevention and treatment of plant diseases and insect pests. Upstream industry is the foundation and precondition for steady
development of industry. Midstream industry refers to the extraction, processing, and production of steviol glycoside. Downstream industry is
involved with the application of S. rebaudiana and steviol glycoside.
The dry extract from the leaves of stevia contains sweet diterpene glycosides (with a sugar equivalent of 50), flavonoids, alkaloids, water-
soluble chlorophylls and xanthophylls, hydroxycynnamic acids (caffeic, chlorogenic, etc.), neutral water-soluble oligosaccharides, free sugars,
amino acids (17 types), lipids, essential oils, and trace elements (Bakal and O’Brien 1986, Crammer and Ikan 1986, Komissarenko 1994). Along
with the sweetener stevioside, stevia also contains significant quantities of chlorogenic acid, which has hypoglycemic effects (Khramov and
Dmitrienko 2000, Gregersen et al. 2004). Kinghorn and Soejarto (1985) listed triterpene, labdane diterpene, sterol, flavonoid glycoside, tannins
and 31 volatile oils as being the main leaf components.
The Paraguayan Indians use the powered leaves and stems of S. rebaudiana in the form of a tea as an oral contraceptive centuries ago.
Not until 1970, when Sumida brought the seeds from Brazil to Japan, did its study reach Japan; some areas in Japan began to cultivate stevia in
1974. Since the 1950s, the Japanese overcame refining problems to eliminate undesirable off-flavors. Following a slow start in the 1970s, stevia
leaf extracts have been used in products in Japan for 20 years and is now used in a wide range of food applications in Japan, and in some SE
Asian and South American countries, while some ingredients of Stevia are commercially used as a low-caloric sweetener, i.e. as a sugar
substitute. The consumption of steviol glycoside increased from about 200 t/year in 2002 (Terai et al. 2002) to 300-500 t/year in Japan (Steviakai
2005). In 2002, market research results indicate that it is most extensively used as a food additive. Two important terms of steviol glycoside
additives are set out in the Food Additives (2002 ed), published by the Japan Food Additives Association, stating that: 1) the content of ST and
R-A should not be <80% of the leaf extract; 2) the purity of R-A prepared by enzyme engineering should not be <80%.
Stevia extracts obtained by various techniques (discussed in some detail below) can have a diversity of chemical composition, therefore, the
additional process steps required in order to reach the pre-established content of the target components will vary immensely. For instance, CO2-
soluble stevia extract can find numerous applications due to its particular composition while the CO2 + water (or ethanol) soluble extract finds
uses in the formulation of sweet food stuff for diets with or without sucrose or glucose. This is possible due to the stability of ST and R-A in
solution (Chang and Cook 1983).

1.4. Balancing potential toxicicity and safety

Some important research has been done to observe the effects of stevia sugars in the human intestinal microflora (Koyama et al. 2003b), the
metabolism of these sugars in humans (Koyama et al. 2002, Gardana et al. 2003) and rats (Koyama et al. 2002); on the gastric mucosa of trout
(Shiozaki et al. 2004); activity against rotavirus (Takahashi et al. 2001). In addition to that, stevia extracts have been used as an ingredient of
various beverages (Yuan and Zhou 2001, Li 2002, Han et al. 2003), germicide for treating and preventing stomach cancer or gastric ulcer (Sato
2001), against rotavirus (Takashi et al. 2001), health-drinks (Otsuka Yakuhin Kogyo KK 1998, Li 2000 2002, Lu 2000, Yuan and Zhou 2001,
Wang et al. 2002), medicine (He and Li 1997, Zhu 1997, Li et al. 2000, Lu 2000, Ji 2003, Sangi 2004), pest repellent (Sato et al. 2001),
preparation for dioxin decomposition (Kim and Kim 2001), antimicrobial agent (Sato et al. 2001, Koyama et al. 2002 2003b), in the formulation of
cosmetics and toiletries (Frey 2000), formulation of nutrient solution (Li and Chen 1995). Among these patents there is a very curious one where
stevioside is used in the formulation of a functional food claimed to improve drinking beyond capacity (Li 2004).
Since 1999, Japan had fervently applied to JECFA (Joint Expert Committee on Food Additives, an international scientific group administered
by the United Nations and the World Health Organization) for the use of steviol glycoside. Finally, in 2004, JECFA granted stevia a temporary
“acceptable daily intake” of up to 2 mg/kg of body weight (or 127 mg for a 140-pound person). But there are certain paradoxes between research

Floriculture, Ornamental and Plant Biotechnology Volume IV ©2006 Global Science Books, UK
Meireles et al. 418               Stevia

results concerning the safety aspect of steviol glycosides. We will review them below.
Some experiments identified fertility problems resulting from the administration of STs (Yodyingyuad and Bunyawong 1991); another
concern is the carcinogenicity of ST (Xili et al. 1995). Chromosomal damage was observed in animal test subjects (Temcharoen et al. 2000).
Planas and Kuc (1968) reported that female rats fed a 5% aqueuous extract of stevia displayed a severely reduced fertility which was not entirely
reversed. Rec-assay and reversion tests of crude extracts of stevia containing about 18% ST of purified extracts of stevia with about 55% ST,
and of stevia with about 55% ST, and of stevia crystals (95-98% St) were all negative. Pezzuto et al. (1985) claimed steviol to be mutagenic.
But more experiments around the world (but primarily in Japan) argued that STs are safe, confirming that ST and R-A are expelled from
body without any structural change, and have no acute toxicity to animals (e.g. Shiozu 1996, Takahashi 1997, Tomita and Sato 1997, Toyoda
and Matsui 1997, Akao 2000, Kakegawa 2001, Yasukawa et al. 2002, Koyama et al. 2003a 2003b, Kawa et al. 2004). Also no evidence of
chronic toxicity to reproduction, variation, and heredity was observed. In Brazil and Japan, steviol glycoside has been ratified as a food additive
for over 30 years. Instead of saccharine, the dosage of natural glycosides is recently increasing in many countries. Oliveira-Filho et al. (1989)
noted no adverse effects of S. rebaudiana on contraception in female rats while DuBois and Stephenson (1984), using data from animal feeding
trials in human trials, indicated that stevia leaf extracts are safe for human consumption.
Increasingly more experiments showed that STs could be used as therapeutic agents for some diseases. Mizushina et al. (2005) reported
the anticarcinogenic activity of isosteviol, which potently inhibited both mammalian DNA polymerases (Pols) and human DNA topoisomerase II
(Topo II), preventing the growth of human cancer cells. Chan et al. (2000) showed that orally-administered ST is a well tolerated and effective
modality that may be considered as an alternative or supplementary therapy for patients with hypertension. Yamada (1985) fed rats with low
concentrations of stevia extracts in their diet over a long period of time; results showed that stevia extracts had no significant chronic toxicity on
rats after hematological and blood biochemical analyses, or organ weight, macroscopic or microscopic observations. An experiment conducted in
mice (Abdula et al. 2004) showed that stevia is excreted by renal tubular epithelium and induces diuresis and natriuresis and a fall in renal
tubular reabsorption of glucose. R-A stimulates insulin secretion in a glucose-dependent manner (3.3 to 16.7 mmol/L) and only potentiated
insulin secretion at glucose >6.6 mmol/L. The effect of R-A is critically dependent on the presence of extra cellular Ca2+, i.e., R-A-induced insulin
stimulation at high glucose disappears in the absence of extracellular Ca2+. In conclusion, R-A possesses insulin tropic effects and may serve a
potential role in the treatment of type 2 diabetes mellitus (Abdula et al. 2004).
Isosteviol is a derivative of ST, commonly used as a noncaloric sugar substitute in Japan and Brazil. In this study the role of potassium
channels in the vasodilator effect of isosteviol was investigated using potassium channel blockers on isosteviol-induced relaxation of isolated
aortic rings prepared from Wistar rats. Vasodilatation induced by isosteviol is related to the opening of SK(Ca) and K(ATP) channels. Steviol
glycosides (ST, R-A, rebaudioside C, and dulcoside A) showed strong inhibitory activity against 12-O-tetradecanoylphorbol-13-acetate-induced
inflammation in mice. Takahashi et al. (2001) performed a binding assay with radiolabeled purified viruses indicating that extracts from S.
rebaudiana may bind to 37 kD VP7 and interfere with the binding of VP7 to the cellular receptors by steric hindrance, which results in the
blockade of the virus attachment to cells. Aliev and Faizullaeva (2000) suggested the use of dry stevia extract as an immunomodulant,
antioxidant, anti-inflammatory, antisclerotic, and antidiabetic remedy. It was reported that the extracts of stevia leaves could inhibitate the pyloric
Spirillum cultivated on Muller-Hinton agar. Liu et al. (2003) confirmed, by intravenous injection, that ST is an effective antihypertensive natural
product, whose mechanism of action is related to Ca2+.
It is interesting some researchers attempted to spray a solution containing stevia glycosides of some concentration to the leaves of
soybeans, watermelons and several other horticultural products, demonstrating that their yield and quality improved (Dong 2004). This may have
great applications for the floricultural industry as a foliar spray without any harmful side-effects. These studies also emphasize the need, despite
the more wide-spread use of STs, to determine their safety in parallel.

1.5. Conventional breeding, harvest conditions, and pests

Seeds and vegetative stem cuttings of Stevia can be used to propagate this plant at a planting density of 120,000-150,000 plants/ha, but the
former can produce more leaves and total weight per plant than seed-based propagation. Plant density drops every year, decresing to about
75,000 plants/ha in the 4th year. In the South of China, plant roots stored in a warm place re-sprout in spring the following year, between April and
July, at which time cuttings can be prepared. These cuttings (terminal 5-7 cm; most leaves removed) are simpy inserted into soil (2-3 cm deep),
under moist soil and low light intensity. Plants root after about 15 days. Since stevia plants are drought-sensitive, they should be watered
regularly, and planted in a mixture of sand clay (LEd2), hen manure (10% v/v), and lime (Carneiro et al. 1997). Fertilization affects ST yields.
Katayama et al. (1976) observed that stevia needed approximately 105 kg N, 23 kg P and 180 kg K per ha. In China, hen manure, livestock
manure or thoroughly decomposed bean cake are used as organic fertilizer; inorganic fertilizers use Ca(H2PO4)2, urea and KCL.
Large-scale propagation of the crop is mostly based on seeds, though seed germination is often poor and rates of less than 50% are
common. The crop’s flowering depending on the daylength sensitvity of the cultivars used for seed production. 1,000-seed weights for stevia
seed usually range between 0.15-0.30 g, depending on plant density, with seed yields of up to 8.1 kg/ha possible (Carneiro et al. 1997). The
same authors showed that the ideal medium for greenhouse cultivation was a mixture of sand clay (LEd-2) soil, laying hen manure (10% v/v) and
lime. The concentration of STs in the leaves of stevia increases when the plants are grown under long days (Metivier and Viana 1979); since
glycoside synthesis is reduced just before flowering, delaying flowering under long days will result in higher steviol glycoside yields. Nakamura
and Tamura (1985) found that total glycoside concentrations at the seedling and harvest stages were not correlated, suggesting that early
selection for total glycosides would not be effective. In contrast, leaf yield and leaf-to-stem ratio were heritable, allowing for selection for these
traits. Some procedures were developed to improve leaf yield and R-A concentration in the leaves with a resulting total sweet glycoside
concentration was reported to be as high as 20.5%, and R-A:St ratio of 9:1 (Mori et al. 1972, Shizhen 1995).
Ermakov and Kochetov (1996) showed the importance of photoperiod and irradiance, demonstrating that greatest vegetative mass could be
produced in a long day (18 hours) and at low irradiation (50 W/m2).
Under warm (>22°C) and humid (>90%) conditions, S. rebaudiana is easily susceptible to Sclerotinia scerotiorum K.F. Disease symptoms in

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Meireles et al. 419                 Stevia

4-week-old plants are dark brown lesions on aerial stems, leaves turn black and downward curling caused by the lesions (Chang et al. 1997).
The infection of stevia by Septoria steviae results in angular shiny, olive-gray lesions, often bound by a vein, and surrouded by a chlorotic halo
(Lovering and Reeleder 1996). Seedling blight, mosaic and Stevia black blotch are the main diseases in Shandong China; control measures
developed include: chlorothalonil for seedling blight, comlex mildethane for Stevia black blotch, and some anti-viral chemicals for mosaic (Zhu et
al. 2002).

1.6. Tissue culture and bioreactors

Several reports have been published on the in vitro culture of Stevia. Clonal propagation using stem segments with two axillary buds is the
easiest form of in vitro propagation (Kornilova and Kalashnikova 1996, Patil et al. 1996). Stevia was tissue cultured on an MS medium
supplemented with 0.5 mg/L BA, 0.5 mg/L NAA and 3% (w/v) sucrose on an agar-base (0.9%, w/v; Shen and Zhong 1996). Swanson et al.
(1992) reported that STs could be produced from all tissue cultured organs. Yamazaki et al. (2000) used shoot tip culture in liquid medium to
produce ST while Sikach (1998) showed that modifications of the medium resulted in different levels of the diterpene glycosides, or that
maximum production occurred under continuous white light (3,000 lux; Sugiyama et al. 1998). Liquid medium supplemented with 0.1 or 1 mg/L
BAP was found to be suitable for callus induction from anthers of greenhouse plants, while transfer of callus onto the same medium (solidified)
resulted in shoot formation; shoots could be readily rooted on 0.1 mg/L NAA medium (Flachsland et al. 1996). Root cultures demonstrate a
photosynthetic capacity (Flores et al. 1993). Colchicine-induction of cell suspension cultures resulted in high levels of polyploidy (diploid,
tetraploid, aneuploid) cells (Handro et al. 1993). Somatic embryogenesis was obtained from floret explants cultured on MS supplemented with
2,4-D (9.05 and 18.1 μM) and kinetin (0-9.29 μM), with callus forming initially at the base of the corolla and ovary (Bespalhok and Hattori 1997).
Bioreactors have been successfully been used to mass-produce Stevia. Akita et al. (1994) showed how 64.6 kg of shoots could be formed
from an initial 460 g of initial shoot primordia in a 500 L bioreactor. A 3% sucrose solution supllemented with 0.1 mg/L BA or in conjuction with
NAA resulted in increased root development, while the addition of GA3 lengthened shoots and roots in a roller bioreator (Bondarev et al. 2003).

2. THE CHEMISTRY OF STEVIA

2.1. Biochemical considerations

Ten sweet steviol glycosides have been isolated and identified from S. rebaudiana leaves (Bridel and Lavieille 1931, Crammer and Knox 1986,
Kinghorn et al. 1994, Geuns 2003, Dacome et al. 2005). These glycosides occur almost exclusively in the leaves, with small amounts in the stem,
and are non-detectable in the roots (Brandle and Rosa 1992). These glycosides taste intensely sweet and have a similar biosynthetic pathway to
those of gibberellic acid (GA), a polycyclic diterpene whose biosynthesis is ubiquitous among plants. Steviol is also a polycyclic diterpene and it
shares many structural features with GA. The tetracyclic diterpeneskeletons of steviol and GA are nearly identical. The initial steps leading to the
formation of GA result from the two-step rotonation-initiated cyclization of geranylgeranyl diphosphate (GGDP) to (±)-kaurene via the action of
two terpene cyclases (±)-copalyl diphosphate synthase (CPS) and (±)-kaurene synthase (KS) (Hanson and de Oliveira 1993, Totté et al. 2000;
Figs. 2, 3). All diterpenes are derived from GGDP and are prevalent throughout the plant kingdom (Hedden and Kamiya 1997). CPS and KS are
part of the steviol glycoside biosynthetic pathway and S. rebaudiana has recruited two genes to secondary metabolism from a highly regulated

geranylgeranyl diphosphate

-(-)copalyl diphosphate synthase

-(-)copalyl diphosphate

Steviol Sterebin D Sterebin E Sterebin F

(-)-kaurene synthase

(-)-kaurene

R=R1=H Sterebin A
R= Ac, R1=H Sterebin B
(-)-kaurene oxidase
R=H, R1=Ac Sterebin C

(-)-kaurenoic acid

Sterebin

GA aldehde steviol
12

Fig. 2 The synthetic pathway of steviol. Fig. 3 The structure of eight sweet glycosides and other diterpenes in the leaves of Stevia rebaudiana

Floriculture, Ornamental and Plant Biotechnology Volume IV ©2006 Global Science Books, UK
Meireles et al. 420               Stevia

pathway involved in hormone biosynthesis. The steps encoding steviol from GGDP, and the gene encoding CPS have been identied and
characterized (Kim et al. 1996a 1996b, Brandle 1999).
Diterpene biosynthesis generally occurs in plastids of plant cells and there is good evidence that steviol biosynthesis conforms to this
pattern and is localized in leaf chloroplasts. High levels of HMG-CoA reductase activity can be extracted from isoloted stevia chloroplast stroma
while, in contrast, the UDO-glucosyl transferases performing the glycosylations on the steviol skeleton are operationally soluble enzymes,
indicating that these reaction happen outside of the chloroplast (Kim et al. 1996a 1996b). Steviol glycosides are transported to the cell vacuole
where they are stored. The glycosides accumulate in stevia leaves where they may comprise from 10-20% of the leaf dry weight. The conditions
that favour the skeleton formation of such high diterpene producers are unknown. Like other plant secondary metabolites, the steviol glycosides
may function in a defensive capacity as feeding deterrents or anti-microbial agents against specific herbivores, pests, or pathogens. This may
allow stevia extracts to be applied as a foliar spray for ornamental greenhouse cultures.

2.2. Extraction and separation methodologies

Since France pharmaceutist Bridel and Lavieille firstly abstracted stevioside using pure ethanol as solvent in 1931, many methods have been
developed to extract Stevia glycosides but often use ST and/or R-A as an external standard (Payzant et al. 1998). Since the configuration and
polarity of glycosides have tiny differences between them, they are difficult to separate. The extraction and purification of STs are associated
almost exclusively with the use of organic solvents, such as methanol, ethanol or ether, and many require that the STs be absorbed at first on a
resin (Dacome et al. 2005) with subsequent elution with an organic solvent. We discuss briefly some of the advances in methodology below.
High Performance Liquid Chromatography (HPLC) analysis is a simple, accurate, highly sensitive and reproducible method for
determination of glycosides of Stevia (Kasai et al. 1987, Liu and Liu 1995, Dacome et al. 2005). Ahmed and Dobberstein (1982a 1982b) applied
an oxygen-containing organic stationary phase covalently bonded to a silicon atom in an inorganic support, and by changing the solvent, could
collect individually eluate fractions rich in respective glycosides. Exchange resins may be used: firstly a strongly acidic ion exchange resin, such
as Dowex 50W, then a weakly basic ion exchange resin, such as Dowex WGR, are used to treat the solution obtained from dried stevia leaves;
this solution is then concentrated and filtered; finally 75% of stevioside compounds may be obtained. Rongfu et al. (2001) developed an
adsorbent with a quaternary ammonium group, which possess defined different adsorption-adsorption properties for stevia glycosides and
coloured matter in the extract, and could be used to produce high quality stevia glycosides. Countercurrent Chromatography is a system in which
a fluid is bound to a stationary phase, while another exists as the mobile phase, and can be used from a capillary array to a microchip channel,
the sample being absorbed by the mobile phase. No case studies have been demonstrated yet. Yoda and Marques (2003) could isolate S.
rebaudiana glaucosides using CO2 supercritical fluid extraction. Thin layer chromatography (TLC) and capillary electrophoresis are very simple
methods for the detection of STs (Metivier and Viana 1979, Tanaka 1982, Kinghorn et al. 1982 1984, Fujinuma et al. 1986, Nikolova-Damyanova
et al. 1994, Mauri et al. 1996, Dacome et al. 2005). The levels of compounds in Stevia plants have been determined enzymatically (Mizukami et
al. 1982) and by near infrarred reflectance specroscopy (Nishiyama et al. 1992). Separations have been performed using silica gel (Nikolova-
Damyanova et al. 1994), hydrokyapite (Kasai et al. 1987), hydrophilic (Hashimoto et al. 1978), and size exclusion columns (Ahmed and
Dobberstein 1982a 1982b), amino-bonded columns being the most frequently used for the analysis of ST.
Several processes with a view to improving the extraction and purification of these important secondary metabolites are discussed in 3.

3. WHAT THE LITERATURE REVEALS: PAPERS VS PATENTS

One of the other purposes of this chapter was to compile recent and relevant information available for designing the process to obtain stevia
extracts. Thus, a literature search is extremely important to answer the following questions: What are the current applications of stevia extracts?
Is there any possibility to find new applications of stevia extracts? To answer these two questions a search was done in Web of Science SCI-
Expanded database. Because, products containing stevia and/or stevia extracts are found in several food or drug stores around the world and
are extensively used as an alternative calorie-free additives to other sweet sugars such as sucrose, glucose, fructose, and so on, the techniques
to cultivate stevia and to obtain stevia extracts must be available for industrial use; therefore, a search for patents can be helpful in
understanding the uniqueness of stevia and its products, therefore, a search in the Derwent Innovation Index databases was also done. The
following queries were used: i) “glycoside AND extraction”, ii) “stevia”, iii) “stevia AND extraction”, iv) “stevioside AND extraction”, v) “steviol AND
extraction”. The entire databases timespan (1945-2005 and 1966-2005, respectively) were searched; the words could be any where in the
documents. Table 1 shows the number of scientific papers and patents. Using the word stevia by itself resulted in a higher number of patents
(837) than scientific papers (329). These numbers demonstrate that stevia has found applications in various fields. The association of the word
“extraction” decreased considerably the numbers of scientific papers and patents; and, when extraction was associated to glycoside the
numbers of matches were larger than when the word extraction was associated to stevia, stevioside, or steviol. From this prospective study we

Table 1 Numbers of scientific papers and patents obtained from a search the Web of Science databases

Query Scientific papers Scientific papers Patents

Time span 1945-2005 2000-2005 1966-2005

stevia 329 87 837

steviol AND extraction 2 2 1

stevioside AND extraction 12 9 26

stevia AND extraction 17 10 42

glycoside AND extraction 108 57 172

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learned that glycoside-rich extracts have been prepared for a variety of applications and from several glycoside-rich plants (Zoecklein et al. 2000,
Gow et al. 2003 2004, Yu et al. 2003 2005, Zhuang 2005) including S. rebaudiana Table 1 shows that the number of patents related to stevia is
roughly 2.5 times larger than that of scientific papers; comparing the numbers of patent and scientific papers related to glycoside extraction,
which are present in several other plants besides stevia, the number of glycoside patents is about 1.5 times larger than the number of scientific
papers. These results confirm the uniqueness of stevia and stevia products. There are several interesting scientific papers and patents related to
the genetic improvement of stevia cultivars (Brandle and Brandle 1999, Ta Stevia KK 2002, Morita Kagaku Kogyo KK 2003, Megeji et al. 2005).
The most used process to obtain stevia extracts is the one that employs hot water, with a variety of possibilities as suggested in the
following patents: Daisel Ltd. (1978), Sunpak KK (1987), Dainippon Ink & Chem. Inc. (1995), Zubtsov et al. (2000) among others. When using
water, ethanol, hexane or other solvents, besides the stevia glycosides these solvents co-extract other substances; therefore, to obtain
commercial or standardized stevia-sugar a number of other processes including centrifugation, filtration, evaporation, crystallization are involved
in the production of stevia extracts. Now and then, new processes are made available, such as supercritical fluid extraction (SFE) (Chugai
Pharm Co Ltd. 1978, Kienle 1989, Pasquel et al. 2000, Choi et al. 2002, Yoda et al. 2003, Volikov et al. 2005).
In the next section a summary of the available processes will be presented. The current and alternative processes for the required
clarification of the aqueous and/ or alcoholic stevia extracts will also be discussed. A brief description of the SFE process will be presented along
with the more important process parameters.

3.1. Processes for obtaining stevia extracts: the road ahead Extraction (LPSE): Fixed bed or Stirred tank
•Acetonitrile + Water
•Ethyl alcohol
Natural extracts are mixtures of substances obtained from a plant, in the
•Glycerine
case of vegetable extracts, by one of the following methods: •Halogenated hydrolyzed starch
hydrodistillation (plant material is combined directly with water) or steam •Methanol (Methanol + Chloroform)
distillation (water vapor is injected inside a fixed-bed of plant material) for •Propylene glycol
volatile substances that forms the essential oils, or more adequately •Sorbitol
denoted as odoriferous or volatile oils, which are formed by a mixture of
terpenoids. Conceptually, either one of these two processes can not be Separation:
classified as an extraction process since the soluble substances are •Centrifugation
removed from the vegetable matrix due to the differences in their vapor Solid waste •Decantation Extract
pressure with that of water: as a rule, the vapor pressure of terpenoids are •Filtration
lower (these substances have normal boiling temperature above 100°C)
Clarification:
than the vapor pressure of water. Adsorption
Low pressure solvent extraction (LPSE) is understood as any process Crystallization
done at (or near to) atmospheric pressure and conducted in extracting Desorption
units operating continuously, or in a semi-batch or batch fashion using a Diafiltering
Ion-exchange
variety of organic solvents of a wide range of polarities. Another extraction
Liquid-liquid extraction
technique is SFE, in which the solvent is a pressurized gas, generally CO2 Membrane
for several applications in the food, cosmetic, and pharmaceutical Osmosis
industries. Precipitation
The mixture of solute, denoted as the extract, may or may not be Spray-dryer
Waste Ultrafiltration
standardized in terms of a target or key-component depending on the
Vacuum evaporation
application. The extract can be commercialized as a liquid, solid, or semi-
solid mixture with an assigned degree of purity, generally expressing the
content of the reference compound. Nowadays, societies are increasingly Stevia sugar
demanding the use of clean technologies. Thus, to produce a good quality
Fig. 4 Organogram of the conventional process for producing stevia
natural extract requires the use of a plant material cultivated using extracts. The extraction can be performed using one or a combination of the
environmentally safe cultivation techniques and the use of solvents solvents indicated. Separation and clarification can be done using a
recognized as safe or GRAS (generally recognized as safe) such as water, combination of the indicated processes.
ethanol, their mixtures modified or not by the addition of salts. For SFE,
because of the non-toxicity, low cost, low critical temperature (31.2°C) and relatively low critical pressure (73.4 bar), extraction with CO2 has
been considered a green or clean technique.
Fig. 4 shows an organogram indicating the major steps required to prepare stevia extracts by LPSE. In extraction step two types of
equipment can be use: a fixed bed extractor or a stirred tank reactor; several solvent can be used such as water, methanol, ethanol, and so on.
Separate of the solid waste and the extract solution can be accomplished by centrifugation, filtration, decantation, and similar processes. The
most difficult step is the clarification of the glycoside-rich fraction that is done by a combination of processes; some of them are listed in Fig. 4.
Water has been used as a solvent by Shiozaki et al. (2004) and numerous patents have been assigned (Dainippon Ink & Chem Inc. 1995,
Zubtsov et al. 2000, Ippolito et al. 2001, Goo and Lee 2003, Lisitsin and Khakimov 2003, Volikov et al. 2005). Also for water combined with other
solvents such as acetonitrile/water (1:1) (Starratt et al. 2002). Other solvents such as methanol associated or not with water have also been
used (Fuji Chem Ind Co Ltd 1976, Kumar et al. 2000), methanol-chloroform (Banshu Chomiryo KK 1977), ethyl alcohol (Dmitrienko and Sukhinin
2000), isopropyl alcohol or dioxin (Toyo Ink MFG Co. 1977), glycerine, sorbitol or propylene glycol (Sun Star Hamigaki 1980); halogenated
hydrolyzed starch was also used as solvent (Sun Star Hamigaki 1978). Purification or standardization can be accomplished by several
processes such as ultrafiltration (Kumar et al. 2000), liquid-liquid extraction with fatty alcohols (Yuan 1997) or n-hexane (Fuji Chem Ind Co Ltd
1976), adsorption (Sunpak KK 1987), precipitation with cationic high molecular mass coagulant or surfactant (Banshu Chomiryo KK 1977),
calcium hydroxide (Seikagaku Tama 1975); selective adsorption followed by desorption in a weakly basic anion-exchange resin (Snayo-Kokusa-

Floriculture, Ornamental and Plant Biotechnology Volume IV ©2006 Global Science Books, UK
Meireles et al. 422               Stevia

c
>P

c
P

P <P
T, P

P
Temperature H ea t in
ingg

EXTRACTION

Decom pr ession
Com pr ession

T, P SEPARATION
CO 2

Con den sa t ion

Entropy
Ex tra c t

Fig. 5 Flow diagram for the SFE process over the TS diagram indicating the behavior of the solvent in each step of the process.

ku Pulp 1977), crystallization (Ippolito et al. 1998). Montovaneli et al. (2004) successfully used zeolites for the clarification of aqueous stevia
extracts. Zhang et al. (2000) used membrane based process to purify stevia extracts; Shi et al. (2002) prepared a bifunctional polymeric
adsorbent and use it for the purification of stevia glycosides. Some of the solvents listed previously can not be classified as GRAS solvent, thus,
any substitution of a non-GRAS solvent by a GRAS solvent can be viewed as an improvement in the process.
Fig. 5 shows a SFE flow diagram over the temperature/entropy (TS) diagram indicating the behavior of the solvent in each step of the
process. The process consists of the following steps: (1) liquid CO2 is pumped by a high pressure pump up to the extracting pressure, (2) is
heated to the extraction temperature and fed into the extractor vessel containing the plant, (3) the solvent solubilize the soluble material, and (4)
is carried to the separator vessels. The solute/solvent mixture is separated by decreasing the pressure/heating the expansion valve. The entire
process is very similar to the operation of a refrigerant cycle. Volikov et al. (2005), Kienle (1989), Pasquel et al. (2000), and Yoda et al. (2003)
used SFE to remove the CO2-soluble substances associated to the bitter taste of stevia extracts. Yoda et al. (2003) reported that the stevia
extract obtained with CO2 contained sesquiterpenes (nerolidol 1.7%; sphatulenol 2.8%, epi--cadinol 0.4%; khusinol 0.8%), alcohol (octadecanol
3.9%), diterpenes (manoyl oxide 1.9%; 3-keto-manoyl oxide 0.6%; jhanol 6.4%; 3--acetoxy-manol 1.8%; austroinulin 30.0%) hydrocarbons (n-
tetracosane 13.5%; n-pentacosane 9.2%; n-octacosane 3.4%), traces of -sitosterol, and of the triterpenes -amyrine, -amyrine. Subsequently,
the stevia glycosides were extracted from the solid residue by SFE with CO2 and the co-solvents were water, ethanol, or the mixture water and
ethanol (Pasquel et al. 2000); Yoda et al. (2003) just used water as a co-solvent; Kienle (1989) suggested a similar process. Pasquel et al.
(2000) reported that water is the preferred co-solvent to be associated with CO2. Pasquel et al. (2000) and Yoda et al. (2003) demonstrated that
the glycoside-rich fraction obtained by SFE with CO2 + water (or ethanol) is a clear limpid liquid, the simple evaporation of the water resulted in
stevia-sugar with almost no bitter taste, rich in R-A, and ready for use. Using the two-step process we obtain 2 stevia extracts: (1) a volatile
fraction rich in austroinulin (30%) and (2) a glycoside fraction rich in R-A.
Other results for stevia glycosides extraction and purification were reported by Bondarev et al. (2003/4), Gardana et al. (2003), Kolb et al.
(2001), Minne et al. (2004), and Vanek et al. (2001).

4. FUTURE PERSPECTIVES

The benefits of Stevia as a low-calorie natural sweetener with wide-ranging, safe applications is unquestionable. Research should first be
focussed towards varietal improvement of S. rebaudiana. Since the content of glycosides in dry leaves is relatively low (<20% of total mass),
improved varieties should seek to surpass this amount. Moreover, the R-A content should surpass that of ST, and be 70% of the total glycoside,
which can then reduce the production and processing costs. This would also serve to increase the genetic base of ST, which is at present very
narrow. The second suggestion is to optimize the manufacture of steviol glycosides. At present, the most used method is the colophon method,
with a degree of crosslinking >40, its features being fast absorption and fast elution. With the rapid development of modern industry and
biotechnology, new techniques should be explored. ST and R-A isolation techniques, transforming ST into R-A by enzyme engineering, as well
as cultivation of the variety only containing ST or R-A are getting more important than ever. Transgenic technologies that engineer genes
responsible for key enzymes (Figs. 2, 3) for increased in planta production (in or ex vitro) might never be approved by increasingly scrutinous
governments and societies since Stevia is a consumable, unlike ornamentals. Tissue culture, however, holds great promise as a technique that
will evolve to take Stevia and its glycoside research into a new dimension.

Floriculture, Ornamental and Plant Biotechnology Volume IV ©2006 Global Science Books, UK
Meireles et al. 423                 Stevia

ACKNOWLEDGEMENTS
MAA Meireles is grateful to Dr S. Quispe-Condori for drawing Fig. 5.

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