Steviol and Glycosides

Cassia Lux, Philipp Martschin, Sarah Moschner, Johannes Nette | June 25, 2015

Contents

1 Structure 1

2 Occurrence and Production 1 2.1 Occurrence ...... 1 2.2 Extraction ...... 2 2.3 Synthesis ...... 2 2.3.1 Biosynthesis ...... 2 2.3.2 Synthetic Synthesis ...... 3

3 Toxicity and pharmacological Properties 4

4 Use of the Glycoside 4

5 Economics 5

1 Structure Steviol is a naturally occurring chiral diterpene from the group Kaurene. By substitution of the Hydrogen from the hydroxyl or the carboxylate, Steviol can be transformed into a wide variety of glycosides. Two prominent examples are Steviosid and Rebaudiosid A. In 1956, the structure of the steviosid was determined by Erik Vis and Hewitt G. Fletscher.

Figure 1: Steviol

2 Occurrence and Production

2.1 Occurrence Steviol is found in the leaves of the Stevia Plant. The genus Stevia contains more that 240 natural species. They can be found in the warm regions of Latin America and in parts of the United States. Because of its sweet qualities, it is commonly called sugar leaf and is used as a sweetener for centuries. In 1899 Moisés Santiago Bertoni ﬁrst described and observed the plant, noticing its sweet taste.

1 Steviol and Glykosides

Figure 2: The cultivated Stevia Rebaudiana1

Because of the growing popularity of the steviol glycosides, the species Stevia Rebaudiana is being kultivated in vast parts of Asia, North and South America, as well as in some countries in Europe, such as Spain and England.

2.2 Extraction Steviol and the other occurring glycosides can be extracted from the leaves of the plant. Using the method of Digestion, the extracts can be safely extracted, without altering them, from the tough leaves of the plant. The extract agent is normally water, but Ethanol is used as well. The extracted solution is cleansed with inorganic salts, which precipitate the pigments and impurities. Due to the insufﬁcient cleansing and the loss of the raw product, this method is not the only one used. With C4 – C8 alcohols, the diterpenpycosides can be extracted from the aqueous solution. Another option, to remove the impurities, is activated carbon, which adsorbs these. Unfortunately the coal also adsorbs Stevioside leading to a loss in product. After drying, the crystallization of stevioside can be triggered by the resumption of Methanol. This method is very useful because it can be applied directly on the cleansed extract. With usage of the column chromotog- raphy and a Chloroform/Methanol/Water eluent, the stevioside and the diterpenes can be separated. Steviol itself is not directly extractable from the plant, but it can be formed by enzymatic hydrolysis from the retrieved glycosides.

2.3 Synthesis

2.3.1 Biosynthesis The cells in the green tissue of the Stevia plant, contain the important enzymes and genes for the formation of diterpene. 15 genes are needed for the biosynthesis of Steviol, which is the basic structure of every glycoside found in Stevia. The Biosynthesis can be divided into two parts, the Methylerythritol phosphate path (MEP-Path) and the formation of Steviol and its glycosides from geranylgeranyl diphosphate (GGDP), which is also called the GA-Path because it’s similar to the synthesis of gibberallic acid.

MEP-Path The smallest components in the Biosynthesis are Pyruvate and Glyceraldehyde-3-phosphate. The following reactions are catalyzed by the genes. In the last step, Isopentylpyrophosphate (IPP) and Dimethylallypyrophosphate (DMAPP) are formed in a ratio of 5:1. These two substances are required for the synthesis of GGDP.

1 "Stevia." Specialty Cropportunities. Ministry of Agriculture, Food & Rural Affairs; Ontario, 17 Oct. 2012. Web. 20 June 2015.

2 Steviol and Glykosides

Figure 3: The MEP-Pathway2

GA-Pathway The formed GGDP is converted into the cyclic system Copalyldiphosphate. This molecule already owns a similar skeletal structure of Steviol. The catalytic reaction of CDP leads to the formation of Kaurene and eventually to Steviol. Finally, Enzymes transform Steviol into the large variety of glycosides.

Figure 4: The GA-Pathway

2.3.2 Synthetic Synthesis The total synthetic synthesis of Steviol has always been described as very difﬁcult, due to the enormity of steps, the need of catalystic genes and the low yield of product. Although, enzymes were sucessfully extracted,isolated and cloned, an easier synthesis was searched. The ﬁrst approach to a solution was made by the german chemist Reinhard Hoffman. He introduced "overbred" intermediates. These intermediates have the basic skeleton of the desired product, but posess to many bonds and residues. These intermediates should be easily to synthesise, and the removal of the extra bonds should lead to a high yield of the desired product. The american chemist Phil Baran and his team adopted this idea and used it to create a complete synthesis for Steviol. Using a linear carbon compound with an epoxid, a decalin can be created with the correct stereochemistry for the Steviol. Next, the hydroxyl formed from the epoxide is removed and with the use of light an addition of cyclobutane is preformed

2 Brandle, J.E., and P.G.Telmer. "Steviol Glycoside Biosynthesis." Everstevia. Science Direkt, 6 Oct. 2006. Web. 20 June 2015.

3 Steviol and Glykosides with an excellent yield. The resulting product is the first "overbred" intermediate. Proceeding, the olefin is transformed to a ketone using ozone, the high-energy Cyclobutane is fragmented and a low-energy Bicyclooctane is created. With zinc as a reducing agent, an unstable five-ring is formed, which is reacted with acetic anhydride to form the second "overbred" intermediate. For this intermediate, there are two possible fragmentations. Only one of them leads to the desired produkt, so the conditions of this step have to be set for the precursor of steviol to be formed in a excellent yield. With the method of Baran, only about 20% of the products are byproduct. In three further steps, Steviol is formed.

Figure 5: The total synthetic Synthesis3

3 Toxicity and pharmacological Properties

Toxicity tests have shown that stevioside is not toxic. The acute toxicity was tested on rabbits, guinea pigs and poultry with only negative results. A sample of a leaf containing 50% stevioside was fed to rats and the lethal dose of 3.4 g/kg was discovered. Concerning the topic of endangerment for the fertility, no effect has been observed. Steviol slightly promotes growth in plants and is converted by a mutant of the fungus Gibberella fuijkuroi in 1,3-Hydroxygibberelline. A test has shown, that metabolized Steviol in the organism of a mouse has a mutagenic effect. Today it is classiﬁed as harmless, as long as it’s consumed in the right amounts. Stevioside is about 200 times sweeter than sucrose, while Rebaudiosid A is approximatly 250 sweeter. Steviol, on the other hand, has no sweetness at all. None of them have a natural ﬂavor, but Stevioside and Rebaudiosid have a faint licorice-like aftertaste.

4 Use of the Glycoside

Fructose and sugar substitutes like sorbitol and xylitol are degraded by the body’s metabolism. In contrast, synthetic sweeteners are not absorbed and they have no calories. Therefore they are recommended in case of obesity. A prominent example is the new "Coke life". The main components, which are responsible for the sweetness, are Stevio- side, Rebaudioside A and C and dulcoside A. According to the World Health Organization, the global obesity epidemic is in many parts of the world one of the biggest problems to the public health. For example, the epidemic spread of diabetes mellitus is caused by obesity. With decreas- ing physical activity and therefore less daily energy needs in the modern life, the necessary nutrients have to be less concentrated in calories. Added sugar provides energy, but has little to no nutrients and degrades the nutrient-calorie ratio. One of the main reasons for the recommended reduction in the consumption of sugar is namely its contribution to the recording of unnecessary calories, as well as the fact that sugar is a "nutrient robbers" and can lead to the develop- ment of caries. In the traditional medicine, in Paraguay it’s used to treat diabetes and used as a contraceptive.

3 Docherty, Paul. "Steviol." RSC RSS. Royal Society of Chemistry, 2 Sept. 2013. Web. 21 June 2015.

4 Steviol and Glykosides

5 Economics Surveys show that in 2012 the stevia will replace the sugar by 40%, the aspartame by 57.2%, and sucralose by 52.4%. It is expected, that the world’s demand for leaves of the stevia plant will exceed 6-8 million tonnes per year in the next decade. One group of exporters are China and India, which provide a very pure reﬁning, Exports of Paraguay and Brazil are dry extracts and prepared products such as drinks, teas, sweeteners for food or pharmaceutical products. The marketing of synthetic sweeteners provided 47.5 billion USD between 07.2007 and 07.2008, and the commercialization of Stevia 5.5 Mrd.USD. It is to be expected that in 2015 a turnover of USD 8.2 billion is reached. In Japan, stevia is seen as the main sweetener of the future. Until 2005, China was the largest producer of stevia leaves (75% of world production). It is estimated that in 2007, the production in China has amountede to 5,000 tons per year and that it has expanded its capacity to 11789 tonnes per year in 2009. In October 2010, 35 countries have placed Stevia sweetener products on the market.

References [1] Peters, Eike. "Süßstoffe." Stromberg Gymnasium. Web. 20 June 2015. www.stromberg-gymnasium.de/ unterricht/faecher/ch/sueszstoffe_06/Sueszstoffe-Schriftliche_Ausarbeitung.pdf

[2] Nowotny, Markus. Entwicklung Und Bewertung Von Verfahren Zur Gewinnung Der Süßstoffe Aus Stevia Rebaudi- ana. Stuttgart: Grauer, 1995. Print.

[3] Docherty, Paul. "Steviol." RSC RSS. Royal Society of Chemistry, 2 Sept. 2013. Web. 21 June 2015.

[4] Brandle, J.E., and P.G.Telmer. "Steviol Glycoside Biosynthesis." Everstevia. Science Direkt, 6 Oct. 2006. Web. 20 June 2015

[5] Kumari, Madhumita, and Sheela Chandra. "Stevia Rebaudiana: Beyond Sweetness." Handbook of Medicinal Plants and Their Bioactive Compounds. Department of Biotechnology, Birla Institute of Technology, 2014. Print.

[6] Foerster, H. "Steviol Glucoside Biosynthesis." MetaCyc. Boyce Thompson Institute, 26 June 2012. Web. 20 June 2015.

[7] Lebensmittelchemie, Baltes, Springer Lehrbuch, 4.Auﬂage

[8] : Ikhlas A. Khan, Ehab A. Abourashed, Leung’s Encyclopedia of Common Natural Ingredients, 3. Auﬂ. Wiley, S.576ff

[9] Römpp Lexikon Chemie, 10. Auﬂage, 1996-1999: Band, S. 4247

[10] Klaus Roth: Chemische Leckerbissen, Wiley-VCH,Weinheim,2014,1,128-134