Stevia Plant

Egypt

The chemistry of the diterpene glycosides sweeteners

                The sweet diterpene glycosides of stevia have been the subject of a number of reviews (Kinghorn and Soejarto 1985, Crammer and Ikan 1986, and Hanson and De Oliveira 1993). Although interest in the chemistry of the sweet principles dates from very early in the century, significant progress towards chemical characterization was not made until 1931, with the isolation of stevioside (Bridel and Lavieille 1931a). Treatment of this substance with the digestive juice of a snail yielded three moles of glucose and one mole of steviol, while acid hydrolysis gave isosteviol (Bridel and Lavieille 1931b). Isosteviol was also obtained when steviol was heated in dilute sulfuric acid. Subsequent studies have led to the isolation of seven other sweet glycosides of steviol. Typical proportions, on a dry weight basis, for the four major glycosides found in the leaves of wild stevia plants is 0.3 % dulcoside, 0.6% rebaudioside C, 3.8 % rebaudioside A and 9.1 % stevioside.

1- Source : J.E. Brandle - brandleje@agr.gc.ca, A.N. Starratt and M. Gijzen Stevia rebaudiana , Its biological, chemical and agricultural properties   http://res2.agr.gc.ca/london/faq/stevia_rev_e.htm

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The main plant chemicals in Stevia include:

Apigenin

 Austroinulin

 Avicularin

 beta-sitosterol

 caffeic acid

 campesterol

 caryophyllene

 centaureidin

 chlorogenic acid

 chlorophyll

 cosmosiin

 cynaroside

 daucosterol

 diterpene glycosides

 dulcosides A-B

 foeniculin

 formic acid

 gibberellic acid

 gibberellin

 indole-3-acetonitrile

 isoquercitrin

 isosteviol

 jhanol

 kaempferol

 kaurene

 lupeol

 luteolin

 polystachoside

 quercetin

 quercitrin

 rebaudioside A-F

 scopoletin

 sterebin A-H

 steviol

 steviolbioside

 steviolmonoside

 stevioside

 stevioside a-3

 stigmasterol

 umbelliferone

xanthophylls

 

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What is mean Stevioides?

                I'm not sure about this, but there is a genus Stevia in the Asteraceae that grows in Paraguay and from which the artificial sweetener Stevia was derived. It was named after Pedro Jaime Esteve (d. 1566), a Spanish botanist and physician. From the form of the name stevioides, I think it likely that it means "like Stevia".

Other Meaning: Resembles Stevia (genus named for Pedro Jaime Esteve, 16th century Spanish physician and botanist)

To see More information Click History of Stevia          .

Steviosides Compounds

No

Name

Sweeting

%

1

Stevioside

150-300

60%

2

Rebandioside A (Dulcoside )

250-400

25 %

3

Rebandioside D

250-450

1

Steviolbioside

100-125

 

15 %

2

Rubusoside

100-120

4

Dulcoside A

50-120

6

Rebandioside B

300-350

7

Rebandioside C

50-120

9

Rebandioside E

150-300

Source : Eng. Mohamed Diaa ElDin Soliman (1997). Stevi Plant, Natural Concentrated sweeteners .Egyptian Society of Sugar Technologists, 28th Annual Conference , Dec. 2-4, 1997 (in Arabic)

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Structure of steviol, isosteviol and stevioside

           The structure, stereochemistry and absolute configuration of steviol and isosteviol were established, through a series of chemical reactions and correlations over 20 years after the pioneering work of Bridel and Lavieille (Mosettig and Nes, 1955; Dolder et al. 1960; Djerassi et al. 1961; Mosettig et al. 1963). Structures of these and other diterpenes and diterpene glucosides are presented in Fig. 1. Concurrent studies on the parent glycoside indicated that one D-glucopyranose residue, hydrolyzed under alkaline conditions yielding steviolbioside, was attached to a carboxyl group (Wood et al. 1955) while the other two were components of a sophorosyl group (Vis and Fletcher 1956) bound to the aglycone through a $-glycosidic linkage (Yamasaki et al. 1976). Support for the proposed stereochemistry was achieved by the synthetic transformation of steviol into stevioside (Ogawa et al. 1980). Earlier, several approaches to the in vitro synthesis of steviol had been reported (Cook and Knox 1970; Nakahara et al. 1971; Mori et al. 1972; Ziegler and Kloek 1977). Recently, spectroscopic data concerning stevioside and steviolbioside were published (Van Calsteren et al. 1993).

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Other diterpenoid glycosides

                Further investigation of extracts of S. rebaudiana leaves resulted in the isolation and identification of seven other sweet diterpenoid glycosides. Kohda et al. (1976) obtained the first two of these, rebaudiosides A and B, from methanol extracts together with the major sweet substance stevioside and steviolbioside, a minor constituent which was first prepared from stevioside by alkaline hydrolysis (Wood et al. 1955). Subsequently, it was suggested that rebaudioside B was an artifact formed from rebaudioside A during the isolation (Kaneda et al. 1977; Sakamoto et al. 1977b). Stevioside has been converted by enzymatic and chemical procedures to rebaudioside A (Kaneda et al. 1977). Further fractionation of leaf extracts led to the isolation and identification, which was aided by 13C NMR spectroscopy, of three other new sweet glycosides named rebaudioside C, D and E (Sakamoto et al. 1977a, 1977b). Both rebaudioside A and rebaudioside D could be converted to rebaudioside B by alkaline hydrolysis showing that only the ester functionality differed (Kohda et al. 1976; Sakamoto et al. 1977b). Dulcosides A and B, the latter having the same structure as rebaudioside C, were reported by another laboratory (Kobayashi et al. 1977).

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Methods of diterpenoid glycoside analysis

                A wide range of analytical techniques have been employed to assess the distribution and level of sweet diterpenoid glycosides in S. rebaudiana. These include thin layer chromatography (Tanaka 1982; Metivier and Viana 1979; Kinghorn et al. 1984; Nikolova-Damyanova et al. 1994), over pressured layer chromatography (Fullas et al. 1989), droplet counter-current chromatography (Kinghorn et al. 1982), and capillary electrophoresis (Liu and Li 1995; Mauri et al. 1996). Stevioside levels have also been determined enzymatically (Mizukami et al. 1982) and by near infrared reflectance spectroscopy (Nishiyama et al. 1992) in plant strains producing mainly stevioside. The most common analytical method, however, has been high performance liquid chromatography. Although separations have been also achieved using silica gel (Nikolova-Damyanova et al. 1994), hydroxyapatite (Kasai et al. 1987), hydrophilic (Hashimoto et al. 1978), and size exclusion (Ahmed and Dobberstein 1982a,1982b) columns, amino bonded columns have been used most frequently for the analysis of the sweet glycosides (Kinghorn et al. 1984; Liu and Li 1995; Makapugay et al. 1984; Striedner et al. 1991). Amino columns have also been used to measure stevioside and related glycosides in foods and beverages (Chang and Cook 1983; Fujinuma et al. 1986; Kitada et al. 1989). In our laboratories, a carbohydrate cartridge column with a propylamine bonded phase, has been used to analyze the diterpenoid glycosides in more than 4000 stevia leaf samples (W.A. Court unpublished). Stevioside and rebaudioside A have also been analyzed by HPLC after conversion to the p-bromophenacyl esters of steviolbioside and rebaudioside B (Ahmed et al. 1980).

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Other constituents

                In addition to the sweet diterpenoid glycosides, several other diterpenes have been isolated from stevia. Since these compounds may be part of the waste stream produced during stevia processing, their availability in large quantities could make them into valuble co-products. The first to be characterized were jhanol and austroinulin, previously obtained from other plants, and 6-O-acetylaustroinulin (Sholichin et al. 1980). Also reported were the triterpenes $-amyrin acetate and three esters of lupeol and the sterols stigmasterol and $-sitosterol, previously isolated from leaves by Nabeta et al. (1976). Jhanol, austroinulin, 6-O-acetylaustroinulin and 7-O-acetylaustroinulin as well as stevioside and rebaudioside A have been obtained from stevia flowers (Darise et al. 1983). Eight additional diterpenes, called sterebins A-H, have been isolated from leaves and identified (Oshima et al. 1986, 1988).

                Other chemical constituents of stevia have been reported. Rajbhandari and Roberts (1983) identified six flavonoid glycosides in an aqueous methanol extract of leaves: apigenin-4'-O-glucoside, luteolin-7-O-glucoside, kaempferol-3-O-rhamnoside, quercitrin, quercetin-3-O-glucoside and quercetin-3-O-arabinoside and 5, 7, 3'-trihydroxy-3, 6, 4'-trimethoxyflavone (centaureidin). The major identified components in the essential oil were the sesquiterpenes $-caryophyllene, trans-$-farnesene, "-humulene, *-cadinene, caryophyllene oxide and nerolidol and the monoterpenes linalool, terpinen-4-ol and "-terpineol (Fujita et al. 1977). Later, Martelli et al. (1985) identified 54 components of a steam distillate of dried leaves from Brazil. Of these, caryophyllene oxide and spathulenol were the main components, totaling 43%. Interestingly, these substances were not the major components in an essential oil preparation from a fresh sample of cultivated stevia plants from Italy.

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Biosynthesis of the sweet glycosides

                Steviol glycosides are derived from the mevalonic acid pathway. This is a fundamental metabolic route that provides the two C5 building block molecules, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), that are required for synthesis of all isoprenoid compounds (Chappell 1995; McGarvey and Croteau 1995).

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Steviol Biosynthesis from Geranylgeranyl Pyrophosphate

                Steviol biosynthesis was first investigated over 30 years ago (Ruddat et al. 1965; Bennett et al., 1967; Hanson and White 1968). This early work established that the initial steps leading to the steviol glycosides from GGPP are identical to those in gibberellin biosynthesis. Thus, GGPP is converted to ent-copalyl pyrophosphate (CPP) by CPP synthase (also called ent-kaurene synthase A) and ent-kaurene is produced from CPP by ent-kaurene synthase (also called ent-kaurene synthase B). Subsequent oxidation of this product at the C-19 position to ent-kaurenoic acid is assumed to occur via the action of one or more P450 monooxygenases that have yet to be identified (Hedden and Kamiya, 1997). At this point the pathways to the steviol glycosides and the gibberellins diverge. Steviol is produced by further hydroxylation of ent-kaurenoic acid at the C-13 position. This ent-kaurenoic acid 13-hydroxylase has been purified from stevia leaf extracts and partially characterized (Kim et al., 1996a). The native enzyme is a 160 kD homotetramer that requires NADPH and O2 for catalysis.

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The Glycan Side Chains

                The two oxygenated functional groups of steviol, the C-19 carboxylate and the C-13 alcohol, provide attachment points for the sugar side chains that determine the identity of the 8 different glycosides identified to date. These glycan side chains are comprised predominately of glucose residues but may also contain rhamnose (Fig. 1). The biosynthetic sequence of glycosylations that give rise to the different glycan side chains is still in the early stages of elucidation. At least three distinct glucosyltransferase activities have been identified (Shibata et al. 1991, 1995). Two of these activities have been purified and characterized. Activity I transfers glucose from UDP-glucose to the 13-hydroxy position of steviol to afford steviolmonoside. Activity IIb has a much broader substrate specificity, using steviol, steviolmonoside, steviolbioside, or stevioside as substrate for further glucosylation by UDP-glucose (Shibata et al. 1995).

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Compartmentation of Biosynthesis and Storage

                Diterpene biosynthesis has been found to occur generally in plastids of plant cells (McGarvey and Croteau 1995; Hedden and Kamiya 1997). 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 isolated stevia chloroplasts and the ent-kaurenoic acid 13-hydroxylase that converts ent-kaurenoic acid to steviol was purified from the chloroplast stroma (Kim et al. 1996a, 1996b). In contrast, the UDP-glucosyl transferases performing the glycosylations on the steviol skeleton are operationally soluble enzymes, indicating that these reactions happen outside of the chloroplast. Steviol glycosides are transported to the cell vacuole where they are stored. The glycosides accumulate in stevia leaves where they may comprise from 10 to 20% of the leaf dry weight. Thus, a large fraction of total plant metabolism is committed to the synthesis of these structurally complex molecules. The conditions that favoured selection of such high diterpene glycoside producers are not known. 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.

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Functional and sensory properties of steviol glycoside sweeteners

                Of the four major sweet diterpene glycoside sweeteners present in stevia leaves only two, stevioside and rebaudioside A, have had their physical and sensory properties well characterized. Stevioside and rebaudioside A were tested for stability in carbonated beverages and found to be both heat and pH stable (Chang and Cook 1983). However, rebaudioside A was subject to degradation upon long term exposure to sunlight. Kinghorn and Soejarto (1985) also cite numerous Japanese studies that demonstrate that stevioside is very stable.

                Phillips (1989) has summarized the early sensory research. Stevioside was between 110 and 270 times sweeter than sucrose, rebaudioside A between 150 and 320, and rebaudioside C between 40 and 60. Dulcoside A was 30 times sweeter than sucrose. Rebaudioside A was the least astringent, the least bitter, had the least persistant aftertaste and was judged to have the most favourable sensory attributes of the four major steviol glycosides (Phillips 1989, Tanaka 1997). Dubois and Stephanson (1984) have also confirmed that rebaudioside A is less bitter than stevioside and demonstrated that the bitter notes in stevioside and rebaudioside A are an inherent property of the compounds and not necessarily the result of impurities in whole plant extracts. Relative to other high potency sweeteners such as apsartame, bitterness tends to increase with concentration for both stevioside and rebaudioside A (Schiffman et al. 1994). Both stevioside and rebaudioside A are synergistic in mixtures with other high potency sweeteners such as aspartame and are good candidates for inclusion in blends (Schiffman et al. 1995). Although specialty applications may exist for the other glycosides, increasing levels of rebaudioside A in stevia leaves is a clear objective for breeding work.

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Common extraction of steviol  glycosides

             Most of the commercial processing of stevia leaves occurs in Japan and there are dozens of patents describing methods for the extraction of steviol glycosides. Kinghorn and Soejarto (1985) have categorized the extraction patents into: those based on solvent (Haga et al. 1976), solvent plus a decolorizing agent (Ogawa 1980), adsorption chromatography (Itagaki and Ito 1979), ion exchange (Uneshi et al. 1977), and selective precipitation of individual glycosides (Matsushita and Kitahara 1981). Phillips (1989) has indicated that the most favoured extraction processes involve four steps: aqueous or solvent extraction, ion exchange, precipitation or coagulation with filtration, then crystallization and drying. New methods based on ultra-filtration have been disclosed recently (Tan and Ueki 1994).

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Conclusion

                Stevia represents a new opportunity for researchers and farmers alike. A great deal of information relating to production practices and disease control is required to optimize annual transplant production for Canada. Such basic things as herbicide and fungicides registration, optimum planting and harvest times, fertilizer recommendations are all essential. Since markets exist for stevia now, production and optimization must occur in parallel. The production of remarkably high levels of one class of secondary metabolite is of significant interest for chemists, biochemists and geneticists and may prove to be a foundation for the production of new metabolites in the future. Because the safety of stevia for human consumption remains controversial, there is a clear need for further experimentation with respect to the metabolic fate of steviol glycosides.

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 References

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  • Ahmed, M. S. and Dobberstein, R. H. 1982a. Stevia rebaudiana. II. High-performance liquid chromatographic separation and quantitation of stevioside, rebaudioside A and rebaudioside C. J. Chromatogr. 236: 523-526.
  • Ahmed, M. S. and Dobberstein, R. H. 1982b. Stevia rebaudiana. III. High-performance liquid chromatographic separation and quantitation of rebaudiosides B, D, and E, dulcoside A, and steviolbioside. J. Chromatogr. 245: 373-376.
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  • Lee, J.I., Kang, K.K., and Lee, E.U. 1979. Studies on new sweetening resource plant stevia (Stevia rebaudiana Bert.) in Korea. I. Effects of transplanting date shifting by cutting and seeding dates on agronomic characteristics and dry leaf yields of stevia (English abstr.). Res. Rep. ORD 21: 171-179.
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  • Matsui, M., Matsui, K., Kawasaki, Y., Oda, Y., Noguchi, T., Kitagawa, Y., Sawada, M., Hayashi, M., Nohmi, T., Yoshihira, K., Ishidate, M., Jr, and Sofuni, T. 1996. Evaluation of the genotoxicity of stevioside and steviol using six in vitro and one in vivo mutagenicity assays. Mutagenesis 11: 573-579.
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